CN102863531A - CD20-resistant monoclonal antibody as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a humanized CD20-resistant monoclonal antibody. A heavy chain variable region of the humanized CD20-resistant monoclonal antibody has an amino acid sequence as follows: VH: SEQ ID:NO.6; and a light chain variable region has an amino acid sequence as follows: VL:SEQ ID:NO.8. The invention further comprises an application of the monoclonal antibody to the aspects of researches as well as diagnosis and treatment. The invention provides the humanized CD20-resistant monoclonal antibody with high specificity. Compared with a positive medicament, the monoclonal antibody has the advantages of better medicinal effect, simple and convenient preparation process, lower cost, wide application range, and capability of meeting the social requirements, and supplies an accurate, easy, convenient and effective monitoring means for the prevention, treatment, early-stage screening and diagnosis work of cancers and eye diseases. Above all, the CD20-resistant monoclonal antibody positively adapts to the working requirements and the requirement of user-friendly service in the fields of modern biology, prevention and health care, clinical diagnosis and the like; the humanized CD20-resistant monoclonal antibody is researched and developed to provide a treatment medicament with higher cost performance for the medicament market in China; and the CD20-resistant monoclonal antibody has a very wide application prospect.

Description

A kind of anti-CD-20 monoclonal antibody and its production and use

Technical field

The invention belongs to the technical fields such as biological medicine, monoclonal antibody technique, antibody humanization's technology, specifically relate to a kind of monoclonal antibody and its production and use, more particularly relate to a kind of for anti-CD-20 monoclonal antibody and its production and use.

Background technology

The mortality ratio of tumour occupies the umber one in the mortality ratio of whole world various diseases, and malignant tumour has become the killer who has a strong impact on human health and life.Non-Hodgkin lymphoma (Non Hodgkin ' s Lymphoma, NHL) is one of common malignant hematologic disease, and is clinical modal lymphsystem malignant tumour, causes (the B cell derived accounts for 85%) by the pernicious growth of bone-marrow-derived lymphocyte, is apt to occur in person between twenty and fifty.In recent years, NHL sickness rate and case fatality rate are year by year ascendant trend, and human health in serious harm.The treatment of non-Hodgkin lymphoma and diagnosis are study hotspots in recent years, and it causes (the B cell derived accounts for 85%) by the pernicious growth of bone-marrow-derived lymphocyte, is apt to occur in person between twenty and fifty.In recent years, NHL sickness rate and case fatality rate are year by year ascendant trend, and human health in serious harm.But the mechanism of non-Hodgkin lymphoma is complicated, be mutual, the coefficient result of many factors, and the research of single mechanism often can not reach satisfied effect, so comprehensive study number of mechanisms, that the multiple medicines thing is united utilization remains further to be carried out.

The cell surface molecule of being expressed by B cell and its corresponding malignant tumour is the important target of immunotherapy.The B cell-specific member CD20 of MS4A gene family prematurity and mature B cell with and the expression (Tedder and Engel (1994) Immunol.Today 15:450-454) of corresponding malignant tumour.Most human B cell pedigree malignant tumours is expressed CD20(Anderson etc., Blood, 198463:1424).Magnetic target therapy for Several Kinds of Malignancy superficial cell surface antigen mark makes great progress in recent years, the clinical effectiveness of using monoclonal antibody treatment non-Hodgkin lymphoma makes substantial progress, so that become the good a kind of new antitumoral method of development prospect for the mab treatment non-Hodgkin lymphoma of CD20.

Anti-CD-20 monoclonal antibody is treatment B the most effective lymphadenomatous medicine, in treatment NHL extremely important status is arranged.CD20 is that a kind of molecular weight is that (the CD20 schematic arrangement is seen Fig. 1 for the phosphorylated protein molecule of 33～37kD, wherein), it is the non-glycosylated quadruple cross-film of bone-marrow-derived lymphocyte phosphorprotein, be positioned at the bone-marrow-derived lymphocyte surface, be bone-marrow-derived lymphocyte Surface Differentiation antigen, and all do not express at its hetero-organization and multipotency B lymphoid stem cell.It mainly participates in regulating propagation and the differentiation of bone-marrow-derived lymphocyte, play an important role in immunity system, CD20 began to express from the pre-B lymphocyte stage, until the plasmocyte stage is just without CD20 expression (Stashenko p., Nsdler LM, Hardy R, et al.Characterization of a human Blymphocyte-specific antigen.J.Immun.1980; 125:1678-1685).After CD20 and the anti-CD20 antibodies combination endocytosis does not occur, thereby cell surface CD20 quantity does not reduce the free CD20 existence of nothing in human serum because of the combination of antibody.CD20 antigen high expression level is in the B lymphoma cell that surpasses 95% normal or worsen, there is not remarkable endocytosis and (the Press OW that comes off, Farr AG, Borroz KI et al.Endocytosis and Degradation of Monoclonal Antibodies Targeting Human B-Cell Malignancies.Cancer Res1989; 49:4906-4912), thus CD20 be the desirable target spot for the treatment of bone-marrow-derived lymphocyte knurl.

The CD20 monoclonal antibody that is used for the treatment of NHL through FDA approval listing has Rituximab, Zevalin, Bexxar etc., Rituximab(Rituximab, trade(brand)name: Rituximab; Rear abbreviation " Rituximab ") be that first is the anti-CD-20 monoclonal antibody class medicine of listing, it is a kind of mosaic human IgG1 anti-humen CD 20 monoclone antibody, can efficiently induce classical pathway that complement (C) activates and to the cytotoxicity (Reff etc. of the Complement Dependent of the lymphoma cell of fresh separated and B clone, Blood, 1994,83:435-445; Golay etc., Blood, 98:3383-3389; The Blood such as Cragg, 2003,101:1045-1052; The J.Immunl.2003 such as Di Gaetano, 171:1581-1587; Bellosillo etc., Blood, 2001,98:2771-2777).Rituximab is also at patient (van der Klok etc., Br.J.Hematol.2001,115:807-811) and primates (Kennedy etc., Blood, 2003,101:1071-1079) vivo activation complement.In addition, the complement that comprises CD59 is transferred expression in the protelytic tumour cell and resistance relevant (Golay etc., Blood, the 98:3383-3389 of anti-CD20 treatment; J.Immunotheraphy, the 200124:263-271 such as Treon).Although many people think that the cytotoxicity of Complement Dependent is that Rituximab eliminates employed main application (Golay etc., Blood, 98:3383-3389 among the human lymphoma cell in external (invitro) and body; The Blood such as Cragg, 2003,101:1045-1052; The J.Immunl.2003 such as Di Gaetano, 171:1581-1587; Golay etc., Blood, 95:3900-3908; The J.Hematol.2001 such as Di Gaetano, 114:800-809; Weiner Blood2003,101:788), but other people find, can not predict result for the treatment of (Weng and the Levy of Rituximab to the susceptibility of complement-mediated cracking and complement inhibitor CD46, CD55 and the expression of CD59 on tumour cell according to tumour cell, Blood, 2001,98:1352-1357).Because the mosaic anti-CD-20 monoclonal antibody different from the isotype of clinical use can not be eliminated normal B cell (Anderson etc. in inhuman or primates, Biochem.Soc.Transac., 1997,25:705-708), and the anti-tumour effect of anti-CD-20 monoclonal antibody partly relies on immuno-stimulating (Clynes etc., Nature Med., 2000 of the Fc acceptor (Fc γ R) by IgG, 6:443-446), so other antibody dependence effect also is important.Use separately efficient the reaching about 50% of non-Hodgkin lymphoma of Rituximab treatment recurrence and refractory, if with chemotherapeutics coupling, the then efficient 90-100% that reaches.Simultaneously, Rituximab can bring a series of untoward reaction, such as: alopecia, feel sick, vomiting, hemocytopenia etc., and again slight heating, shiver etc.But Rituximab still by it in treatment the non-Hodgkin lymphoma good efficacy and the low toxic side effect that show, become one of antitumor drug of U.S.'s sales volume maximum, 2008 annual sales amounts are nearly 3,000,000,000, annual growth 14%.

But these pharmacological agent expenses are higher, cause a lot of patients of China to be difficult to receive treatment.Therefore, the medicine that research good effect, treatment cost is low is significant to the quality of life that improves patients with non Hodgkin lymphoma.In addition, research and develop littlely to people's immunogenicity, expression amount is higher, and anti-CD-20 monoclonal antibody or polyclonal antibody with other good characteristics, more becomes present research emphasis.Therefore, develop the product, particularly medicine of the aspects such as anti-CD20, significant, and have significant Social benefit and economic benefit.

Summary of the invention

The problems referred to above for prior art exists the purpose of this invention is to provide monoclonal antibody of a kind of Humanized anti-CD 20 and its production and use.

For achieving the above object, the technical solution used in the present invention is as follows:

A kind of Humanized anti-CD 20 monoclonal antibody, wherein variable region of heavy chain has following aminoacid sequence VH:SEQ ID:NO.5; Variable region of light chain has following amino acid sequences VL:SEQ ID:NO.7.

Preferably, above-mentioned Humanized anti-CD 20 monoclonal antibody variable region of heavy chain has aminoacid sequence shown in the SEQ ID:NO.6, and variable region of light chain has aminoacid sequence shown in the SEQ ID:NO.8.

The present invention also provides aminoacid sequence and its variable region chain thereof of anti-CD-20 monoclonal antibody, and other protein or fusion expressed product with these chains, particularly, the present invention includes and have the hypervariable region of containing (complementary determining region, any protein of light chain CDR) and heavy chain or protein conjugate and fusion expressed product (being immune conjugate and fusion expressed product), as long as the hypervariable region of this hypervariable region and light chain of the present invention and heavy chain is identical or at least 90% homology, preferably at least 95% homology.

The antigenic structure characteristic of antibody can be described by 3 each the specific zone that are positioned at heavy chain and variable region of light chain, become complementary determining region (complementarity determining region, CDR), should intersegmentally be divided into 4 each frame area (FR), the aminoacid sequence of 4 FR is relatively conservative, does not participate in association reaction directly.These CDR form ring texturees, and the β-pleated sheet structure that the FR by therebetween forms is mutually close on space structure, and the CDR on the CDR on the heavy chain and the corresponding light chain has consisted of the antigen binding site of antibody.Those skilled in the art can determine its CDR district by heavy chain and the sequence of light chain (SEQID NO:1 and SEQ ID NO:3) of ordinary method analysis with antibody of the present invention.The present invention also comprises these identical heavy chain of antibody in CDR district and light chain of antibody, and the antibody that is made of described heavy chain and light chain.

In addition, also find recently the dependency structure that is made of variable region of light chain, compare with corresponding variable region of heavy chain, the kinetics of its combination is less, and the weight chain variable zone of separation self has antigen-binding activity.

The present invention not only comprises complete monoclonal antibody, also comprises having immunocompetent antibody fragment, such as Fab or (Fab ') ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered scFv molecule; Or chimeric antibody, as have the murine antibody binding specificity but keep antibody from people's antibody moiety, the technology that can be used for formation chimeric antibody of the present invention is well known in the art, and antibody and the couplings such as medicine, toxin, cytokine, radionuclide or enzyme, forms immune conjugate.

The present invention also provides the dna molecular of coding said monoclonal antibody or its fragment.The method of the common available PRC TRAP of the Nucleotide full length sequence of monoclonal antibody of the present invention or its fragment, recombination method or synthetic obtains.A kind of feasible method is to synthesize the shorter chamber of relevant sequence, especially fragment length with the method for synthetic.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.In addition, also the encoding sequence of light chain and heavy chain can be merged, form single-chain antibody.

After obtaining relevant sequence, namely can obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell over to again, then separates obtaining relevant sequence from the host cell after the propagation by ordinary method.

A kind of method for preparing said monoclonal antibody, comprise the aminoacid sequence that goes out humanized antibody BB3CD20-7 by computer aided design (CAD), the heavy chain of the synthetic BD31-7 of full gene and chain variable region gene and through gene recombination, constant region of light chain gene splicing heavy with human normal immunoglobulin respectively, be cloned in the carrier for expression of eukaryon, make up respectively light, the heavy chain expression carrier of humanized antibody, then with light, heavy chain expression carrier liposome method cotransfection Chinese hamster ovary celI, the humanized antibody that then screen, culture purified namely gets anti-humen CD 20 of the present invention: BD31-7.

" nucleic acid molecule " described in the present invention refers to polynucleotide, for example deoxyribonucleotide (DNA) and ribonucleotide (RNA), also comprise the DNA that formed by nucleotide analog or equivalent, the analogue of RNA, strand (sense strand or antisense strand) and double stranded polynucleotide.This term also comprises homologous sequence.For a person skilled in the art, obviously, the nucleic acid molecule of any one above-mentioned form has all been contained all above-mentioned equivalents of this " nucleic acid molecule " natch.

In addition, at present can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis, then this dna sequence dna can be introduced in various existing dna moleculars as known in the art (or such as carrier) and the cell.In addition, also can will suddenly change by chemosynthesis and introduce in the protein sequence of the present invention.

The invention still further relates to the carrier that comprises above-mentioned suitable dna sequence dna and suitable promotor or control sequence.These carriers can be used for transforming suitable host cell, with can marking protein.

Above-mentioned host cell can be prokaryotic cell prokaryocyte, such as bacterial cell; Or the eukaryotic cell such as low, such as yeast cell; Or higher eucaryotic cells, thin such as Mammals.Typical example has: the zooblast of the insect cell of the bacterial cell of intestinal bacteria, streptomyces, Salmonella typhimurium, fungal cell such as yeast, fruit bat S2 or Sf9, CHO, COS7 or 293 cells etc.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When host cell was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be received at the exponential growth after date, uses CaCl ₂Method is processed, and used step is known in the art.Another kind method is to use MgCl ₂If necessary, conversion also can be carried out with the method for electroporation.When the host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to the used cell of appealing to, used substratum can be selected the substratum of various routines in the cultivation.Under the condition that is fit to the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (such as temperature inversion or chemical induction), cell is cultivated for some time again.

Recombinant polypeptide in aforesaid method can be in cell or cytolemma express or be secreted into the extracellular.If necessary, can utilize other characteristics its physics, chemistry by the albumen of various separation method separation and purification restructuring.These methods are well-known to those skilled in the art.The example of these methods includes but not limited to: conventional renaturation processes, process the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods with protein precipitant.

A kind of purposes of said monoclonal antibody is for the preparation of the medicine for the treatment of non-Hodgkin lymphoma or for the preparation of the reagent of Diagnosis of malignant B cell lymphoma as activeconstituents.

As further preferred version, be as activeconstituents for the preparation for the treatment of in the medicine of non-Hodgkin lymphoma, rheumatoid arthritis, idiopathic thrombocytopenic purpura and hemolytic anemia and other immune-mediated diseases, optimum is non-Hodgkin lymphoma.

The present invention also provides a kind of pharmaceutical composition for the treatment of non-Hodgkin lymphoma, and it contains above-mentioned monoclonal antibody or immune conjugate, and pharmaceutically acceptable carrier.Usually, but these materials are disposed at nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is generally 5-8, better pH is 6-8, although the pH value can change to some extent with the character that is formulated thing and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intraperitoneal, intravenously or topical.

The preparation method of pharmaceutical composition of the present invention can be with existing commercially available above-claimed cpd, or the extract that contains above-claimed cpd that from biology, extracts, by proportioning, adopt the ordinary method of this area to obtain, namely get described anti-CD-20 monoclonal antibody composition.Each raw material of addressing among the present invention or reagent is commercially available getting all.

In concrete use, anti-CD-20 monoclonal antibody composition of the present invention can directly use separately, can also use with other many chemical substances.No matter whether these chemical substances have biological activity or have the function for the treatment of disease, comprise subsidiary function such as collaborative amplification, antagonism or alleviate the side effect etc. of anti-CD-20 monoclonal antibody composition, these chemical substances are to comprise in pharmaceutically acceptable carrier, natural product, chemical synthetic drug or the human medication etc. one or more; Preferably include in pharmaceutically acceptable carrier etc. one or more.

Pharmaceutical composition of the present invention contains the pharmaceutical composition of the above-mentioned monoclonal antibody of the present invention of safe and effective amount, and pharmaceutically acceptable carrier or vehicle or thinner.This class carrier includes, but are not limited to: one or more in solvent, dispersion medium, afterbirth, antiseptic-germicide and anti-mycotic agent, isotonic agent or the absorption delay agent etc. that any He all physiology is suitable for.The example of pharmaceutically acceptable carrier comprises one or more water, salt solution, phosphate-buffered saline, damping fluid, glucose, glycerine or ethanol etc. and in the composition one or more thereof.For example be prepared by ordinary method with physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as injection, solution should be made under aseptic condition.

As used herein, term " monoclonal antibody specific of anti-CD20 ", " IDEC-C2B8 ", " CD20 monoclonal antibody ", " CD20 antibody " etc. are used interchangeably, and all refer to the antibody that the aminoacid sequence by the aminoacid sequence of heavy chain of antibody element and light chain of antibody element consists of; This term also comprises the fusion rotein that forms with GST etc., if in this fusion rotein antibody moiety still keep with CD20 in conjunction with active.

Described " pharmaceutically acceptable carrier " comprises one or more in any He all physiology applicable solvent, dispersion medium, afterbirth, antiseptic-germicide and anti-mycotic agent, isotonic agent or the absorption delay agent etc.The example of pharmaceutically acceptable carrier comprises one or more water, salt solution, phosphate-buffered saline, glucose, glycerine or ethanol etc. and in the composition one or more thereof.In many cases, in said composition, preferably include isotonic agent, for example, sugar, such as in the polyvalent alcohol of N.F,USP MANNITOL, sorbyl alcohol, sorbyl alcohol or the sodium-chlor etc. one or more.Pharmaceutically acceptable carrier can also comprise a small amount of auxiliary substance, and such as in wetting agent or emulsifying agent, sanitas or the damping fluid etc. one or more, they have strengthened validity period or the effectiveness of this anticomplement or antioxidizing composition.

From concrete classification, said pharmaceutically acceptable carrier refers to the pharmaceutical carrier of pharmaceutical field routine, comprises lubricant, such as in talcum powder, polyoxyethylene glycol or the Magnesium Stearate etc. one or more; Disintegrating agent is such as in Microcrystalline Cellulose, Xylo-Mucine or the low-substituted hydroxypropyl cellulose etc. one or more; Weighting agent is such as in starch, dextrin or the lactose etc. one or more; Tackiness agent is such as in pregelatinized Starch, derivatived cellulose, alginate, gelatin, polyvinylpyrrolidone, polyvinylpyrrolidone or the hydroxypropylcellulose etc. one or more; Osmotic pressure regulator is such as in sodium-chlor, glucose, sucrose, sorbyl alcohol or the N.F,USP MANNITOL etc. one or more; PH adjusting agent, one or more in the acid such as example hydrochloric acid, sodium hydroxide or the alkali; Solvent, as in water, damping fluid, ethanol or the propylene glycol etc. one or more etc.; Oxidation inhibitor and complexing agent are such as among S-WAT, the EDTA etc. one or more; Tensio-active agent is such as quaternary ammonium compound, cetyl alcohol etc.; Absorption carrier is such as in kaolin or the soap clay etc. one or more; The macromolecular scaffold agent is such as in cyclodextrin, polyoxyethylene glycol, the poloxamer etc. one or more; In thinner such as starch, Icing Sugar, dextrin, Microcrystalline Cellulose, N.F,USP MANNITOL, lactose and the soybean wet goods one or more; In stablizer such as Xylo-Mucine or the cyclodextrin etc. one or more; In sanitas such as ethyl p-hydroxybenzoate or the Sodium Benzoate etc. one or more.In addition, can also in this pharmaceutical composition, add other auxiliarys, such as in flavouring agent and/or sweeting agent such as sucrose, fructose and the aspartame etc. one or more.

Compared to existing technology, the present invention has following unusual effect:

The present invention studies the CD20 monoclonal antibody targetedly, has found a kind of new anti-CD-20 monoclonal antibody composition, has made beyond thought achievement; Simultaneously, the present invention is the activity of Effect of Anti CD20 monoclonal antibody combination targetedly also, and its pharmacological action is stronger, uses safety, has brought into play to greatest extent effect.The present invention resists the CD20 monoclonal antibody combination and has expanded new medicinal use, also provides a kind of new medicament sources for diagnosis, detection, protection, treatment and research anti-CD-20 monoclonal antibody.

This pharmaceutical composition is used for the treatment of the malignant tumour of B cell induction, particularly non-Hodgkin lymphoma, can significantly kill and wound the B lymphoma cell, and result for the treatment of is remarkable, and toxic side effect is little, has overcome the side effect that existing common drug causes.The present invention also provides a kind of new pharmaceutical means for the malignant tumour for the treatment of and diagnosis B cell induction.Anti-CD-20 monoclonal antibody composition of the present invention pharmacological action is stronger, successful.

In a word; active adaption of the present invention modern medical service and the need of work of scientific research field and the needs of human nature service; provide new medicine and preparation source for researching and developing new anti-CD-20 monoclonal antibody product; has important value to developing the existing medicine of China; be to originate for the safety of the aspects such as diagnosis, detection, protection, treatment and research B cell malignancies, have important value to improving and improving existing medical level.

Description of drawings

Fig. 1 is CD20 molecular structures schematic diagram, and wherein Fig. 1 (A) is three kinds of topological frameworks of CD20, and Fig. 1 (B) is the cross-film structure of CD20 molecule;

Fig. 2 has embodied the ADCC experimental result of monoclonal antibody BD31-7 to the Dauli cell;

Fig. 3 has embodied the ADCC experimental result of monoclonal antibody BD31-7 to the Raji cell;

Fig. 4 has embodied the CDC experimental result of monoclonal antibody BD31-7 to the Dauli cell;

Fig. 5 has embodied the CDC experimental result of monoclonal antibody BD31-7 to the Raji cell;

Fig. 6 has embodied monoclonal antibody BD31-7 and has induced Dauli cell apoptosis assay result;

Fig. 7 has embodied monoclonal antibody BD31-7 and has induced Raji cell apoptosis assay result.

Embodiment

Below in conjunction with embodiment and Comparative Examples to the present invention do further in detail, intactly explanation.Should be understood that following embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as people such as Smabrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Ratio and per-cent are based on weight, unless stated otherwise.

Used animal, plant and instrument, reagent and preparation thereof etc. all are from commercially available in the experiment of following examples.

Raji (Human 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, Promega company product

Human myeloma IgG1, K, Sigma company product

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

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

Rituximab, Roche company product

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

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

With lymphocyte separation medium (ancient cooking vessel state biotech development company product) separating health human lymphocyte, 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) report sunlight sequence designs respectively primer 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 respectively nucleotide sequence and the aminoacid sequence of CH (CH).SEQ ID NO:3 and SEQ ID NO:4 have shown respectively nucleotide sequence and the aminoacid sequence of constant region of light chain (CL).Correct clone in this example is denoted as pGEM-T/CH and pGEM-T/CL.

Preparation and the screening of embodiment 1 mouse monoclonal antibody

(1) the preparation human B lymphocyte specific membrane antigen CD20 gene of antigen is transcribed amplification acquisition the group by PCR from people's gene, and primer is given birth to worker bio-engineering corporation by Shanghai and synthesized, and sequence is as follows:

GSP1-H，5’-ACC TGG GTAGGT GCG GAC ACC ACT-3’；

GSP2-H，5’-CAC ACT TTC ACG TCAAGG ACA-3’；

GSP3-H,5’-CTT GTC CAG GGA TCC TAG AGT-3’；

GSP1-L,5’-TTG CAG TCC TGA TCA GGC CAA CT-3’；

GSP2-L,5’-TGT CGT GCA CTG GCAACA ATC TT-3’：

GSP3-L,5’-TTG TTC ATG AAG CTC AAG GCT GA-3’。

Utilize BamHI and Xho I double digestion, gene fragment is connected into prokaryotic expression carrier pGEM-T (U.S. Promega company product), PCR and double digestion check order after identifying positive colony, after sequencing is correct, extract plasmid.Prokaryotic expression carrier pGEM-CD20 transforms the Rosetta bacterial strain, and in 100ml LB substratum, OD600 reaches at approximately 0.5 o'clock, ImM IPTG abduction delivering 4 hours, and CD20 albumen forms inclusion body.After the carrying out ultrasonic bacteria breaking, with 1 * SDS sample-loading buffer dissolving inclusion body, measure the expression amount of target protein.The CD20 albumen for preparing is carried out the SDS-PAGE electrophoresis, the rubber tapping purifying protein.

(2) the CD20 protein dissolution of the immunity of mouse after with purifying got the emulsification of an amount of protein solution and Freund's complete adjuvant (Sigma) mixing at 1 * PBS, gets the female mouse of BALB/c in 8 ages in week, subcutaneous multi-point injection antigen.After 2 weeks, get an amount of egg from solution and the emulsification of Freund's incomplete adjuvant (Sigma) mixing, subcutaneous mouse carries out the immunity second time.Every 3 weeks, carry out third and fourth time immunity later on.Rear 3 days of the 4th immunity got spleen and carried out cytogamy.

(3) screening of cytogamy and positive colony

Crane one and put to death mouse, with its body surface of 70% alcohol disinfecting.Take out spleen under the aseptic condition, in the sterilizing stainless steel net, in spleen, inject the 3ml serum-free medium with syringe, suction obtains cell suspension for several times repeatedly, and cell suspension is injected the 50ml centrifuge tube, adds the 20ml serum-free medium, blow and beat for several times gently, 1000rpm/min is centrifugal.With serum-free medium that cell is resuspended, with murine myeloma cell SP210 with suitable proportion mixing gently, in 37 ℃ of water-baths, add PEG1400, water-bath 1.5min.Add serum-free medium 35ml, centrifugal.Precipitation is resuspended with the RPM1-1640 that contains 20%FCS, and every hole 200uL all assigns to 96 porocyte culture plates, puts into 37 ℃ of 5%CO ₂Cultivate in the incubator.After 24 hours, the nutrient solution HAT that brings Selection In changed one time fresh medium in per 3 days.

After 14 days, get cells and supernatant, ELISA identifies positive colony.Repeatedly carry out subclone and identify that the positive colony cell becomes system, continues vitro culture 6 months.Select 7 plant height efficient expression cell strains, respectively called after 31-1,31-2,31-3,31-4,31-5,31-6,31-7.

(4) evaluation of monoclonal antibody

Serum 1:1000-1:50000 dilution, 100ng CD20 loading is carried out the specificity that Western Blot analyzes antibody.The CD20 gene is connected into expression vector pLent17.3/V5-TOPO by the TOPO cloning process, carrier for expression of eukaryon pLent17.3/V5-CD20 utilizes Lipofectamine2000 (1nvitrogen) mediation transient transfection 293T cell, identified by immunofluorescence antibody is carried out in serum 1:100 dilution.Measure as can be known, the antibodies specific of 21-7 cell strain is best, and can identify the antigen of the endogenous expression of cell.

(5) acquisition of variable region of mab sequence

Utilize Trizol (Invitrogen) method to extract total RNA of hybridoma cell strain 21-7, reverse transcription becomes cDNA with SuperScript II Reverse Transcriptase (lnvitrogen) then to use the oligo-dT primer.According to the FR1 region amino acid sequence of mouse antibodies heavy chain and light chain, the synthetic Oligonucleolide primers that annexs is as just same primer.Reverse primer can obtain whole variable region amplifications according to the constant region design of mouse antibodies.After the PCR reaction was finished, the dna fragmentation with heavy chain of antibody and light chain was connected in the TA cloning vector respectively, and picking the clone check order.

The gained antibody sequence is as follows:

The variable region of heavy chain nucleotide sequence:

CAGGTACAACTACAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAGCAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCAGGAAATGGTTTCACTTCCTACAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAAGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACAAGGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCA；

The weight chain variable region amino acid sequence:

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGFTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYKGGDWYFNVWGAGTTVTVSA；

The variable region of light chain nucleotide sequence:

CAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTACATCCACTGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGTAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGACTAGTCGCCCACCCACGTTCGGTGGTGGG ACCAAGCTGGAGA TCAAA；

The light chain variable region amino acid sequence:

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSRPPTFGGGTKLEIK。

Humanization and the expression and purification of embodiment 2 mouse monoclonal antibodies

(1) humanization of mouse monoclonal antibody is compared antibody aminoacid sequence and the people's antibody aminoacid sequence of mouse, obtain the framework of people's subclass consensus sequence of highest homology sequence, be people's light chain VL κ subgroup II (VL κ II) and heavy chain VH subgroup I (VHI), as the humanization framework.According to the people's such as kabat the definition CDR1:24-34 with the L chain, CDR2:50-56, CDR3:89-97; The CDR1:26-35 of H chain, CDR2:50-65, CDR3:95-102 (sequences of proteins of immunological Interest, (5th), Public Health Service, National Institutes of Health, Bethesda, MD (1991).Utilize computer to make up the stereographic map model (Carter etc. in mouse monoclonal antibody VL-VH district, Proc.Natl.Acad.Sci.USA89:4285-4289 (1992) Eigenbrot.J.Mol.Biol.) 229:969-995 (1993)), determines which murine antibody framework region residue is introduced humanized antibody.Wherein heavy chain removes CDR1, CDR2, and outside the CDR3, the humanization framework is used murine antibody amino acid instead in the H73 site, light chain CDRL1, CDRL2, CDRL3 uses the murine antibody aminoacid sequence.

(2) after the structure of humanized antibody IgG and expression and purification humanized antibody heavy chain and the full gene of light chain variable region nucleotide sequence synthesize, be cloned into respectively carrier A bVec1-hIgG1, AbVec1-hIgKappa.Transform DH5Q, extract the positive colony plasmid.Utilize transfection reagent Lipofectamine2000 (Invitrogen), with the plasmid co-transfection people 293A cell that builds.After cultivating 24h, the DMEM substratum is replaced by minimum medium, and within 5 days every day gather in the crops supernatant.Use ProteinA-Sepharose CL-4B (Amersham) antibody purification after supernatant merges, and identify with the SDS-PAGE electrophoresis.Evaluation product through affinity chromatography passes through sieve chromatography again, obtains highly purified monoclonal antibody, then utilizes EZQ ProteinQuantitation Kit (Invitrogen) antagonist quantitative, to carry out next step experiment.

Embodiment 3 Humanized monoclonal antibodies CD20 avidity are analyzed

The Scatchard method is measured the avidity of monoclonal antibody 31-7, BD31-7, Rituximab.Table 1 has shown the result who measures, and experimental result illustrates that two strain monoclonal antibodies and VEGF have stronger binding affinity.

Table 1 monoclonal antibody avidity result

Embodiment 4, detection monoclonal antibody BD31-7 are to the lethal effect of B cell lymphoma cell

1. the cytotoxicity experiment that relies on of antibody

1) experiment purpose: observe the cytotoxicity (ADCC) that can BD31-7 induce the antibody dependence of Raji cell and Daudi cell.

2) experimental technique

PBS with new preparation is mixed with the solution that concentration is respectively 0.8,4,20 μ g/ml with BD31-7, and the Rituximab injection liquid is mixed with the solution for later use that concentration is 20 μ g/ml.Cell counting Raji=5.1 * 10 ⁵Individual/ml, Daudi=4.9 * 10 ⁵Individual/ml, PBMC=1 * 10 ⁸Individual/ml.

Cell is fully outstanding even, by every hole 800 μ l with Raji or Daudi cell suspension kind in 12 well culture plates, 37 ℃, contain in the incubator of 5% carbonic acid gas and hatch.

Grouping arranges such as table 2:

Each grouping of table 2 arranges

Press shown in the table 2, each hole of 12 orifice plates of containing 800 μ l cell suspensions in every hole adds BD31-7 or Rituximab or the equal-volume PBS of respective concentration, establishes three multiple holes for every group.Cell is put into incubator hatched 1 hour, add again PBMC(effect target ratio in each respective aperture: 25:1) or equal-volume PBS, continue to hatch 5 hours aftertreatment samples.Process front 45 minutes maximum LDH of release of clockwise cell of sample and organize the lysate that every hole adds the outfit of 100 μ l test kits.Every hole is got 1ml and is placed in the 1.5mlEP pipe 1000 to leave the heart 5 minutes when processing sample, gets 50 μ l supernatants in 96 porocyte culture plates, according to CytoTox

On-radiation cytotoxicity detection kit specification sheets, every hole adds the substrate mixture 50 μ l that prepare, and lucifuge 30 minutes adds 50 μ l stop buffers again in every hole, survey the OD value at the 470nm place with microplate reader immediately.

The cytotoxicity calculation formula:

Killing activity %=100 * (the spontaneous release LDH group of experimental port OD value-cell OD value)/(the maximum spontaneous release LDH group of the LDH group OD value-cell OD value that discharges of cell)

3) statistical study

Statistical test is checked with t, and p＜0.05 is for there being significant difference, and p≤0.01 is for having highly significant difference, p〉0.05 be there was no significant difference.

4) experimental result

A) Daudi cell ADCC experimental result:

As shown in Figure 2, compare the cytotoxicity (ADCC) (P＜0.01) that BD31-7 lower concentration (0.08 μ g/ml), middle concentration (0.4 μ g/ml), high density group (2 μ g/ml) and Rituximab group (2 μ g/ml) all can significantly induce the antibody of Daudi cell to rely on negative control group; The BD31-7 of same concentrations and Rituximab(2 μ g/ml) induce the ADCC effect there was no significant difference (P〉0.05) of Daudi cell.

B) Raji cell ADCC experimental result:

As shown in Figure 3, compare with negative control group, BD31-7 lower concentration (0.08 μ g/ml), middle concentration (0.4 μ g/ml), high density group (2 μ g/ml) and Rituximab group (2 μ g/ml) all can significantly induce cytotoxicity (ADCC) that the antibody of Raji cell relies on (during the BD31-7 lower concentration, P＜0.05, P＜0.01 when concentration and high density among the BD31-7); The BD31-7 of same concentrations and Rituximab(2 μ g/ml) induce the ADCC effect there was no significant difference (P〉0.05) of Raji cell.

2. the cytotoxicity experiment of Complement Dependent

1) experiment purpose: observe the cytotoxicity (CDC) that can BD31-7 mediate the Complement Dependent of Raji cell and Daudi cell.

2) experimental technique

PBS with new preparation is mixed with the solution that concentration is respectively 0.8,4,20 μ g/ml with BD31-7, and the Rituximab injection liquid is mixed with the solution for later use that concentration is 20 μ g/ml.Cell counting Raji=4.5 * 10 ⁵Individual/ml, Daudi=5.3 * 10 ⁵Individual/ml.

Cell is fully outstanding even, by every hole 800 μ l with Raji or Daudi cell suspension kind in 12 well culture plates, 37 ℃, contain in the incubator of 5% carbonic acid gas and hatch.Grouping setting sees Table 3.

Table 3 experiment grouping arranges

	Drug level (μ g/ml)	Experimental system arranges
			Negative control group	0	800 μ l cells+100 μ lPBS+100 μ l complements
The BD31-7 low dose group	0.08	800 μ l cells+100 μ lBD31-7+100 μ l complements
			Dosage group among the BD31-7	0.4	800 μ l cells+100 μ lBD31-7+100 μ l complements
The BD31-7 high dose group	2	800 μ l cells+100 μ lBD31-7+100 μ l complements
			Positive drug group (Rituximab)	2	800 μ l cells+100 μ lRituximab+100 μ l complements
The spontaneous release LDH group of cell	0	800 μ l cells+200 μ lPBS
			The maximum LDH group that discharges of cell	0	800 μ l cells+100 μ lPBS+100 μ l lysates

Press shown in the table 3, each hole of 12 orifice plates of containing 800 μ l cell suspensions in every hole adds HO2 or Rituximab or the equal-volume PBS of respective concentration, establishes three multiple holes for every group.Cell is put into incubator hatched 1 hour, in each respective aperture, add complement or equal-volume PBS again, continue to hatch 5 hours aftertreatment samples.Process front 45 minutes maximum LDH of release of clockwise cell of sample and organize the lysate that every hole adds the outfit of 100 μ l test kits, continue to hatch.When processing sample, get 1ml from every hole and place in the 1.5mlEP pipe 1000 to leave the heart 5 minutes, get 50 μ l supernatants in 96 orifice plates, according to CytoTox

On-radiation cytotoxicity detection kit specification sheets, every hole adds the substrate mixture 50 μ l that prepare, and lucifuge 30 minutes adds 50 μ l stop buffers again to every hole, survey the OD value at the 470nm place with microplate reader immediately.

The cytotoxicity calculation formula:

Killing activity %=100 * (the spontaneous release LDH group of experimental port OD value-cell OD value)/(the maximum spontaneous release LDH group of the LDH group OD value-cell OD value that discharges of cell).

3) statistical study

Statistical test is checked with t, and p＜0.05 is for there being significant difference, and p≤0.01 is for having highly significant difference, p〉0.05 be there was no significant difference.

4) experimental result

A) Daudi cell CDC experimental result:

As shown in Figure 4, compare with negative control group, BD31-7 lower concentration (0.08 μ g/ml), middle concentration (0.4 μ g/ml), high density group (2 μ g/ml) and Rituximab group (2 μ g/ml) all can significantly be induced the cytotoxicity (CDC) (P＜0.01) of the Complement Dependent of Daudi cell; The BD31-7 of same concentrations and Rituximab(2 μ g/ml) induce the CDC effect there was no significant difference (P〉0.05) of Daudi cell.

B) Raji cell apoptosis assay result

As shown in Figure 5, compare with negative control group, BD31-7 lower concentration (0.08 μ g/ml), middle concentration (0.4 μ g/ml), high density group (2 μ g/ml) and Rituximab group (2 μ g/ml) all can significantly be induced the cytotoxicity (CDC) (P＜0.01) of the Complement Dependent of Raji cell; The BD31-7 of same concentrations and Rituximab(2 μ g/ml) induce the CDC effect there was no significant difference (P〉0.05) of Raji cell.

3. cell apoptosis assay

1) experiment purpose: observe CD20 monoclonal antibody BD31-7 and can significantly induce B lymphoma cell apoptosis.

2) experimental technique

PBS with new preparation is mixed with the liquid that concentration is respectively 0.8,4,20 μ g/ml with BD31-7, and the Rituximab injection liquid is mixed with the solution for later use that concentration is 20 μ g/ml.Cell counting Raji=5.9 * 10 ⁵Individual/ml, Daudi=6.2 * 10 ⁵Individual/ml.

Cell is fully outstanding even, by every hole 900 μ l with Raji or Daudi cell suspension kind in 12 well culture plates, 37 ℃, contain in the incubator of 5% carbonic acid gas and hatch.Grouping setting sees Table 4.

Each grouping of table 4 experiment arranges

Press shown in the table 4, each hole of 12 orifice plates of containing 900 μ l cell suspensions in every hole adds HO2 or Rituximab or the equal-volume PBS of respective concentration, establishes three multiple holes for every group.In incubator, hatch 16 hours aftertreatment samples.When processing sample, every hole is got 1ml and is placed in the 1.5mlEP pipe 1000 to leave the heart 5 minutes, abandon supernatant, add gently re-suspended cell of 1mlPBS, 1000 left the heart 5 minutes, abandon supernatant, according to Annexin V-FITC cell apoptosis detection kit specification sheets, add 195 μ lAnnexin V-FITC in conjunction with liquid re-suspended cell gently, add 5 μ lAnnexin V-FITC, mixing gently, room temperature lucifuge 10 minutes, 1000 left the heart 5 minutes, abandoned supernatant, add 190 μ lAnnexin V-FITC in conjunction with liquid re-suspended cell gently, add 10 μ l propidium iodide staining fluids, mixing gently, the ice bath lucifuge is placed, carry out immediately flow cytometer and detect, the early apoptosis rate and late period the apoptosis rate sum count apoptosis rate.

3) 3. statistical study

Statistical test is checked with t, and p＜0.05 is for there being significant difference, and p≤0.01 is for having highly significant difference, p〉0.05 be there was no significant difference.

4) experimental result

A) Daudi cell apoptosis assay result:

As shown in Figure 6, compare with negative control group, BD31-7 lower concentration (0.08 μ g/ml), middle concentration (0.4 μ g/ml), high density group (2 μ g/ml) and Rituximab group (2 μ g/ml) all can significantly induce the Daudi apoptosis (when BD31-7 lower concentration and middle concentration, P＜0.05; BD31-7 high density and Rituximab, P＜0.01); The BD31-7 of same concentrations and Rituximab(2 μ g/ml) induce Daudi cells apoptosis there was no significant difference (P〉0.05).

B) Raji cell apoptosis assay result:

As shown in Figure 7, compare with negative control group, BD31-7 lower concentration (0.08 μ g/ml), middle concentration (0.4 μ g/ml), high density group (2 μ g/ml) and Rituximab group (2 μ g/ml) all can significantly induce the Raji apoptosis (when BD31-7 lower concentration, high density and Rituximab, P＜0.01; Concentration among the BD31-7, P＜0.05); The BD31-7 of same concentrations and Rituximab(2 μ g/ml) induce Raji cells apoptosis there was no significant difference (P〉0.05).

Experiment conclusion

By ADCC, CDC and cell apoptosis assay, draw to draw a conclusion: people mouse chimeric mAb BD31-7 all can significantly induce the cytotoxicity (ADCC) of Raji Human B lymphoma cell and the dependence of Daudi Human B lymphoma cell generation antibody, cytotoxicity (CDC) and the apoptosis of Complement Dependent in external lower concentration (0.08 μ g/ml), middle concentration (0.4 μ g/ml), high density (2 μ g/ml) and Rituximab high density (2 μ g/ml), thereby significantly kills and wounds the B lymphoma cell; The BD31-7 of same concentrations and Rituximab(2 μ g/ml) induce the effect there was no significant difference of Raji Human B lymphoma cell and Daudi Human B lymphoma cell ADCC, CDC and apoptosis.

Be necessary at last in this explanation to be: above embodiment only is used for technical scheme of the present invention is described in more detail; can not be interpreted as limiting the scope of the invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.

Sequence table

＜110〉the rich fertile biotechnology company limited in Shanghai

＜120〉vascular endothelial growth factor Humanized monoclonal antibodies

<170>Patentln version3.3

<210>1

<211>990

<212>DNA

＜213〉nucleotide sequence of human antibody heavy chain's constant region (CH)

<400>1

GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC

ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGA

ACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCT

TCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG

TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC

CCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACT

CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT

CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT

CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT

GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAAGA

GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG

ACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA

GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA

GGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCC

TGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG

AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTC

CGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGC

AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT

ACACGCAGAAGAGCCTCTCCCTGTCTCCCGGTAAA

<210>2

<211>330

<212>PRT

＜213〉aminoacid sequence of human antibody heavy chain's constant region (CH)

<400>2

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP

PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE

VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT

PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

<210>3

<211>321

<212>DNA

＜213〉nucleotide sequence of human antibody light chain constant region (CL)

<400>3

CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT

CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG

TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA

GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA

AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA

GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT

<210>4

<211>106

<212>PRT

＜213〉aminoacid sequence of human antibody light chain constant region (CL)

<400>4

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

<210>5

<211>363

<212>DNA

＜213〉humanized antibody BD31-7 variable region of heavy chain nucleotide sequence

<400>5

CAGGTGCAGCTGCAGCAGCCGGGCGCGGAACTGGTGCGCCCGGGCGCGAGCGTGAA

AATGAGCTGCAAAGCGAGCGGCTATAGCTTTAGCAGCTATAACATTCATTGGGTGAAA

CAGACCCCGGGCCGCGGCCTGGAATGGATTGGCGCGATTTATCCGGGCAACGGCGATA

CCAGCTATAACCAGAAATTTAAGGCAAAGCGACCCTGACCGCGGATAAAAGCAGCA

GCACCGCGTATATGCAGCTGAGCAGCCTGACCAGCGAAGATAGCGCGATTTATTATTG

CGCGCGCAGCACCTATTATTATGGCGCGTATTATTTTGATTATTGGGGCCAGGGCACCA

CCCTGACCGTGAGCAGC

<210>6

<211>121

<212>PRT

＜213〉humanized antibody BD31-7 weight chain variable region amino acid sequence

<400>6

QVQLQQPGAELVRPGASVKMSCKASGYSFSSYNIHWVKQTPGRGLEWIGAIYPGNGDTS

YNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAIYYCARSTYYYGAYYFDYWGQGTTL

TVSS

<210>7

<211>318

<212>DNA

＜213〉humanized antibody BD31-7 variable region of light chain nucleotide sequence

<400>7

CAGATTGTGCTGAGCCAGAGCCCGGCGATTCTGAGCGCGAGCCCGGGCGAAAAAGTG

ACCATGACCTGCCGCGCGAGCAGCAGCGTGAGCTATATTCATTGGTTTCAGCAGAAAC

CGGGCAGCAGCCCGAAACCGTGGATTTATGCGACCAGCAACCTGGCGAGCGGCGTGC

CGGTGCGCTTTAGCGGCAGCGGCAGCGGCACCAGCTATAGCCTGACCATTAGCCGCGT

GGAAGCGGAAGATGCGGCGACCTATTATTGCCAGCAGTGGACCAGCAACCCGCCGAC

CTTTGGCGGCGGCACCAAACTGGAAATTAAACGC

<210>8

<211>106

<212>PRT

＜213〉humanized antibody BD31-7 light chain variable region amino acid sequence

<400>8

QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMHWFQQKPGSSPKPWIYATSNLASGVPVR

FSGSGSGTSYSLTISRMEAEDAATYYCQQWSSNPPTFGAGTKLELKR

Claims (4)

1. Humanized anti-CD 20 monoclonal antibody, it is characterized in that: its variable region of heavy chain has following aminoacid sequence VH:SEQ ID:NO.6; Variable region of light chain has following amino acid sequences VL:SEQ ID:NO.8.

2. the nucleic acid molecule of a separation, it is characterized in that: described nucleic acid molecule contains the nucleotide sequence of the described variable region of mab of coding shown in the SEQ ID NO.5, and the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in the SEQ ID NO.7.

3. described nucleic acid molecule according to claim 2 is characterized in that: its monoclonal antibody claimed in claim 1 of encoding.

4. pharmaceutical composition is characterized in that: it with the described Humanized anti-CD 20 monoclonal antibody of claim 1～3 as main active principle and pharmaceutically acceptable carrier.

A kind of method for preparing Humanized anti-CD 20 monoclonal antibody claimed in claim 1 is characterized in that: in host cell, express nucleic acid molecule claimed in claim 2, and from the host cell culture separation and purification antibody.

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