CN102050878A - Anti-human CD20 humanized antibody and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the biotechnology field and in particular discloses an anti-human CD20 humanized antibody hu3D9 and a preparation method and application thereof. The amino acid sequences of the hypervariable region of the heavy chain of the anti-human CD20 humanized antibody hu3D9 are CDR1:NYWMQ, CDR2:AIYPGDGDTRYTQKFKG and CDR3:EGAYGYDDGMDY. The amino acid sequences of the hypervariable region of the light chain of the anti-human CD20 humanized antibody hu3D9 are CDR1:RASQSIRNNLH, CDR2:YASQSIS and CDR3:QQSNSWPLT. The anti-human CD20 humanized antibody hu3D9 disclosed by the invention retains the affinity and specificity of the original mouse antibody, has certain biological functions and can be used for preparing the drugs for treating lymphoma in a high CD20 expression state.

Description

Anti-humen CD 20 humanized antibody, Preparation Method And The Use

Technical field

The present invention relates to biological technical field, relate in particular to a kind of antibody, Preparation Method And The Use.

Background technology

Non-Hodgkin lymphomas (NHL) is modal lymphsystem malignant tumour, and good sending out in person between twenty and fifty is wherein most for B cell source, accounts for 85%.Under therapeutic state not, low and pernicious NHL of part moderate such as small lymphocytic lymphoma and the lymphadenomatous growth forms of folliculus type often are inert process, and median survival interval is 5～9 years.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.The NHL of some high malignancies then mortality ratio is high.

In recent years, obtained huge progress and become a kind of very rising methods of treatment at the antigen magnetic target therapy of cell surface molecule, wherein be widely used and fruitful be monoclonal antibody preparation [the Maloney DG.Immunotherapy for non-Hodgkin ' s lymphoma:monoclonal antibody and vaccines.J Clin Oncol.2005Sep 10 of anti-CD20; 23 (26): 6421-6428].

CD20 antigen is a desirable target spot of non-associativity monoclonal antibody therapy, because this antigen has clone-specific, only is expressed in all pre B cells and mature B cell, and is not expressed in hemopoietic stem cell, plasmocyte and other hematopoietic cells system; Simultaneously, the CD20 molecule increases unusually at the expression amount on B lymphoma cell surface, and phosphorylation increases, and is closely related with the generation of B cell lymphoma.The CD20 molecule all has expression in surpassing 95% B cellularity NHL, and antigen molecule relatively exposes on film, and is approaching easily, combines the no remarkable internalization in back with monoclonal antibody and comes off, can therefore not become the desirable target spot of treatment B cell lymphoma because of antigenic modulation taking place with combining of antibody yet.

CD20 monoclonal antibody therapy has obtained gratifying effect in the treatment B cell lymphoma, at present anti-CD20 antibodies mainly contains following severally, is divided into two big classes according to difference in functionality of its performance antitumor action: (1) is mainly by CDC, ADCC (CDCC) effect performance function: C2B8,1F5,2H7,2F2 (2) mainly pass through apoptosis, ADCC (CDCC) and act on the performance function: B1,11B8.American I DEC pharmaceuticals is that first is used for the treatment of lymphadenomatous antibody through drugs approved by FDA at the chimeric anti-CD-20 monoclonal antibody Rituximab-C2B8 of the people mouse of B cell lymphoma production.Approval listing in 1997.Mabthera is a kind of mosaic type IgG1 immunoglobulin (Ig), and it is the gene product of expressing behind the hamster ovum by transfection genes involved structure.Mabthera contains 1328 amino acid, and the about 144KD of molecular weight is made of variable region Fab and human IgG1's antibody stable region Fc fragment of mouse-anti CD20 monoclonal antibody.

After entering human body, the mouse resource monoclonal antibody that hybridoma technology is produced to cause human antimouse antibody immunne response human antimouse antibody response (HAMA) [Winter G, Harris WJ.Humanized antibodies.Immunol Today.1993 Jun; 14 (6): 243-6].Therefore, how to make up the anti-immunogenic antibody that reduces mouse source antibody by genetic engineering technique, the incidence that reduces the HAMA reaction effectively is the problem that those skilled in the art is eager to solve.

Humanized antibody [Ishida T, Tmai K.The expression technology of chimeric and humanized antibody.Nippon Rinsho, 2002,60 (3): 439-444Ishida T, Tmai K.The expression technology of chimeric and humanized antibody.Nippon Rinsho, 2002,60 (3): 439-444] be for overcome mouse source monoclonal antibody in clinical application defective and the novel gene engineered antibody that grows up.First-generation humanized antibody is that the constant region of the variable region of mouse monoclonal antibody and people's antibody is formed chimeric antibody, because the antigen avidity of antibody is by its variable region decision, therefore the avidity of chimeric antibody keeps finely, and immunogenicity has also obtained reduction to a certain degree simultaneously.The variable region of antibody is made up of hypervariable region (CDR) district and framework region (FR) district, and CDR is the zone of alterable height, and directly mediate antibody combines with antigenic.The FR district is conservative relatively, is keeping the locus in CDR district as support, and it is to produce immunogenic main region in the variable region.Owing to also keeping the mouse variable region on the chimeric antibody, still usually having intensive HAMA reaction during clinical application.Therefore, in order to reduce the immunogenicity of chimeric antibody as far as possible, people consider mouse CDR district is grafted directly in the FR district in the human antibody variable region, obtain CDR grafted antibody, just humanized antibody.But, simple CDR transplants the avidity that often reduces even lose original antibody, this is because the FR district not only provides the space conformation environment of CDR, sometimes also participate in mutually combining of antigen-antibody directly, have only to become mouse source residue could recover the activity of antibody some important residue reverse mutation among the FR.(C，Queen，W，P，Schneider，H，E，Selick，P，W，Payne，N，F，Landolfi，J，F，Duncan，N，M，Avdalovic，M，Levitt，R，P，Junghans，T，A，Waldmann，A?humanized?antibody?that?binds?to?the?interleukin?2?receptor，Proc.Natl.Acad.Sci.USA?86(1989)10029-10033.V.L.Pulito，V.A.Roberts，J.R.Adair，A.L.Rothermel，A.M.Collins，S.S.Varga，C.Martocello，M.Bodmer，L.K.Jolliffe，R.A.Zivin，Humanization?and?molecular?modeling?of?the?anti-CD4?monoclonal?antibody，OKT4A，J.Immunol.156(1996)2840-2850.K.Nakamura，Y.Tanaka，K.Shitara，N.Hanai，Construction?of?humanized?anti-ganglioside?monoclonal?antibodies?with?potent?immune?effector?functions，Cancer?Immunol.Immunother.50(2001)275-284.)

In sum, obtain active high mouse source monoclonal antibody and to utilize further the be restored humanized antibody of antibody activity of its CDR be the problem that those skilled in the art puts forth effort to solve always.

Summary of the invention

One object of the present invention is to provide the anti-humen CD 20 humanized antibody, by mouse CDR district is transplanted to the human antibody hypervariable region, and simultaneously the amino acid of human antibody framework region is retrofited and obtains avidity and keep good antibody.

Therefore, the invention provides a kind of anti-humen CD 20 humanized antibody, its heavy chain hypervariable region aminoacid sequence is CDR1:NYWMQ, CDR2:AIYPGDGDTRYTQKFKG and CDR3:EGAYGYDDGMDY, and light chain hypervariable region aminoacid sequence is CDR1:RASQSIRNNLH; CDR2:YASQSIS and CDR3:QQSNSWPLT;

Anti-humen CD 20 humanized antibody provided by the invention, its weight chain variable region amino acid sequence is SEQ ID NO:10, the light chain variable region amino acid sequence is SEQ ID NO:12, more specifically, Humanized anti-CD 20 antibody provided by the invention, its heavy chain amino acid sequence is SEQ ID NO:6, and light-chain amino acid sequence is SEQ ID NO:8

Further, anti-humen CD 20 humanized antibody of the present invention is made up of heavy chain and light chain, and wherein heavy chain comprises variable region of heavy chain and human immunoglobulin heavy chain's constant region or its fragment; The albumen light chain comprises variable region of light chain and human normal immunoglobulin constant region of light chain or its fragment.

The human normal immunoglobulin constant region can be selected from various types of immunoglobulin (Ig)s, and preferred human immunoglobulin heavy chain's constant region or its fragment are γ type (IgG3), and aminoacid sequence is shown in SED ID NO.2; Human normal immunoglobulin constant region of light chain or its fragment are κ type or λ type, and the aminoacid sequence of preferred human normal immunoglobulin constant region of light chain is shown in SED ID NO.4.

Another object of the present invention is to provide the nucleic acid molecule of the above-mentioned anti-humen CD 20 humanized antibody of coding, wherein the nucleotides sequence of encoding heavy chain variable region is classified SEQ ID NO:9 as, the nucleotides sequence of encoded light chain variable region is classified SEQ ID NO:11 as, the nucleotides sequence of encoding heavy chain is classified SEQ ID NO:5 as, the nucleotides sequence of coding light chain is classified SEQ ID NO:7 as, is also contained among the present invention by this nucleic acid molecule host transformed.

The present invention further provides the preparation method of the humanized antibody of above-mentioned anti-humen CD 20, comprise the aminoacid sequence that goes out humanized antibody by computer aided design (CAD), full gene synthetic heavy chain and chain variable region gene and, constant region of light chain gene splicing heavy with human normal immunoglobulin respectively through gene recombination, be cloned in the carrier for expression of eukaryon, make up light, the heavy chain expression carrier of humanized antibody respectively, then with light, heavy chain expression carrier liposome method cotransfection Chinese hamster ovary celI, the humanized antibody that screen then, culture purified promptly gets anti-humen CD 20: hu3D9.

The antibody that utilization of the present invention obtains has carried out a series of experiments, and exo-antigen shows that in conjunction with the determination of activity result hu3D9 can combine with the Burkitt lymphoma cell line Raji specificity of high expression level CD20 well.Competition suppresses experimental result and shows that hu3D9 has kept the avidity and the specificity of former mouse source antibody, the extracorporeal biology Function detection its have must biological function, the survival rate test shows that the survival time of the mouse of injection hu3D9 group obtains significant prolongation, hu3D9 can be used for treating the lymphoma disease of high expression level CD20, preferred, hu3D9 can be used for the treatment of non-Hodgkin lymphomas.

Description of drawings

Fig. 1: the molecular simulation structural representation of 3D9 monoclonal antibody: FR district residue represents that with grey (light color) band CDR district residue represents that with black (dark color) band 11 are positioned at around the CDR district

The interior FR district residue of distance is represented with the bat shape;

Fig. 2: the comparison chart of the heavy chain of humanized antibody hu3D9 (Fig. 2-1) and light chain (Fig. 2-2) aminoacid sequence and correlated series, wherein 3D9VH and 3D9VL represent the heavy chain of mouse resource monoclonal antibody 3D9 and the variable region of light chain respectively; The variable region of light chain of selecting the variable region of heavy chain of human immunoglobulin heavy chain III subgroup and people's antibody I g κ chain I subgroup is respectively as the framework region of humanized antibody hu3D9 heavy chain and light chain; Hu3D9VHa represents different humanized antibody variable region of heavy chain with hu3D9VHb, and hu3D9VLa represents different humanized antibody variable region of light chain respectively with hu3D9VLb; Dash represents and human immunoglobulin heavy chain III subgroup or the identical amino acid of the Ig κ chain corresponding residue of I subgroup that what represent in the parantheses is the CDR district; Amino acid whose numbering according to Kabat is numbered [E.A.Kabat, T.T.Wu, H.M.Perry, K.S.Gottesman, C.Foeller, Sequences of Proteins of Immunological Interest, Fifth ed., United States Department of Health and Human Services, Bethesda, MD, 1991.];

The antigen-binding activity experimental result of the humanized antibody of Fig. 3: 3D9;

Fig. 4: competition suppresses experimental result, Fig. 4-1 Dauli cell, Fig. 4-2 Raji cell;

Fig. 5: CDC experimental result, Fig. 5-1 Dauli cell, Fig. 5-2 Raji cell;

Fig. 6: antibody is to the ADCC exercising result of Dauli and Raji cell, Fig. 6-1 Dauli cell, Fig. 6-2 Raji cell;

Fig. 7: antibody induction Dauli and Raji apoptosis experimental result, Fig. 6-1 Dauli cell, Fig. 6-2 Raji cell;

Fig. 8: the survival rate curve of female BALB/C mice.

Embodiment

Following examples only further specify the present invention, should not be construed as limitation of the present invention

Raji (people B lymphoma cell, ATCC, CCL-86)

The pGEM-T carrier, U.S. Promega company product

PcDNA3.1 (+), American I nvitrogen company product

The T4DNA ligase enzyme, American I nvitrogen company product

PcDNA3.1/ZEO (+) carrier, American I nvitrogen company product

COS-1 cell (ATCC CRL 1650)

CHO-K1 cell (ATCC CRL-9618)

PGEM-T easy carrier, U.S. Promega company product

Human myeloma IgG1, κ, Sigma company product

HRP-goat-anti people kappa, Southern Biotechnology Associates company product

Human IgG is the humanized antibody trastuzumab of anti-people her2, Roche company product

Rituximab, Roche company product

Daudi (people B lymphoma cell, ATCC, CCL-213)

People's antibody is light, the clone of weight chain constant area gene

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 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.

Preparation-cytogamy hybridoma of [embodiment 1] anti-humen CD 20 monoclone antibody 3D9 prepares monoclonal antibody

Raji cellular immunization BALB/c mouse (available from available from the Shanghai Experimental Animal Center) with high expression level CD20, make bone-marrow-derived lymphocyte in its spleen can produce the antibody of anti-humen CD 20, the splenocyte and the NS-1 (BALB/c mouse myeloma cell) that get immunity back mouse merge, cultivate through the HAT selectivity, through cultivating, filter out the anti-humen CD 20 positive colony, after cloning, filter out subclone again, to guarantee that antibody is to be produced by single clone cell, collect single clone cell culture supernatant then behind Protein G column purification, just obtain the monoclonal antibody 3D9 of anti-humen CD 20.

The structure of [embodiment 2] chimeric antibody c3D9

The clone of anti-humen CD 20 monoclonal antibody 3D9 variable region gene

Extract total RNA of the hybridoma 3D9 of 2 * 106 secretion anti-humen CD 20 monoclonal antibodies by " Trizol Reagent " test kit (U.S. Gibco BRL company product) specification sheets.Select antibody (IgG3, κ) appropriate location of heavy chain and constant region of light chain is designed 3 gene-specific primer GSP1, GSP2, GSP3 respectively, and wherein GSP1 distance variable district gene is used for reverse transcription reaction farthest, GSP2 is used for first run pcr amplification, and GSP3 is used for the nido amplification.Primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized, and sequence is as follows: GSP1-H, 5 '-GTA GAG GTC AGA CTG CAG GAC-3 '; GSP2-H, 5 '-CTC AGG GAA ATA GCC CTT GAC-3 '; GSP3-H, 5 '-AGA TCC AGG GGC CAG TGG ATA GAC-3 ' .GSP1-L, 5 '-TTG CTG TCC TGA TCA GTC CAA CT-3 '; GSP2-L, 5 '-TGT CGT TCA CTG CCA TCA ATC TT-3 '; GSP3-L, 5 '-TTG TTC AAG AAG CAC ACG ACT GA-3 '.

According to 5 ' RACE test kit (U.S. Gibco BRL company product) specification sheets is that primer becomes cDNA with total RNA reverse transcription with GSP1, add poly (C) tail for then 3 ' the end of the first chain cDNA, being that primer carries out pcr amplification with GSP2 and AAP behind the tailing, is that primer carries out the nest-type PRC amplification with AUAP and GSP3 for 100 times with the amplified production dilution again.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.The nest-type PRC product reclaims purifying purpose segment and is cloned in the pGEM-T easy carrier after 1% agarose gel electrophoresis separates, the screening positive clone order-checking is analyzed sequencing result.Then with the correct pGEM-T/V that checks order _HBe template, design primer H justice AAG CTT GCC GCCACC ATG GAA TGT AAC TGG ATA C and H antisense GCT AGC TGA GGA GAC GGT GACTG, adopt Onestep RT-PCR reaction amplification VH chain variable region gene and make its 5 ' end contain restriction enzyme sites HindIII, 3 ' end contain restriction enzyme sites Nhe I, reaction conditions is: 50 ℃ 30 minutes; 95 ℃ 15 minutes; 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 50 seconds, 30 circulations; 72 ℃ 10 minutes.Reclaim the PCR product and be cloned in the pGEM-T carrier through the agarose gel electrophoresis purifying, screening positive clone sequence verification, result prove that the sequence of this sequence and 5 ' RACE is in full accord.Correct clone's note in this example is made pGEM-T/VH.

With the correct pGEM-T/V that checks order _LBe template, design primer L justice AAG CTT GCC GCC ACC ATG GAG ACA GAC ACA CTC CT and L antisense TGG TGC AGC CAC AGT CCG TTT TAT TTC CAG adopt Onestep RT-PCR reaction amplification VL gene and make its 5 ' end contain restriction enzyme sites HindIII, 3 ' end contains the complementary sequence of human antibody light chain constant region 5 ' end, and reaction conditions is: 94 ℃ 5 minutes; 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 1 minute, 30 circulations; 72 ℃ 10 minutes.Reclaim the PCR product and be cloned in the pGEM-T carrier through the agarose gel electrophoresis purifying, screening positive clone sequence verification, result prove that the sequence of this sequence and 5 ' RACE is in full accord.Correct clone's note in this example is made pGEM-T/VL.

Make up chimeric antibody c3D9

Plasmid pGEM-T/VH HindIII and Nhe I double digestion that above-mentioned Onestep RT-PCR order-checking is correct, reclaiming the enzyme section that obtains about 440bp through the agarose gel electrophoresis purifying breaks, be connected with the plasmid pGEM-T/CH that cuts with enzyme, selecting correct clone back cuts with HindIII and EcoR I enzyme, reclaim the purpose fragment through the agarose gel electrophoresis purifying, be connected with the T4 dna ligase with the plasmid pcDNA3.1 (+) that cuts with enzyme, be built into carrier for expression of eukaryon pcDNA3.1 (+) (VHCH).

Adopt the Overlapping PCR clone pGEM-T/VL that above-mentioned Onestep RT-PCR order-checking is correct directly with the correct clone pGEM-T/CL fusion of constant region of light chain, reaction conditions is: 50 ℃ 30 minutes; 95 ℃ 15 minutes; 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 50 seconds, 30 circulations; 72 ℃ 10 minutes, obtain PCR product VLCL, its 5 ' end contains restriction enzyme sites HindIII, 3 ' end contains restriction enzyme sites EcoR I.Reclaim the PCR product and be cloned in the pGEM-T carrier screening positive clone order-checking through the agarose gel electrophoresis purifying.The VLCL gene that order-checking is correct downcuts from the pGEM-T carrier with HindIII and the two enzymic digestions of EcoR I, is cloned in pcDNA3.1/ZEO (+) carrier, is built into carrier for expression of eukaryon pcDNA3.1/ZEO (+) (VLCL).

In 3.5cm tissue culture ware, inoculate 3 * 10 ⁵The CHO-K1 cell, cell cultures is carried out transfection when 90%-95% merges: (plasmid pcDNA3.1 (+) is 4 μ g (VHCH) to get plasmid 10 μ g, plasmid pcDNA3.1/ZEO (+) is 6 μ g (VLCL)) and 2 μ l Lipofectamine2000 Reagent (Invitrogen company product) be dissolved in 500 μ l serum-free DMEM substratum respectively, room temperature left standstill 5 minutes, with above 2 kinds of liquid mixing, incubated at room 20 minutes is so that the formation of DNA-liposome complex, therebetween with the blood serum medium that contains in the DMEM substratum replacement culture dish of 3ml serum-free, then the DNA-liposome complex that forms is joined in the plate CO ₂Incubator is cultivated after 4 hours and is added the DMEM perfect medium that 2ml contains 10% serum, places CO ₂Continue in the incubator to cultivate.Cell changed the selection substratum screening resistance clone that contains 600 μ g/ml G418 and 250 μ g/ml Zeocin 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) 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 (Human myeloma IgG1, κ), 37 ℃ of incubation 2h, add HRP-goat anti-human igg (κ) and carry out association reaction, 37 ℃ of incubation 1h add TMB in 37 ℃ of effect 5min, use H at last ₂SO ₄Termination reaction is surveyed A ₄₅₀Value.With the high-expression clone serum free medium enlarged culturing that screening obtains, use Protein A affinity column (GE company product) separation and purification chimeric antibody c3D9.Antibody purification is dialysed with PBS, at last with the uv-absorbing standard measure.

The structure of [embodiment 3] 3D9 humanized antibody

The homology mould of 3D9 monoclonal antibody variable region, mouse source (Fv) three-dimensional structure is built

Utilize the Insight II routine package of Accelrys company to simulate the three-dimensional structure of monoclonal antibody variable region, 3D9 mouse source.At first, (Protein Data Bank searches for 3D9 heavy chain and the proteic template albumen of variable region of light chain respectively in PDB) at the protein structure database with blast program.Choose the highest antibody of homology (PDB NO.2E27) and (PDB NO.1FH5) respectively as the mould modeling plate of 3D9 heavy chain and light chain, homology is respectively 83% and 95%, the three-dimensional structure of utilizing Insight II program mould to build out 3D9, as shown in Figure 1.

The design of 3D9 humanized antibody and structure

Select human immunoglobulin heavy chain III subgroup (heavy chain subgroupIII (humIII)) and Ig κ chain I subgroup (light chain k subgroup I (humkI)) humanization template respectively as 3D9 antibody weight, light chain.We at first are grafted directly to heavy chain and light chain CDR people from the district of 3D9 respectively on From Template human immunoglobulin heavy chain III subgroup and the Ig κ chain I subgroup, constitute CDR grafted antibody, and heavy chain is hu3D9Ha, and light chain is hu3D9La.The variable region amino acid sequence of hu3D9Ha and hu3D9La as shown in Figure 2.Heavy, the chain variable region gene (hu3D9VHa and hu3D9VLa) of the synthetic humanized antibody of full gene is that template is passed through the synthetic humanized antibody heavy chain gene of overlapping PCR with hu3D9VHa gene and pGEM-T/CH carrier then, and 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 humanization heavy chain 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:

ATGGAATGTAACTGGATACTTCCTTTTATTCTGTCAGTAACTTCAGGTGTCTACTCA。Last agarose gel electrophoresis separates pcr amplification product, reclaims the purpose band and is cloned in the pGEMT carrier screening positive clone order-checking.Selecting the correct clone of order-checking cuts with HindIII and EcoR I enzyme, reclaim humanized antibody heavy chain fragment hu3D9VHaCH through the agarose gel electrophoresis purifying, with be connected with the plasmid pcDNA3.1 (+) that EcoR I enzyme is cut with HindIII, be built into humanization heavy chain carrier for expression of eukaryon pcDNA3.1 (+) (hu3D9VHaCH).

With hu3D9VLa gene and pGEM-T/CL carrier is that template is passed through the synthetic humanized antibody light chain gene of overlapping PCR, and reaction conditions is: 95 ℃ 15 minutes; 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 50 seconds, 30 circulations; 72 ℃ 10 minutes, obtain PCR product hu3D9VLaCL, 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:

ATGGTTTTCACACCTCAAATACTTGGACTTATGCTTTTTTGGATTTCAGTCTCCAGAGGT。Selecting the correct clone of order-checking cuts with HindIII and EcoR I enzyme, reclaim humanized antibody light chain segments hu3D9VLaCL through the agarose gel electrophoresis purifying, with be connected with plasmid pcDNA3.1/ZEO (+) carrier that EcoR I enzyme is cut with HindIII, be built into humanization light chain carrier for expression of eukaryon pcDNA3.1/ZEO (+) (hu3D9VLaCL).

In 24 hole tissue culturing plates, inoculate 0.8 * 10 ⁵The COS-1 cell in/hole carries out transfection with the RPMI1640/DMEM mixed culture medium (16/DM substratum) of 10% FCS: get plasmid 1 μ g (light chain expression vector 0.6 μ g when being cultured to the 90-95% degrees of fusion; Heavy chain expression carrier 0.4 μ g) and 2 μ lLipofectamine2000 Reagent be dissolved in 50 μ l serum-free 16/DM substratum respectively, room temperature left standstill 5 minutes, with above 2 kinds of liquid mixing, incubated at room 20 minutes is so that the formation of DNA-liposome complex, replace the blood serum medium that contains in 24 orifice plates with the 16/DM substratum of 0.5ml serum-free therebetween, then the DNA-liposome complex that forms is joined in the hole CO ₂Incubator is cultivated after 4 hours and is added the 16/DM substratum that 0.5ml contains 20% FCS, places CO ₂Continue in the incubator to cultivate, get culture supernatant analysis after 72 hours, adopt ELISA to determine the content of antibody in the culture supernatant: Goat anti-human IgG (Fc) bag quilt is in elisa plate, and 4 ℃ are spent the night, sealed 2 hours in 37 ℃ with 2% BSA-PBS, (Human myeloma IgG1 κ), was hatched 2 hours for 37 ℃ to add culture supernatant to be measured and standard substance, add HRP-goat-anti people kappa and carry out association reaction, hatched 1 hour for 37 ℃, add TMB, use H at last in 37 ℃ of effects 5 minutes ₂SO ₄Termination reaction is surveyed the OD450 value.

With the people Raji cell resuspended one-tenth 1 * 10 of 2%FCS-PBS ⁶Cells/ml, the COS-1 cells and supernatant that adds different dilution transfection humanized antibodies respectively, expression is placed on 4 ℃ and hatches 60min, wash cell 2 times with 2%FCS-PBS, add FITC-goat anti-human IgG (H+L) again and hatch 60min in 4 ℃, wash the fluorescence intensity of analyzing and calculate cell behind the cell with FCM.Found that with the c3D9 chimeric antibody and compare that the activity of the humanized antibody (hu3D9Ha/hu3D9La) that hu3D9Ha and hu3D9La form is almost completely lost (Fig. 3).Therefore, in order to obtain the humanized antibody of high-affinity, we also need mouse source, the FR district residue that may influence the 3D9 antibody binding activity is analyzed and reverse mutation.

By analyzing the three-dimensional structure (Fig. 1) of the 3D9 monoclonal antibody variable region that mould builds, we find around the CDR district

Spatial dimension in may influence original antibody CDR conformation and the FR district residue different with corresponding position in people's From Template has 12, be respectively L3Val, L4Leu, L49Lys, H27Tyr, H30Thr, H43Gln, H48Ile, H67Ala, H69Leu, H71Ala, H73Lys and H78Ala.These mouse source amino-acid residues are retained in the CDR grafted antibody of structure and can obtain humanized antibody (hu3D9Hb/hu3D9Lb).The variable region amino acid sequence of hu3D9Hb and hu3D9Lb as shown in Figure 2, SEQ ID NO:9 and SEQ ID NO:10 have shown the nucleotide sequence and the aminoacid sequence of hu3D9Hb variable region of heavy chain respectively.SEQ ID NO:11 and SEQ ID NO:12 have shown the nucleotide sequence and the aminoacid sequence of hu3D9Lb variable region of light chain respectively.SEQ ID NO:5 and SEQ ID NO:6 have shown nucleotide sequence and the aminoacid sequence of hu3D9Hb respectively.SEQ ID NO:7 and SEQ ID NO:8 have shown nucleotide sequence and the aminoacid sequence of hu3D9Lb respectively.Three CDR region amino acid sequences of hu3D9Hb are respectively (can referring to the HU3D9VHb of Fig. 2-1): HCDR1 (NYWMQ), HCDR2 (AIYPGDGDTRYTQKFKG), HCDR3 (EGAYGYDDGMDY); Three CDR region amino acid sequences of hu3D9Lb are respectively (can referring to the HU3D9VLb of Fig. 2-2): LCDR1 (RASQSIRNNLH), LCDR2 (YASQSIS), LCDR3 (QQSNSWPLT).Heavy, the chain variable region gene (hu3D9VHb/hu3D9VLb) of the synthetic respectively humanized antibody of the method that adopts overlapping PCR, and by the method identical with humanized antibody (hu3D9Ha/hu3D9La) make up light chain expression vector pcDNA3.1/ZEO (+) (hu3D9VLbCL) and heavy chain expression carrier pcDNA3.1 (+) (hu3D9VHbCH).With light, heavy expression vector cotransfection COS-1 cell,, find that it is active similar to the 3D9 chimeric antibody with combining of Raji, then with this humanized antibody (hu3D9Hb/hu3D9Lb) called after hu3D9 with the antigen-binding activity of flow cytometry mensuration antibody.

The stably express and the purifying of [embodiment 4] humanized antibody

In 3.5cm tissue culture ware, inoculate 3 * 10 ⁵The CHO-K1 cell, cell cultures is carried out transfection when 90%-95% merges: (plasmid pcDNA3.1 (+) is 4 μ g (hu3D9VHbCH) to get plasmid 10 μ g, plasmid pcDNA3.1/ZEO (+) is 6 μ g (hu3D9VLbCL)) and 2 μ lLipofectamine2000Reagent (Invitrogen company product) be dissolved in 500 μ l serum-free DMEM substratum respectively, room temperature left standstill 5 minutes, with above 2 kinds of liquid mixing, incubated at room 20 minutes is so that the formation of DNA-liposome complex, therebetween with the blood serum medium that contains in the DMEM substratum replacement culture dish of 3ml serum-free, then the DNA-liposome complex that forms is joined in the plate CO ₂Incubator is cultivated after 4 hours and is added the DMEM perfect medium that 2ml contains 10% serum, places CO ₂Continue in the incubator to cultivate.Cell changed the selection substratum screening resistance clone that contains 600 μ g/ml G418 and 250 μ g/ml Zeocin 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) 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 (Human myeloma IgG1, κ), 37 ℃ of incubation 2h, add HRP-goat-anti people kappa and carry out association reaction, 37 ℃ of incubation 1h add TMB in 37 ℃ of effect 5min, use H at last ₂SO ₄Termination reaction is surveyed the A450 value.With the high-expression clone serum free medium enlarged culturing that screening obtains, use Protein A affinity column (GE company product) separation and purification humanized antibody hu3D9.Antibody purification is dialysed with PBS, at last with the uv-absorbing standard measure.

[embodiment 5] competition suppresses experiment

With dialysis labelling method traget antibody: with the carbonate buffer solution of 0.025M pH9.5 the antibody 3D9 of preliminary making is diluted to 1% concentration, in the dialysis tubing of packing into.With same damping fluid the solution that FITC is made into 0.1mg/ml is contained in small beaker, dialysis tubing is immersed in the FITC solution, stir 24h 4 ℃ of lucifuges.Take out marking fluid in the dialysis tubing, cross post with Sephadex G-50, remove free fluorescein, it is standby to collect fluorescence antibody FITC-3D9.Join target cell Raja (1 * 10 after the unmarked antibody purification of the fluorescent-labeled antibody FITC-3D9 of fixed sub-saturated concentration and serial dilution mixed respectively ⁶/ ml) in, hatch 60min for 4 ℃, 1%FCS-PBS washes cell 2 times, flow cytometer detects also uses the Cellquest software analysis.Human IgG in contrast.Each concentration of competition antibody is established 3 multiple pipes, calculation of half inhibitory concentration IC ₅₀Value, maximum fluorescence intensity is illustrated in the average fluorescent strength that obtains when not competing antibody.

Experimental result is seen Fig. 4, and what the result showed that antibody hu3D9 can block fluorescent-labeled antibody FITC-3D9 and Raja cell fully combines their IC ₅₀Be worth approachingly, show that humanized antibody has and similar specificity and the avidity of former mouse source antibody

Cell killing (CDC) experiment of [embodiment 6] complement-mediated

Wash twice with no phenol red RPMI RPMI-1640 behind the collecting cell, be resuspended in no phenol red RPMI RPMI-1640, adjust cell density to 1 * 106/ml, cell suspension is added 96 porocyte culture plates by 100 μ l/ holes.With no phenol red RPMI1640 nutrient solution with Rituximab and c3D9, hu3D9 is diluted to 100 μ g/ml, 20 μ g/ml, 4 μ g/ml, 0.8 μ g/ml and 0.16 μ g/ml respectively, 1%Triton-X 100 is as positive control, human IgG is as irrelevant antibody control, PBS is as negative control, and blank nutrient solution is as blank.To dilute good antibody sample and the above-mentioned 96 porocyte culture plates of contrast adding then, 20 μ l/ holes, the ratio in 50 μ l/ml cell suspensions adds Freshman serum simultaneously, replenishes no phenol red RPMI1640 nutrient solution to cumulative volume 200 μ l/ holes.More than every group establish 3 multiple holes, CO2 incubator effect 4 hours.With centrifugal 5 minutes of 96 porocyte culture plate 200g, 50 μ l supernatants were drawn to another 96 orifice plate respective aperture in every hole after 4 hours.According to the CytoTox of Promega company

Method in the heterotope method cell killing detection kit adds in colour developing liquid 50 μ l//hole to 96 orifice plate that mixes, room temperature, lucifuge effect 30 minutes.Microplate reader is read the absorbance value of 490nm.

Experimental result is seen Fig. 5, test-results shows: in the cell strain Daudi and Raji of CD20 high expression level, c3D9 and hu3D9 have had very strong CDC activity, and the CDC effect that kills and wounds the strongest .c3D9 of intensity and hu3D9 when the concentration of 10 μ g/ml all is better than Rituximab; And control antibodies human IgG can not induce the CDC effect of Dauli cell;

Cytotoxicity (ADCC) experiment of [embodiment 7] antibody-dependant cell mediation

The separation of peripheral blood mononuclear cell (PBMC)

Aseptic collection venous blood injects the centrifuge tube that contains heparin 20U/ml, gently mixing.In Biohazard Safety Equipment, add equal-volume PBS solution, make hemodilution to improve separating effect with aseptic straw.Get the 15ml centrifuge tube, every pipe adds the lymphocyte separation medium of 6ml room temperature, and the inclination centrifuge tube slowly adds anticoagulation cirumferential blood 6ml/ pipe after the dilution along tube wall.Action is soft, with tamper-proof interface.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, cellular segregation liquid, red corpuscle and GCL from top to bottom.The white layer of plasma layer and cellular segregation liquid intersection ground-glass-like is lymphocyte 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.It is centrifugal to add PBS dilution back in the cell of sucking-off, 200g, and 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 the 15ml centrifuge tube, adds the IL-2 preactivate of 160U/ml, places 37 ℃, and 5% CO2 cell culture incubator is standby.

The preparation of target cell suspension

The cell suspension of logarithmic phase is drawn in the 15ml centrifuge tube, 200g, and centrifugal 5min, supernatant discarded is washed 2 times with PBS, no phenol red RPMI-1640 re-suspended cell, counting, adjusting cell density is 3 * 10 ⁵/ ml is standby.

The effect of cell and antibody

Set substratum background hole, the spontaneous releasing value of effector cell+target cell hole, maximum releasing value hole of effector cell+target cell and experimental port, every kind of situation is all established 3 parallel holes, with no phenol red RPMI-1640 substratum with c3D9, hu3D9 is diluted to concentration and is respectively 100 μ g/ml, 20 μ g/ml, 4 μ g/ml, 0.8 μ g/ml and 0.16 μ g/ml, every pipe adds the target cell of adjusting cell density, behind 4 ℃ of effect 30min, the centrifugal 5min of 200g, PBS washes 2 times, be suspended from 300 μ l and do not have phenol red RPMI1640 nutrient solution, cell suspension after above-mentioned and the antibody effect is added in 96 orifice plates, 100 μ l/ holes add effector cell 100 μ l/ holes, with 40: 1 effect, target is than adding in 96 orifice plates, 37 ℃, develop the color behind 5%CO2 cell culture incubator 12h～24h.

Colour developing, reading

Behind the centrifugal 5min of 96 orifice plate 200g, 50 μ l supernatants are drawn to another 96 orifice plate respective aperture, according to the CytoTox of Promega company in every hole

Method in the heterotope method cell killing detection kit adds in colour developing liquid 50 μ l//hole to 96 orifice plate that mixes, room temperature, and lucifuge effect 30min, microplate reader is read the absorbance value of 490nm.Experimental result is seen Fig. 6.

Test-results shows, c3D9 and hu3D9 when lower concentration to do the time spent not obvious, the ADDC effect that can cause tangible ADCC effect .c3D9 and hu3D9 when the concentration of 10 μ g/ml all is better than Rituximab. and control antibodies human IgG can not induce the ADCC effect of Raji.

The experiment of [embodiment 8] apoptosis

In 96 well culture plates, add the cell that number equates, 5 * 10 ⁵/ hole, antibody is added in the respective fine hilum with the final concentration of 10 μ g/ml, 2 μ g/ml, 0.4 μ g/ml and 0.08 μ g/ml respectively, behind 37 ℃ of effect 18～24h, add 5 μ l Annexin V-FITC, mixing gently, room temperature, lucifuge reaction 15min, PBS goes up machine in resuspended back, flow cytometer check and analysis dyeing situation.Negative control is PBS, and irrelevant antibody control is Human IgG, and experimental result is seen Fig. 7.

Test-results shows, c3D9 and hu3D9 when lower concentration to do the time spent not obvious, can cause that when the concentration of 10 μ g/ml the apoptotic effect of a certain amount of apoptosis .c3D9 and hu3D9 compares no difference of science of statistics with Rituximab. and control antibodies human IgG can not induce the Dauli apoptosis

[embodiment 9] survival rate

With 3.5 * 10 ⁶Raji cell tail vein injection immunity female BALB/C mice in 8-10 age in week, the inoculated tumour cell was injected 100 μ g associated antibodies (hu3D9 antibody, Human IgG, Rituximab) after five days, observed the existing state of mouse every day.

Experimental result is seen Fig. 8, and test-results shows: under Isodose, compare with the mouse of injection rituximab, the survival time of injection of antibodies hu3D9 group mouse has obtained significant prolongation (P＜0.05).

Sequence table

＜110〉Antibodies National Engineering Research Center

＜120〉anti-humen CD 20 humanized antibody, Preparation Method And The Use

<130>human?antimouse?antibody?response(HAMA)[Winter?G，Harris?WJ.Humanized?antibodies.Immunol?Today.1993?Jun；14(6)：243-6]

<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?Ile?Glu?Lys?Thr?Ile?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?Ile?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>1353

<212>DNA

＜213〉humanized antibody h3D9 heavy chain nucleotide sequence

<400>5

gaggtccagc?tggtggaatc?cggcggcggc?ctcgtgcaac?caggtggctc?actccgactg 60

tcgtgtgcgg?ctagtggata?tacctttaca?aattactgga?tgcaatgggt?ccggcaggct 120

cccgggcagg?gcttagagtg?gatcagcgcc?atctaccccg?gcgacggtga?cactcgctat 180

acccagaagt?tcaagggaag?agccaccttg?agcgccgata?aatctaagaa?cactgcatac 240

cttcaaatga?actctctccg?cgccgaagat?accgcagtct?actactgtgc?gcgagagggc 300

gcttatggat?acgacgacgg?tatggattac?tggggtcagg?gcaccctagt?aacagtgtcc 360

tcagctagca?ccaagggccc?atcggtcttc?cccctggcac?cctcctccaa?gagcacctct 420

gggggcacag?cggccctggg?ctgcctggtc?aaggactact?tccccgaacc?ggtgacggtg 480

tcgtggaact?caggcgccct?gaccagcggc?gtgcacacct?tcccggctgt?cctacagtcc 540

tcaggactct?actccctcag?cagcgtggtg?accgtgccct?ccagcagctt?gggcacccag 600

acctacatct?gcaacgtgaa?tcacaagccc?agcaacacca?aggtggacaa?gaaagttgag 660

cccaaatctt?gtgacaaaac?tcacacatgc?ccaccgtgcc?cagcacctga?actcctgggg 720

ggaccgtcag?tcttcctctt?ccccccaaaa?cccaaggaca?ccctcatgat?ctcccggacc 780

cctgaggtca?catgcgtggt?ggtggacgtg?agccacgaag?accctgaggt?caagttcaac 840

tggtacgtgg?acggcgtgga?ggtgcataat?gccaagacaa?agccgcggga?agagcagtac 900

aacagcacgt?accgtgtggt?cagcgtcctc?accgtcctgc?accaggactg?gctgaatggc 960

aaggagtaca?agtgcaaggt?ctccaacaaa?gccctcccag?cccccatcga?gaaaaccatc 1020

tccaaagcca?aagggcagcc?ccgagaacca?caggtgtaca?ccctgccccc?atcccgggat 1080

gagctgacca?agaaccaggt?cagcctgacc?tgcctggtca?aaggcttcta?tcccagcgac 1140

atcgccgtgg?agtgggagag?caatgggcag?ccggagaaca?actacaagac?cacgcctccc 1200

gtgctggact?ccgacggctc?cttcttcctc?tacagcaagc?tcaccgtgga?caagagcagg 1260

tggcagcagg?ggaacgtctt?ctcatgctcc?gtgatgcatg?aggctctgca?caaccactac 1320

acgcagaaga?gcctctccct?gtctcccggt?aaa 1353

<210>6

<211>451

<212>PRT

＜213〉humanized antibody h3D9 heavy chain amino acid sequence

<400>6

Glu?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly

1 5 10 15

Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Asn?Tyr

20 25 30

Trp?Met?Gln?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile

35 40 45

Ser?Ala?Ile?Tyr?Pro?Gly?Asp?Gly?Asp?Thr?Arg?Tyr?Thr?Gln?Lys?Phe

50 55 60

Lys?Gly?Arg?Ala?Thr?Leu?Ser?Ala?Asp?Lys?Ser?Lys?Asn?Thr?Ala?Tyr

65 70 75 80

Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Glu?Gly?Ala?Tyr?Gly?Tyr?Asp?Asp?Gly?Met?Asp?Tyr?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser

115 120 125

Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala

130 135 140

Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val

145 150 155 160

Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala

165 170 175

Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val

180 185 190

Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His

195 200 205

Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser?Cys

210 215 220

Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly

225 230 235 240

Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met

245 250 255

Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His

260 265 270

Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val

275 280 285

His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr

290 295 300

Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly

305 310 315 320

Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile

325 330 335

Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val

340 345 350

Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser

355 360 365

Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu

370 375 380

Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro

385 390 395 400

Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val

405 410 415

Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met

420 425 430

His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser

435 440 445

Pro?Gly?Lys

450

<210>7

<211>642

<212>DNA

＜213〉humanized antibody h3D9 light chain nucleotide sequence

<400>7

gatatcgttc?tgacgcagtc?gccttcttct?ctctccgctt?ccgtcggcga?ccgggtaacc 60

attacctgca?gagcctcaca?gtcgatcagg?aacaacctgc?attggtatca?acagaaaccc 120

ggaaaggccc?caaagctgct?cataaagtac?gccagtcaga?gcattagcgg?ggtgccgtct 180

cgtttttctg?gctccggctc?cggtactgac?ttcactctca?caatctcctc?tctgcagccc 240

gaggattttg?caacgtacta?ctgccaacag?tcaaactcgt?ggcctctgac?cttcggacag 300

gggaccaagg?tggaaatcaa?acgcactgtg?gctgcaccat?ctgtcttcat?cttcccgcca 360

tctgatgagc?agttgaaatc?tggaactgcc?tctgttgtgt?gcctgctgaa?taacttctat 420

cccagagagg?ccaaagtaca?gtggaaggtg?gataacgccc?tccaatcggg?taactcccag 480

gagagtgtca?cagagcagga?cagcaaggac?agcacctaca?gcctcagcag?caccctgacg 540

ctgagcaaag?cagactacga?gaaacacaaa?gtctacgcct?gcgaagtcac?ccatcagggc 600

ctgagctcgc?ccgtcacaaa?gagcttcaac?aggggagagt?gt 642

<210>8

<211>214

<212>PRT

＜213〉humanized antibody h3D9 light-chain amino acid sequence

<400>8

Asp?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Arg?Asn?Asn

20 25 30

Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Lys?Tyr?Ala?Ser?Gln?Ser?Ile?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?Asn?Ser?Trp?Pro?Leu

85 90 95

Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala

100 105 110

Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly

115 120 125

Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala

130 135 140

Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?GIy?Asn?Ser?Gln

145 150 155 160

Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser

165 170 175

Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr

180 185 190

Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser

195 200 205

Phe?Asn?Arg?Gly?Glu?Cys

210

<210>9

<211>363

<212>DNA

＜213〉humanized antibody h3D9 weight chain variable region nucleotide sequence

<400>9

gaggtccagc?tggtggaatc?cggcggcggc?ctcgtgcaac?caggtggctc?actccgactg 60

tcgtgtgcgg?ctagtggata?tacctttaca?aattactgga?tgcaatgggt?ccggcaggct 120

cccgggcagg?gcttagagtg?gatcagcgcc?atctaccccg?gcgacggtga?cactcgctat 180

acccagaagt?tcaagggaag?agccaccttg?agcgccgata?aatctaagaa?cactgcatac 240

cttcaaatga?actctctccg?cgccgaagat?accgcagtct?actactgtgc?gcgagagggc 300

gcttatggat?acgacgacgg?tatggattac?tggggtcagg?gcaccctagt?aacagtgtcc 360

tca 363

<210>10

<211>121

<212>PRT

＜213〉humanized antibody h3D9 weight chain variable region amino acid sequence

<400>10

Glu?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly

1 5 10 15

Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Asn?Tyr

20 25 30

Trp?Met?Gln?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile

35 40 45

Ser?Ala?Ile?Tyr?Pro?Gly?Asp?Gly?Asp?Thr?Arg?Tyr?Thr?Gln?Lys?Phe

50 55 60

Lys?Gly?Arg?Ala?Thr?Leu?Ser?Ala?Asp?Lys?Ser?Lys?Asn?Thr?Ala?Tyr

65 70 75 80

Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Glu?Gly?Ala?Tyr?Gly?Tyr?Asp?Asp?Gly?Met?Asp?Tyr?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

115 120

<210>11

<211>324

<212>DNA

＜213〉humanized antibody h3D9 light chain variable region nucleotide sequence

<400>11

gatatcgttc?tgacgcagtc?gccttcttct?ctctccgctt?ccgtcggcga?ccgggtaacc 60

attacctgca?gagcctcaca?gtcgatcagg?aacaacctgc?attggtatca?acagaaaccc 120

ggaaaggccc?caaagctgct?cataaagtac?gccagtcaga?gcattagcgg?ggtgccgtct 180

cgtttttctg?gctccggctc?cggtactgac?ttcactctca?caatctcctc?tctgcagccc 240

gaggattttg?caacgtacta?ctgccaacag?tcaaactcgt?ggcctctgac?cttcggacag 300

gggaccaagg?tggaaatcaa?acgc 324

<210>12

<211>108

<212>PRT

＜213〉humanized antibody h3D9 light chain variable region amino acid sequence

<400>12

Asp?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Arg?Asn?Asn

20 25 30

Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Lys?Tyr?Ala?Ser?Gln?Ser?Ile?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?Asn?Ser?Trp?Pro?Leu

85 90 95

Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg

100 105

Claims (9)

1. anti-humen CD 20 humanized antibody, its heavy chain hypervariable region aminoacid sequence is CDR1:NYWMQ, CDR2:AIYPGDGDTRYTQKFKG and CDR3:EGAYGYDDGMDY; Light chain hypervariable region aminoacid sequence is CDR1:RASQSIRNNLH; CDR2:YASQSIS and CDR3:QQSNSWPLT.

2. the described anti-humen CD 20 humanized antibody of claim 1, its weight chain variable region amino acid sequence is SEQ ID NO:10, the light chain variable region amino acid sequence is SEQ ID NO:12.

3. the described anti-humen CD 20 humanized antibody of claim 2, its heavy chain amino acid sequence is SEQ ID NO:6, light-chain amino acid sequence is SEQ ID NO:8.

4. nucleic acid molecule, the arbitrary described anti-humen CD 20 humanized antibody of coding claim 1～3.

5. the described nucleic acid molecule of claim 4, wherein the nucleotides sequence of encoding heavy chain variable region is classified SEQ ID NO:9 as, and the nucleotides sequence of encoded light chain variable region is classified SEQ ID NO:11 as.

6. the described nucleic acid molecule of claim 5, wherein the nucleotides sequence of encoding heavy chain is classified SEQ ID NO:5 as, and the nucleotides sequence of coding light chain is classified SEQ ID NO:7 as.

7. the preparation method of the humanized antibody of the arbitrary described anti-humen CD 20 of claim 1～3, comprise the aminoacid sequence that goes out humanized antibody by computer aided design (CAD), full gene synthetic heavy chain and chain variable region gene and, constant region of light chain gene splicing heavy with human normal immunoglobulin respectively through gene recombination, be cloned in the carrier for expression of eukaryon, make up light, the heavy chain expression carrier of humanized antibody respectively, then will be light, the heavy chain expression carrier is with liposome method cotransfection Chinese hamster ovary celI, screen then, culture purified promptly.

8. the purposes of the humanized antibody of the arbitrary described anti-humen CD 20 of claim 1～3 in the lymphoid tumor medicament of preparation treatment high expression level CD20.

9. the described purposes of claim 8, wherein the lymphoma of high expression level CD20 is the non-Hodgkin lymphomas.

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