CN102167744A - Human anti-CD20 monoclonal antibody and preparation method and application thereof - Google Patents
Human anti-CD20 monoclonal antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology and particularly discloses a human anti-CD20 monoclonal antibody and a preparation method and application thereof. In the preparation method, a large-capacity natural human phage antibody library is constructed and the antibody library is screened to obtain a human anti-CD20 antibody 3C5, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8. In addition, the invention also discloses a preparation method of the antibody 3C5, a nucleotide sequence coding the antibody 3C5 and an expression vector and host cell containing the nucleotide sequence. Compared with the other anti-CD20 antibodies, the human monoclonal antibody 3C5 has higher antibody affinity and ADCC and CDC activities; and the antibody 3C5 can be used to prepare anticancer medicaments.
Description
Technical field
The present invention relates to biological technical field, more specifically, the invention discloses a kind of total man's resource monoclonal antibody, Preparation Method And The Use.
Background technology
Malignant tumour is the disease of world today's serious harm human health, is in second in death due to the various diseases.Non-Hodgkin lymphoma (Non Hodgkin ' s lymphoma NHL) is clinical modal lymphsystem malignant tumour, and what wherein the B cell was originated accounts for 85%, good sending out in person between twenty and fifty, and sickness rate and case fatality rate are ascendant trend year by year.According to state of an illness characteristics, NHL can be divided into basic, normal, high three degree.The wherein low and part moderate NHL course of disease is made slow progress, and is referred to as inertia NHL.They are to chemotherapy sensitivity first, but are easy to recurrence or resistance, and when chemotherapy or radiotherapy once more, curative effect obviously reduces, thereby are considered to the malignant tumour that is difficult to cure.In recent years, the clinical experimental study of monoclonal antibody and targeted therapy NHL thereof has been obtained major progress, wherein be widely used and fruitful be the monoclonal antibody preparation of anti-CD20.
CD20 antigen is a kind of B cell differentiation antigen, only is positioned at pre B cell and mature B cell, and it is expressed in the B cell lymphoma more than 95%, and surface density is very high, and does not express in hemopoietic stem cell, plasma cell and other healthy tissuess.What is more important, the CD20 molecule is with after monoclonal antibody combines, and do not have remarkable internalization and comes off, so become desirable target spot [the Sacchi S of treatment B cell lymphoma, Federico M, Dastoli G, Fiorani C, Vinci G, Clo V, Casolari B.Treatment of B-cellnon-Hodgkin ' s lymphoma with anti CD 20monoclonal antibody Rituximab.CritRev Oncol Hematol, 2001,37 (1): 13-25].
Adopt anti-CD20 antibodies treatment non-Hodgkin lymphoma to obtain curative effect preferably at present.Rituxan (Rituximab, C2B8) with CD20 the monoclonal antibody of target spot for U.S. Gene science company development, it is a kind of people mouse mosaic gene engineered antibody, the variable region gene of mouse monoclonal antibody and constant region gene [the Reff ME of people's antibody have been comprised, Carner K, Chambers KS, Chinn PC, LeonardJE, Raab R, Newman RA, Hanna N, Anderson DR.Depletion of B cells in vivo bya chimeric mouse human monoclonal antibody to CD20.Blood.1994,83 (2): 435-45].Rituxan has obtained the approval listing of FDA in November, 1997, clinical treatment [the Leget GA that is used for recurrent or intractable minuent or folliculus non-Hodgkin lymphoma, CzuczmanMS.Use of rituximab, the new FDA-approved antibody.Curr Opin Oncol.1998; 10:548-551].Although C2B8 antibody has demonstrated curative effect preferably in clinical treatment, treatment does not produce reaction to C2B8 to still have 52% patient.Therefore, seek more to be effective to treat the lymphadenomatous CD20 antibody drug of B and seem particularly urgent.
Up-to-date experimental data shows that to common realization of the cell of Rituximab tolerance be: CD20 expresses attenuating; The Bcl-2 expression amount such as increases at [Title:Development of Rituximab-ResistantLymphoma Clones with Altered Cell Signaling and Cross-Resistance toChemotherapy.Author:Jazirehi, A.R. in the cell; Vega, M.I.; Bonavida, B.Source:CancerResearch, 2007,67 (3): 1270-1281; Title:Acquirement of Rituximab Resistance inLymphoma Cell Lines Is Associated with Both Global CD20Gene and ProteinDown-Regulation Regulated at the Pretranscriptional and PosttranscriptionalLevels.Author:Czuczman, M.S.; Olejniczak, S.; Gowda, A.Source:ClinicalCancer Research, 2008,14 (5): 1561-1570].And the raising of affinity of antibody can strengthen the specificity of antibody, improves detection sensitivity, and the function that strengthens antibody reduces the consumption of antibody simultaneously.Therefore, be extensive use of on the basis of the Rituximab with better result of treatment clinically, make up higher humanization or the total man source IDEC-C2B8 of humanization degree, further improve the avidity of Rituximab, security and the validity of improving clinical treatment are extremely important.
Summary of the invention
The present invention has made up jumbo natural human source phage antibody library, and therefrom screening has obtained a strain total man source anti-CD20 antibodies 3C5.
More specifically, total man disclosed by the invention source anti-CD20 antibodies, weight chain variable region amino acid sequence are shown in the SEQ ID NO:6, and the light chain variable region amino acid sequence is shown in the SEQ ID NO:8;
Above-mentioned total man disclosed by the invention source anti-CD20 antibodies, heavy chain amino acid sequence is shown in the SEQ ID NO:10, light-chain amino acid sequence is shown in the SEQ ID NO:12;
The invention also discloses a kind of nucleotide sequence, the total man source anti-CD20 antibodies of encoding above-mentioned;
Above-mentioned nucleotide sequence disclosed by the invention, the nucleotides sequence of the total man source anti-CD20 antibodies variable region of heavy chain of wherein encoding is classified as shown in the SEQ ID NO:5, and the nucleotides sequence of coding total man source anti-CD20 antibodies variable region of light chain is classified as shown in the SEQ ID NO:7;
Above-mentioned nucleotide sequence disclosed by the invention, the nucleotides sequence of the total man source anti-CD20 antibodies heavy chain of wherein encoding is classified as shown in the SEQ ID NO:9, and the nucleotides sequence of coding total man source anti-CD20 antibodies light chain is classified as shown in the SEQ ID NO:11;
The invention also discloses a kind of expression vector, contain above-mentioned nucleotide sequence, be pcDNA3.1/ZEO (+) or pcDNA3.1 (+);
The invention also discloses above-mentioned expression vector transformed host cells, be the CHO-K1 cell;
The present invention further discloses the preparation method of above-mentioned total man source antibody, comprise from the screening of phage human antibody library obtaining the anti-CD20 single-chain antibody in high-affinity total man source; Total man source anti-CD20 complete antibody molecule Construction of eukaryotic; The expression of total man source anti-CD20 complete antibody molecule in Chinese hamster ovary celI; The purifying of total man source anti-CD20 complete antibody molecule.
The present invention discloses the purposes of above-mentioned total man source antibody in preparation treatment antitumor drug at last, and these tumours are the lymphoma of high expression level CD20, are preferably the non-Hodgkin lymphomas.
The antibody that utilization of the present invention obtains has carried out a series of experiments, experimental result shows: with chimeric antibody rituximab and Chinese patent application number be 01132226.8 denomination of invention for CD 20 antagonizing Chimeric antibody C4E5 disclosed in " Humanized anti-CD 20 monoclonal antibody " and Humanized anti-CD 20 antibody H9D3 by comparison, the antibody that the present invention obtains has higher affinity of antibody and stronger ADCC and CDC activity.
Description of drawings
Fig. 1. anti-CD20 antibodies 3C5 induces the CDC effect experiment result of CD20 positive cell Raji (Fig. 1-1), Namalwa (Fig. 1-2) and Daudi (Fig. 1-3);
Fig. 2. anti-CD20 antibodies 3C5 induces the ADCC effect experiment result of CD20 positive cell Raji (Fig. 2-1), Namalwa (Fig. 2-2) and Daudi (Fig. 2-3).
Embodiment
Following examples, experimental example only are further detailed the present invention, should not be construed as limitation of the present invention.
The preparation of embodiment antibody
(1) clone of light, the weight chain constant area gene of people's antibody
Separate healthy human lymphocyte with lymphocyte separation medium (ancient cooking vessel state biotech development company product), extract total RNA with Trizol reagent (Invitrogen company product), according to document (Cell, 1980,22:197-207) and document (Nucleic Acids Research, 1982,10:4071-4079) Bao Dao sequence designs primer respectively and adopts RT-PCR reaction amplification heavy chain of antibody and constant region of light chain gene.The PCR product reclaims and is cloned in the pGEM-T carrier (Promega company product) through the agarose gel electrophoresis purifying, confirms to have obtained correct clone after the sequence verification.SEQ ID NO:1 and SEQ ID NO:2 have shown the nucleotide sequence and the aminoacid sequence of CH (CH) respectively.SEQ ID NO:3 and SEQ ID NO:4 have shown the nucleotide sequence and the aminoacid sequence of constant region of light chain (CL) respectively.Correct clone's note in this example is made pGEM-T/CH and pGEM-T/CL.
(2) preparation of eDNA
Collect each 20ml of peripheral blood of 50 healthy people, mix, with lymphocyte separation medium (medical courses in general institute Tianjin blood grind produce) separation mononuclearcell.From isolating human peripheral lymphocyte, extract total RNA of cell with Trizol reagent (Invitrogen company).Go out cDNA with cDNA reverse transcription test kit (Shanghai Shenergy Biocolor BioScience ﹠ Technology Company) reverse transcription.Above step is carried out according to the specification sheets that producer provides.
(3) design of primers
Reference (Immunotechnology, 1998,3:271-278) design and synthesize VHBack, VHFor, VLBack and the VLFor primer of human cloning antibody heavy chain variable region (VH) and variable region of light chain (VL) gene.VHBack, VHFor, the sequence of VLBack and VLFor is seen Immunotechnology, 1998,3:271-278.Wherein 5 ' end at the VHBack primer adds the sequence atg gcc cag ccg gcc atg gcc that contains Sfi I site, 5 ' end at the VHFor primer adds sequence gcc agaacc acc gcc gcc gga gcc acc acc gcc, 5 ' end at the VLBack primer adds sequence tcc ggc ggcggt ggt tct ggc gga ggc gga tct, holds at 5 ' of VLFor primer to add the sequence atg cgg ccg c that contains Not I site.
(4) structure of phage antibody library and screening
CDNA in the employing (2) and the primer in (3) utilize recombinant Phage antibodysystem test kit (Amersham Biosciences company) to make up the phage single-chain antibody storehouse, with specific antigens elutriation are carried out in the library then.Antibody library makes up and the elutriation method is carried out with reference to recombinant Phageantibody system test kit specification sheets, the specific antigens " CD20 antigen peptide-KLH " that is used for elutriation is the part fragment of people CD20 molecular film outskirt, be NIYNCEPANPSEKNSPSTQYCYSIQ totally 25 amino acid whose peptide sections link to each other with KLH, wherein the C of the 5th of peptide section and the 21 's C forms intrachain disulfide bond, and CD20 antigen peptide-KLH is the synthetic preparation of Shanghai section peptide bio tech ltd.Through repeatedly having obtained a strain anti-humen CD 20 single-chain antibody 3C5ScFv behind the elutriation antibody library, the order-checking back obtains its gene order.SEQ ID NO:5 and SEQ IDNO:6 have shown nucleotide sequence and the aminoacid sequence of 3C5ScFv variable region of heavy chain VH respectively.SEQID NO:7 and SEQ ID NO:8 have shown nucleotide sequence and the aminoacid sequence of 3C5ScFv variable region of light chain VL respectively.
(5) expression of total man source antibody in eukaryotic cell
With 3C5ScFv gene and pGEM-T/CH is masterplate, and by the synthetic total man source of overlapping PCR heavy chain of antibody gene, reaction conditions is: 95 ℃ 15 minutes; 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 50 seconds, 30 circulations; 72 ℃ 10 minutes.And making 5 of this total man source heavy chain of antibody gene ' end contain restriction enzyme sites HindIII and signal peptide gene sequence, 3 ' end contains translation stop codon TAA and restriction enzyme sites EcoR I.The signal peptide gene sequence is (ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAAT ATCCAGAGGA).Last agarose gel electrophoresis separates pcr amplification product, reclaims the purpose band and is cloned in the pGEM-T carrier (Promega company product) the screening positive clone order-checking.Selecting the correct clone of order-checking cuts with HindIII and EcoR I enzyme, reclaim total man source heavy chain of antibody fragment 3C5VHCH through the agarose gel electrophoresis purifying, with be connected with the plasmid pcDNA3.1 (+) (Invitrogen company) that EcoR I enzyme is cut with HindIII, be built into total man source heavy chain carrier for expression of eukaryon pcDNA3.1 (+) (3C5VHCH).
With 3C5ScFv gene and pGEM-T/CL carrier is template, and by the synthetic full humanization light chain of antibody gene of overlapping PCR, reaction conditions is: 95 ℃ 15 minutes; 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 50 seconds, 30 circulations; 72 ℃ 10 minutes, obtain the PCR product, its 5 ' end contains restriction enzyme sites HindIII and signal peptide gene sequence, 3 ' end contains translation stop codon TAA and restriction enzyme sites EcoR I.The signal peptide gene sequence is (ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAAT ATCCAGAGGA).Selecting the correct clone of order-checking cuts with HindIII and EcoR I enzyme, reclaim total man source light chain of antibody fragment 3C5VLCL through the agarose gel electrophoresis purifying, with be connected with plasmid pcDNA3.1/ZEO (+) (Invitrogen company) carrier that EcoR I enzyme is cut with HindIII, be built into total man's endogenous light chain carrier for expression of eukaryon pcDNA3.1/ZEO (+) (3C5VLCL).
In 3.5cm tissue culture ware, inoculate 3 * 10
5CHO-K1 cell (ATCC CRL-9618), cell cultures is carried out transfection when 90%-95% merges: get plasmid 10 μ g (plasmid pcDNA3.1 (+) is 4 μ g (3C5VHCH), and plasmid pcDNA3.1/ZEO (+) is 6 μ g (3C5VLCL)) and 20 μ l Lipofectamine2000Reagent (Invitrogen company product) and carry out transfection by Lipofectamine2000Reagent test kit specification sheets.Cell changed the DMEM substratum screening resistance clone that contains 600 μ g/ml G418 (Invitrogen company product) and 250 μ g/ml Zeocin (Invitrogen company product) after 24h was carried out in transfection.Get cells and supernatant and detect the screening high-expression clone with ELISA: goat anti-human igg (Fc) (KPL company) bag quilt is in elisa plate, 4 ℃ are spent the night, seal 2h with 2%BSA-PBS in 37 ℃, add resistance clone culture supernatant to be measured or standard substance Humanmyeloma IgG1, κ (Sigma), 37 ℃ of incubation 2h, ((SouthernBiotechnology Associates company) carries out association reaction to add HRP-goat anti-human igg (κ), 37 ℃ of incubation 1h, add TMB colour developing liquid in 37 ℃ of effect 5min, use the H2SO4 termination reaction at last, survey the A450 value.With the high-expression clone serum free medium enlarged culturing that screening obtains, use ProteinA affinity column (GE company product) separation and purification total man source antibody 3C5.Antibody purification is dialysed with PBS, at last with the uv-absorbing standard measure.SEQ ID NO:9 and SEQ ID NO:10 have shown heavy chain nucleotide sequence and the aminoacid sequence of total man source antibody 3C5 respectively.SEQ ID NO:11 and SEQ ID NO:12 have shown light chain nucleotide sequence and the aminoacid sequence of total man source antibody 3C5 respectively.
Experimental example
Chimeric antibody C4E5, humanized antibody H9D3 are with reference to disclosed method preparation in the Chinese patent application number 01132226.8.
Experimental example 1.CD20 affinity of antibody detects
The avidity of CD20 antibody is measured [Cragg MS by the radioimmunoassay method, Morgan SM, Chan HT, Morgan BP, Filatov AV, Johnson PW, French RR, Glennie MJ (2003) Complement-mediated lysis by anti-CD20mAb correlates with segregation intolipid rafts.Blood 101 (3): 1045-1052].Each CD20 antibody (total man source antibody 3C5; Chimeric antibody C4E5; Humanized antibody H9D3; Rituximab (C2B8, Roche); Negative control antibody Trastuzumab Genentech) carries out mark by iodine pearl method (iodobead method).The antibody and Daudi (ATCC CCL-213) cell of mark 125 iodine were hatched 37 ℃ 2 hours.With the antibody of cell bonded and free iodine labeling by centrifugation, detect the radioactivity of the antibody that is combined in 125 iodine labelings on the cell.Each CD20 affinity of antibody constant is tried to achieve (seeing Table 1) by Hill equation fitting of a curve titration binding curve.The KD value of total man source antibody 3C5 is 1.96 ± 0.15, and less than C4E5, the KD value of H9D3 and Rituximab (P＜0.01), the avidity that total man source antibody 3C5 and CD20 are described be than C4E5, H9D3 and Rituximab height.
The avidity competition of table 1. anti-CD20 antibodies is in conjunction with experimental result
ND represents that avidity is too weak so that can't detect; M is the mean value of KD, and SD is an experimental error, and experimental error derives from three independently experiments.
Experimental example 2. anti-CD20 antibodies 3C5 are to lethal effect (CDC) experiment of the complement dependence of target cell
Anti-CD20 antibodies can be induced the lethal effect of CD20 positive cell Raii, Namalwa and Daudi cell by the lethal effect of complement dependence.The Raji of logarithmic phase (ATCC CCL-86), Namalwa (ATCC CRL-1432) and Daudi (ATCC CCL-213) cell, PBS washes 2 times, with the resuspended Raji of no phenol red RPMI-1640 (Gibco), Namalwa and Daudi cell, adjusting cell density is 2 * 10
5/ mL is standby.With no phenol red RPMI-1640 nutrient solution total man source anti-CD-20 monoclonal antibody 3C5, humanized antibody H9D3, chimeric antibody C4E5, Rituximab (Roche) and Transtuzumab (Genentech) being diluted to concentration is 10 μ g/ml, and make the multiple proportions serial dilution, the sample that dilution is good adds 96 porocyte culture plates respectively, multiple hole is established in 100 μ l/ holes.Adjust Raji, Namalwa and the Daudi cell suspension of density, add 5 μ l Freshman serum as complement with per 100 μ l cell suspensions.The cell suspension that will contain Freshman serum adds respectively in 96 orifice plates of each sample that contains serial dilution, 100 μ l/ holes.37 ℃, 5%CO2 cell culture incubator effect 4h.Catalyst in the heterotope method cell killing detection kit (Roche) (Roche, 1644793) is used the 1ml dissolved in distilled water.With 1: 45 ratio catalyst and Dye solution (being Roche, the composition in 1644793 test kits) are mixed.The centrifugal 5min of 96 orifice plate 200g, 50 μ l supernatants are drawn to another 96 orifice plate respective aperture in every hole.Add the colour developing liquid 50 μ l/ holes that mix, room temperature lucifuge effect 30min.Microplate reader is read the photoabsorption of 490nm.With the sample concentration is X-coordinate, and absorbance value is an ordinate zou, the results are shown in Figure 1, and experimental result also sees the following form.
Raji
Daudi
Namalwa
Experimental result shows, total man of the present invention source anti-CD-20 monoclonal antibody 3C5 induces the EC50 value of CD20 positive cell Raji (Fig. 1-1), Namalwa (Fig. 1-2) and Daudi (Fig. 1-3) CDC effect minimum, therefore the avidity of itself and CD20 is the highest, induces the biological activity of generation the strongest.
Experimental example 3. anti-CD20 antibodies 3C5 are to cytotoxicity (ADCC) experiment of the antibody dependent cellular mediation of target cell
Anti-CD20 antibodies can be induced the lethal effect of CD20 positive cell Raji, Namalwa and Daudi cell by the cytotoxicity of antibody dependent cellular mediation.The Raji of logarithmic phase (ATCCCCL-86), Namalwa (ATCC CRL-1432) and Daudi (ATCC CCL-213) cell, PBS washes 2 times, with the no phenol red resuspended Raji of RPMI-1640 nutrient solution, Namalwa and Daudi cell, adjusting cell density is 3 * 10
5/ mL is standby.Aseptic collection venous blood injects the aseptic 15mL centrifuge tube that contains heparin (Tianjin Biochemical Pharmaceutical Factory) 20U/mL, gently mixing.Add equal-volume PBS solution.Get the 15mL centrifuge tube, every pipe adds the lymphocyte separation medium (ancient cooking vessel state biology) of 6mL room temperature, slowly adds anticoagulation cirumferential blood 6mL/ pipe after the dilution along tube wall.20 ℃, the centrifugal 30min of 800g.Brake is closed natural reduction of speed.Be divided into three layers in the pipe, be followed successively by plasma layer, cell layering liquid, red corpuscle and GCL from top to bottom.The white layer of plasma layer and cell layering liquid intersection ground-glass-like is lymph and mononuclear cell layer.Insert the ground-glass-like layer gently with suction pipe, slowly the sucking-off peripheral blood mononuclear cell is put into another 15mL centrifuge tube.Cell adding PBS dilution back in the centrifuge tube of sucking-off is centrifugal, 200g, centrifugal 5min washes 2 times altogether.Adjusting cell density with no phenol red RPMI-1640 nutrient solution is 6 * 10
6/ mL is suspended from and places 37 ℃ in the 15mL centrifuge tube, 7%CO
2Cell culture incubator is standby.With no phenol red RPMI-1640 nutrient solution total man source anti-CD-20 monoclonal antibody 3C5, humanized antibody H9D3, chimeric antibody C4E5, Rituximab (Roche) and Transtuzumab (Genentech) being diluted to concentration is 10 μ g/ml, and 2 multiple proportions serial dilution, the antibody that dilution is good adds the 1.5ml centrifuge tube, 300 μ L/ pipe.Every pipe adds Raji, Namalwa and the Daudi cell of adjusting cell density, 300 μ L/ pipe.Behind 4 ℃ of effect 30min, the centrifugal 5min of 200g washes 2 times with PBS.Cell precipitation is resuspended in 300 μ L not to be had in the phenol red RPMI-1640 nutrient solution.To add with the cell suspension after the antibody effect in 96 orifice plates, 100 μ l/ holes, each concentration is established multiple hole.Every hole adds effector cell 100 μ l/ holes, 37 ℃, 5%CO2 cell culture incubator 7h with 20: 1 effects, target ratios respectively.With the Catalyst 1ml dissolved in distilled water in the heterotope method cell killing detection kit (Roche).With 1: 45 ratio catalyst is mixed with Dye solution.The centrifugal 5min of 96 orifice plate 200g, 50 μ l supernatants are drawn to another 96 orifice plate respective aperture in every hole.Add the colour developing liquid 50 μ l/ holes that mix, room temperature lucifuge effect 30min.Microplate reader is read the photoabsorption of 490nm.With the sample concentration is X-coordinate, and absorbance value is an ordinate zou, the results are shown in Figure 2, and experimental result also sees the following form.
Raji
Daudi
Namalwa
Experimental result shows, total man of the present invention source anti-CD-20 monoclonal antibody 3C5 induces the EC50 value of CD20 positive cell Raji (Fig. 2-1), Namalwa (Fig. 2-2) and Daudi (Fig. 2-3) ADCC effect minimum, therefore the avidity of itself and CD20 is the highest, induces the biological activity of generation the strongest.
SEQUENCE?LISTING
＜110〉hundred Mai Bo pharmaceutcal corporation, Ltds
＜120〉a kind of total man source anti-CD-20 monoclonal antibody, Preparation Method And The Use
<160>12
<170>PatentIn?version?3.2
<210>1
<211>990
<212>DNA
＜213〉nucleotide sequence of human antibody heavy chain's constant region (CH)
<400>1
gctagcacca?agggcccatc?ggtcttcccc?ctggcaccct?cctccaagag?cacctctggg 60
ggcacagcgg?ccctgggctg?cctggtcaag?gactacttcc?ccgaaccggt?gacggtgtcg 120
tggaactcag?gcgccctgac?cagcggcgtg?cacaccttcc?cggctgtcct?acagtcctca 180
ggactctact?ccctcagcag?cgtggtgacc?gtgccctcca?gcagcttggg?cacccagacc 240
tacatctgca?acgtgaatca?caagcccagc?aacaccaagg?tggacaagaa?agttgagccc 300
aaatcttgtg?acaaaactca?cacatgccca?ccgtgcccag?cacctgaact?cctgggggga 360
ccgtcagtct?tcctcttccc?cccaaaaccc?aaggacaccc?tcatgatctc?ccggacccct 420
gaggtcacat?gcgtggtggt?ggacgtgagc?cacgaagacc?ctgaggtcaa?gttcaactgg 480
tacgtggacg?gcgtggaggt?gcataatgcc?aagacaaagc?cgcgggaaga?gcagtacaac 540
agcacgtacc?gtgtggtcag?cgtcctcacc?gtcctgcacc?aggactggct?gaatggcaag 600
gagtacaagt?gcaaggtctc?caacaaagcc?ctcccagccc?ccatcgagaa?aaccatctcc 660
aaagccaaag?ggcagccccg?agaaccacag?gtgtacaccc?tgcccccatc?ccgggatgag 720
ctgaccaaga?accaggtcag?cctgacctgc?ctggtcaaag?gcttctatcc?cagcgacatc 780
gccgtggagt?gggagagcaa?tgggcagccg?gagaacaact?acaagaccac?gcctcccgtg 840
ctggactccg?acggctcctt?cttcctctac?agcaagctca?ccgtggacaa?gagcaggtgg 900
cagcagggga?acgtcttctc?atgctccgtg?atgcatgagg?ctctgcacaa?ccactacacg 960
cagaagagcc?tctccctgtc?tcccggtaaa 990
<210>2
<211>330
<212>PRT
＜213〉aminoacid sequence of human antibody heavy chain's constant region (CH)
<400>2
Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys
1 5 10 15
Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr
20 25 30
Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser
35 40 45
Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser
50 55 60
Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr
65 70 75 80
Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys
85 90 95
Lys?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys
100 105 110
Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro
115 120 125
Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys
130 135 140
Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp
145 150 155 160
Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu
165 170 175
Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu
180 185 190
His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn
195 200 205
Lys?Ala?Leu?Pro?Ala?Pro?lle?Glu?Lys?Thr?lle?Ser?Lys?Ala?Lys?Gly
210 215 220
Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu
225 230 235 240
Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr
245 250 255
Pro?Ser?Asp?lle?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn
260 265 270
Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe
275 280 285
Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn
290 295 300
Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr
305 310 315 320
Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys
325 330
<210>3
<211>318
<212>DNA
＜213〉nucleotide sequence of human antibody light chain constant region (CL)
<400>3
actgtggctg?caccatctgt?cttcatcttc?ccgccatctg?atgagcagtt?gaaatctgga 60
actgcctctg?ttgtgtgcct?gctgaataac?ttctatccca?gagaggccaa?agtacagtgg 120
aaggtggata?acgccctcca?atcgggtaac?tcccaggaga?gtgtcacaga?gcaggacagc 180
aaggacagca?cctacagcct?cagcagcacc?ctgacgctga?gcaaagcaga?ctacgagaaa 240
cacaaagtct?acgcctgcga?agtcacccat?cagggcctga?gctcgcccgt?cacaaagagc 300
ttcaacaggg?gagagtgt 318
<210>4
<211>106
<212>PRT
＜213〉aminoacid sequence of human antibody light chain constant region (CL)
<400>4
Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln
1 5 10 15
Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr
20 25 30
Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser
35 40 45
Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser?Thr
50 55 60
Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu?Lys
65 70 75 80
His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser?Pro
85 90 95
Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
100 105
<210>5
<211>378
<212>DNA
＜213〉total man source antibody 3C5 weight chain variable region nucleotide sequence
<400>5
caggtgcagc?tgcaggagtc?gggcccagga?ctggtgaagc?cttcggacac?cctgtccctc 60
acctgcgctg?tctctggtta?ctccatcagc?agtagtaact?ggtggggctg?gatccggcag 120
cccccaggga?agggactgga?gtggattggg?tacatctatt?atagtgggag?cacctactac 180
aacccgtccc?tcaagagtcg?agtcaccatg?tcagtagaca?cgtccaagaa?ccagttctcc 240
ctgaagctga?gctctgtgac?cgccgtggac?acggccgtgt?attactgtgc?gagaaacggt 300
acaactggaa?cgacagatta?ctactactac?tacggtatgg?acgtctgggg?gcaagggacc 360
acggtcaccg?tctcctca 378
<210>6
<21l>126
<212>PRT
＜213〉total man source antibody 3C5 weight chain variable region amino acid sequence
<400>6
Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Asp
1 5 10 15
Thr?Leu?Ser?Leu?Thr?Cys?Ala?Val?Ser?Gly?Tyr?Ser?Ile?Ser?Ser?Ser
20 25 30
Asn?Trp?Trp?Gly?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp
35 40 45
Ile?Gly?Tyr?Ile?Tyr?Tyr?Ser?Gly?Ser?Thr?Tyr?Tyr?Asn?Pro?Ser?Leu
50 55 60
Lys?Ser?Arg?Val?Thr?Met?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser
65 70 75 80
Leu?Lys?Leu?Ser?Ser?Val?Thr?Ala?Val?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Arg?Asn?Gly?Thr?Thr?Gly?Thr?Thr?Asp?Tyr?Tyr?Tyr?Tyr?Tyr?Gly
100 105 110
Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser
115 120 125
<210>7
<21l>333
<212>DNA
＜213〉total man source antibody 3C5 light chain variable region nucleotide sequence
<400>7
gaaattgtgt?tgacgcagtc?tccaggcacc?ctgtctttgt?ctccagggga?aagagccacc 60
ctctcctgca?gggccagtca?gagtgttagc?agcagctact?tagcctggta?ccagcagaaa 120
cctggccagg?ctcccaggct?cctcatctat?ggtgcatcca?gcagggccac?tggcatccca 180
gacaggttca?gtggcagtgg?gtctgggaca?gacttcactc?tcaccatcag?cagactggag 240
cctgaagatt?ttgcagtgta?ttactgtcag?cagtatggta?gctcacctcc?aatgtacact 300
tttggccagg?ggaccaagct?ggagatcaaa?cgt 333
<210>8
<211>111
<212>PRT
＜213〉total man source antibody 3C5 light chain variable region amino acid sequence
<400>8
Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Gly?Thr?Leu?Ser?Leu?Ser?Pro?Gly
1 5 10 15
Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Ser
20 25 30
Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu
35 40 45
Ile?Tyr?Gly?Ala?Ser?Ser?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser
50 55 60
Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Leu?Glu
65 70 75 80
Pro?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Ser?Ser?Pro
85 90 95
Pro?Met?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg
100 105 110
<210>9
<21l>1368
<212>DNA
＜213〉heavy chain nucleotide sequence of total man source antibody 3C5
<400>9
caggtgcagc?tgcaggagtc?gggcccagga?ctggtgaagc?cttcggacac?cctgtccctc 60
acctgcgctg?tctctggtta?ctccatcagc?agtagtaact?ggtggggctg?gatccggcag 120
cccccaggga?agggactgga?gtggattggg?tacatctatt?atagtgggag?cacctactac 180
aacccgtccc?tcaagagtcg?agtcaccatg?tcagtagaca?cgtccaagaa?ccagttctcc 240
ctgaagctga?gctctgtgac?cgccgtggac?acggccgtgt?attactgtgc?gagaaacggt 300
acaactggaa?cgacagatta?ctactactac?tacggtatgg?acgtctgggg?gcaagggacc 360
acggtcaccg?tctcctcagc?tagcaccaag?ggcccatcgg?tcttccccct?ggcaccctcc 420
tccaagagca?cctctggggg?cacagcggcc?ctgggctgcc?tggtcaagga?ctacttcccc 480
gaaccggtga?cggtgtcgtg?gaactcaggc?gccctgacca?gcggcgtgca?caccttcccg 540
gctgtcctac?agtcctcagg?actctactcc?ctcagcagcg?tggtgaccgt?gccctccagc 600
agcttgggca?cccagaccta?catctgcaac?gtgaatcaca?agcccagcaa?caccaaggtg 660
gacaagagag?ttgagcccaa?atcttgtgac?aaaactcaca?catgcccacc?gtgcccagca 720
cctgaactcc?tggggggacc?gtcagtcttc?ctcttccccc?caaaacccaa?ggacaccctc 780
atgatctccc?ggacccctga?ggtcacatgc?gtggtggtgg?acgtgagcca?cgaagaccct 840
gaggtcaagt?tcaactggta?cgtggacggc?gtggaggtgc?ataatgccaa?gacaaagccg 900
cgggaggagc?agtacaacag?cacgtaccgt?gtggtcagcg?tcctcaccgt?cctgcaccag 960
gactggctga?atggcaagga?gtacaagtgc?aaggtctcca?acaaagccct?cccagccccc 1020
atcgagaaaa?ccatctccaa?agccaaaggg?cagccccgag?aaccacaggt?gtacaccctg 1080
cccccatccc?gggaggagat?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc 1140
ttctatccca?gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac 1200
aagaccacgc?ctcccgtgct?ggactccgac?ggctccttct?tcctctatag?caagctcacc 1260
gtggacaaga?gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct 1320
ctgcacaacc?actacacgca?gaagagcctc?tccctgtccc?cgggtaaa 1368
<210>10
<211>456
<212>PRT
＜213〉heavy chain amino acid sequence of total man source antibody 3C5
<400>10
Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Asp
1 5 10 15
Thr?Leu?Ser?Leu?Thr?Cys?Ala?Val?Ser?Gly?Tyr?Ser?Ile?Ser?Ser?Ser
20 25 30
Asn?Trp?Trp?Gly?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp
35 40 45
Ile?Gly?Tyr?Ile?Tyr?Tyr?Ser?Gly?Ser?Thr?Tyr?Tyr?Asn?Pro?Ser?Leu
50 55 60
Lys?Ser?Arg?Val?Thr?Met?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser
65 70 75 80
Leu?Lys?Leu?Ser?Ser?Val?Thr?Ala?Val?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Arg?Asn?Gly?Thr?Thr?Gly?Thr?Thr?Asp?Tyr?Tyr?Tyr?Tyr?Tyr?Gly
100 105 110
Met?Asp?Val?Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala?Ser
115 120 125
Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
130 135 140
Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro
145 150 155 160
Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val
165 170 175
His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser
180 185 190
Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile
195 200 205
Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Arg?Val
210 215 220
Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala
225 230 235 240
Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro
245 250 255
Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val
260 265 270
Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val
275 280 285
Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln
290 295 300
Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln
305 310 315 320
Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala
325 330 335
Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro
340 345 350
Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Glu?Glu?Met?Thr
355 360 365
Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser
370 375 380
Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr
385 390 395 400
Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr
405 410 415
Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe
420 425 430
Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys
435 440 445
Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys
450 455
<210>11
<211>651
<212>DNA
＜213〉the light chain nucleotide sequence of total man source antibody 3C5
<400>11
gaaattgtgt?tgacgcagtc?tccaggcacc?ctgtctttgt?ctccagggga?aagagccacc 60
ctctcctgca?gggccagtca?gagtgttagc?agcagctact?tagcctggta?ccagcagaaa 120
cctggccagg?ctcccaggct?cctcatctat?ggtgcatcca?gcagggccac?tggcatccca 180
gacaggttca?gtggcagtgg?gtctgggaca?gacttcactc?tcaccatcag?cagactggag 240
cctgaagatt?ttgcagtgta?ttactgtcag?cagtatggta?gctcacctcc?aatgtacact 300
tttggccagg?ggaccaagct?ggagatcaaa?cgtactgtgg?ctgcaccatc?tgtcttcatc 360
ttcccgccat?ctgatgagca?gttgaaatct?ggaactgcct?ctgttgtgtg?cctgctgaat 420
aacttctatc?ccagagaggc?caaagtacag?tggaaggtgg?ataacgccct?ccaatcgggt 480
aactcccagg?agagtgtcac?agagcaggac?agcaaggaca?gcacctacag?cctcagcagc 540
accctgacgc?tgagcaaagc?agactacgag?aaacacaaag?tctacgcctg?cgaagtcacc 600
catcagggcc?tgagctcgcc?cgtcacaaag?agcttcaaca?ggggagagtg?t 651
<210>12
<211>217
<212>PRT
＜213〉light-chain amino acid sequence of total man source antibody 3C5
<400>12
Glu?Ile?Val?Leu?Thr?Gln?Ser?Pro?Gly?Thr?Leu?Ser?Leu?Ser?Pro?Gly
1 5 10 15
Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Ser
20 25 30
Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu
35 40 45
Ile?Tyr?Gly?Ala?Ser?Ser?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser
50 55 60
Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Leu?Glu
65 70 75 80
Pro?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Ser?Ser?Pro
85 90 95
Pro?Met?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Thr
100 105 110
Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu
115 120 125
Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro
130 135 140
Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly
145 150 155 160
Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr
165 170 175
Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His
180 185 190
Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser?Pro?Val
195 200 205
Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
210 215
Claims (10)
1. total man source anti-CD20 antibodies, its weight chain variable region amino acid sequence is shown in SEQ ID NO:6, and the light chain variable region amino acid sequence is shown in SEQ ID NO:8.
2. the described total man of claim 1 source anti-CD20 antibodies, its heavy chain amino acid sequence is shown in SEQ IDNO:10, and light-chain amino acid sequence is shown in SEQ ID NO:12.
3. nucleotide sequence, the arbitrary described total man of coding claim 1～2 source anti-CD20 antibodies.
4. the described nucleotide sequence of claim 3, the nucleotide sequence of the total man source anti-CD20 antibodies variable region of heavy chain of wherein encoding are shown in SEQ ID NO:5, and the nucleotide sequence of coding total man source anti-CD20 antibodies variable region of light chain is shown in SEQ ID NO:7.
5. the described nucleotide sequence of claim 4, the nucleotide sequence of the total man source anti-CD20 antibodies heavy chain of wherein encoding are shown in SEQ ID NO:9, and the nucleotide sequence of coding total man source anti-CD20 antibodies light chain is shown in SEQ ID NO:11.
6. an expression vector contains the arbitrary described nucleotide sequence of claim 3～5, is pcDNA3.1/ZEO (+) or pcDNA3.1 (+).
7. a host cell transforms the described expression vector of claim 6, is the CHO-K1 cell.
8. the arbitrary described total man of claim 2～3 source anti-CD20 antibodies is preparing the purposes for the treatment of in the antitumor drug.
9. the described purposes of claim 8, wherein tumour is the lymphoma of high expression level CD20.
10. the described purposes of claim 9, wherein the lymphoma of high expression level CD20 is the non-Hodgkin lymphomas.
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| CN102863531A (en) * | 2012-07-31 | 2013-01-09 | 张爱晖 | CD20-resistant monoclonal antibody as well as preparation method and application thereof |
| CN103641917A (en) * | 2013-12-09 | 2014-03-19 | 江苏众红生物工程创药研究院有限公司 | Anti-CD26 antibody and application thereof |
| CN103694352A (en) * | 2013-12-19 | 2014-04-02 | 江苏众红生物工程创药研究院有限公司 | CD26 antibody and preparation method thereof |
| CN103724431A (en) * | 2014-01-16 | 2014-04-16 | 江苏众红生物工程创药研究院有限公司 | Humanized anti-CD26 antibody and application thereof |
| CN104341503A (en) * | 2013-07-29 | 2015-02-11 | 西藏海思科药业集团股份有限公司 | Human antibody with low immunogenicity for Mongoloid and Caucasian and CD20 resistance |
| WO2015042807A1 (en) * | 2013-09-25 | 2015-04-02 | 北京安保康生物医药科技有限公司 | Fully humanized anti-cd20 monoclonal antibody and uses thereof |
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| JP2022506430A (en) * | 2018-10-31 | 2022-01-17 | ジョイント・ストック・カンパニー “バイオキャド” | Monoclonal antibody that specifically binds to CD20 |
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| CN104341503A (en) * | 2013-07-29 | 2015-02-11 | 西藏海思科药业集团股份有限公司 | Human antibody with low immunogenicity for Mongoloid and Caucasian and CD20 resistance |
| CN105143269B (en) * | 2013-09-25 | 2020-05-22 | 北京安保康生物医药科技有限公司 | Fully human monoclonal antibody against CD20 and application thereof |
| WO2015042807A1 (en) * | 2013-09-25 | 2015-04-02 | 北京安保康生物医药科技有限公司 | Fully humanized anti-cd20 monoclonal antibody and uses thereof |
| CN105143269A (en) * | 2013-09-25 | 2015-12-09 | 北京安保康生物医药科技有限公司 | Fully humanized anti-CD20 monoclonal antibody and uses thereof |
| CN103641917A (en) * | 2013-12-09 | 2014-03-19 | 江苏众红生物工程创药研究院有限公司 | Anti-CD26 antibody and application thereof |
| CN103694352A (en) * | 2013-12-19 | 2014-04-02 | 江苏众红生物工程创药研究院有限公司 | CD26 antibody and preparation method thereof |
| CN103694352B (en) * | 2013-12-19 | 2015-12-09 | 江苏众红生物工程创药研究院有限公司 | A kind of anti-CD26 antibody and preparation method thereof |
| CN103724431A (en) * | 2014-01-16 | 2014-04-16 | 江苏众红生物工程创药研究院有限公司 | Humanized anti-CD26 antibody and application thereof |
| CN103724431B (en) * | 2014-01-16 | 2016-03-30 | 江苏众红生物工程创药研究院有限公司 | A kind of people source anti-CD 26 antibodies and application thereof |
| CN108456660A (en) * | 2017-02-17 | 2018-08-28 | 浙江特瑞思药业股份有限公司 | Produce high expression, the strain of high stability Chinese hamster ovary celI and its construction method of Rituximab |
| JP2022506430A (en) * | 2018-10-31 | 2022-01-17 | ジョイント・ストック・カンパニー “バイオキャド” | Monoclonal antibody that specifically binds to CD20 |
| CN110386983A (en) * | 2019-08-12 | 2019-10-29 | 西南医科大学 | The anti-CD20 recombinant antibodies of full source of people |
| CN110386983B (en) * | 2019-08-12 | 2021-02-05 | 西南医科大学 | Fully human anti-CD 20 recombinant antibody |
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