CN1225480C

CN1225480C - Anti CD52 monoclonal antibody, coding sequence and use thereof

Info

Publication number: CN1225480C
Application number: CNB02155174XA
Authority: CN
Inventors: 马菁
Original assignee: Individual
Current assignee: Shanghai Zhangjiang Biotechnology Co ltd
Priority date: 2002-12-18
Filing date: 2002-12-18
Publication date: 2005-11-02
Anticipated expiration: 2022-12-18
Also published as: CN1508155A

Abstract

The present invention provides an anti-CD52 specific monoclonal antibody which has the advantages of good stability and strong specificity and is especially suitable for preventing and treating graft rejective reactions and treating chronic lymphocytic leukemia. The present invention also provides a preparation method of the monoclonal antibody, and a medical composition with the monoclonal antibody.

Description

Anti-CD52 monoclonal antibody, its encoding sequence and application

Technical field

The present invention relates to medical field.More specifically, the present invention relates to monoclonal antibody specific and the application thereof of anti-CD52.Antibody of the present invention is for the prevention and the treatment and very useful of graft-rejection.

Background technology

At present, organ transplantation technique become organ function depleted whole latter stage effectively, treatment means routinely.But the problem of organ transplantation maximum is an immunological rejection.In organ transplantation, particularly in kidney, liver, heart, lung and the bone marrow transplantation, reduce immune graft-rejection and be very important.Developed many immunosuppressor at present, and also obtained success clinically, such as comprising steroid, imuran and cyclosporin A.But they all must control in strictness, to avoid producing unnecessary side effect.Also having a difficult point in addition, is exactly that immunosuppressive action makes transplant organ easily by bacterium or virus infection, and this bacterioid or virus infection under normal circumstances are to be eliminated by the immunity system of individuality.

Except using immunosuppressor, can also use some monoclonal antibodies (MAbs) to suppress immune response.These monoclonal antibodies are usually according to corresponding C D number name, are described as " anti-CD3 " as the antibody of CD3.

CD52 is called campath-1 antigen usually again, and this antigen is a kind of antigen that is present in bone-marrow-derived lymphocyte and T lymphocytic cell surface.Be directed to the antigenic anti-CD52 monoclonal antibody of CD52 and can temporarily remove lymphocyte in the blood in vivo, make immunity system be in failure state.This can not suffer the immune rejection of patient after making that organ is implanted in patient's body at once.Immunity system can " be thought " transplanted organ by mistake to be original integral part in patient's body one, in the bimestrial recovery process gradually.

In addition, anti-CD52 monoclonal antibody can also cause the molten born of the same parents in antibody dependent ground in vivo or kill cytosis, thereby removes the malignant lymphatic cell in blood, marrow and other the influenced organs.

At present, though obtained the antigenic monoclonal antibody of multiple anti-CD52 both at home and abroad, characteristics such as the specificity of these monoclonal antibodies are not ideal enough.Therefore, this area also needs development littler to people's immunogenicity, and expression amount is higher, and the anti-CD52 monoclonal antibody with other good characteristics, thereby develops the medicine that has significant curative effect for chronic lymphocytic leukemia.

Summary of the invention

Purpose of the present invention provides a specific specificity anti-CD52 monoclonal antibody.

Another object of the present invention provides the anti-CD52 MONOCLONAL ANTIBODIES SPECIFIC FOR of a kind of described specificity method.

Another object of the present invention provides a kind of pharmaceutical composition that contains described anti-CD52 monoclonal antibody.

In a first aspect of the present invention, provide a kind of monoclonal antibody V _HChain, its complementary determining region CDR have the aminoacid sequence of the CDR of the group of being selected from down:

CDR1 shown in the SEQ ID NO:5,

CDR2 shown in the SEQ ID NO:6 or 16,

CDR3 shown in the SEQ ID NO:7 or 17.

Preferably, described monoclonal antibody V _HChain has the aminoacid sequence shown in SEQ ID NO:2 or 12.

In a second aspect of the present invention, provide a kind of monoclonal antibody V _LChain, its complementary determining region CDR have the aminoacid sequence of the CDR of the group of being selected from down:

CDR1 shown in the SEQ ID NO:8 or 18,

CDR2 shown in the SEQ ID NO:9 or 19,

CDR3 shown in the SEQ ID NO:10 or 20.

Preferably, described monoclonal antibody V _LChain has the aminoacid sequence shown in SEQ ID NO:4 or 14.

In a third aspect of the present invention, provide a kind of monoclonal antibody, its V _HChain has the aminoacid sequence shown in SEQ ID NO:2 or 12, and its V _LChain has the aminoacid sequence shown in SEQ ID NO:4 or 14 respectively.

Preferably, described monoclonal antibody is a humanized antibody.

In a fourth aspect of the present invention, a kind of dna molecular is provided, its coding is selected from down the protein of group: the monoclonal antibody V that the present invention is above-mentioned _HChain, monoclonal antibody V _LChain or monoclonal antibody.

Preferably, described dna molecular has the dna sequence dna of the group of being selected from down: SEQ ID NO:1,3,11 or 13.

In a fifth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains monoclonal antibody and pharmaceutically acceptable carrier, the V of described monoclonal antibody _HChain and V _LChain has the CDR shown in SEQ ID NO:5-7 and the SEQ ID NO:8-10 respectively.Preferably, the V of described monoclonal antibody _HChain has the aminoacid sequence shown in SEQID NO:2 or 12, and its V _LChain has the aminoacid sequence shown in SEQ ID NO:4 or 14 respectively.

In another preference, anti-CD 52 antibody of the present invention or its pharmaceutical composition are used for the treatment of chronic lymphocytic leukemia.

Embodiment

The inventor is by the screening human antibody library, successfully obtained monoclonal antibody to the CD52 high specific, and unique artificial design has been carried out in antagonism CD52 Humanized monoclonal antibodies gene and antibody variable region site, promptly make up the antibody library in total man source, the high antibody of screening expression amount, and it IgG1-Fc with the people source is linked to each other, so this antibody is the total man source, thereby is more suitable in mammalian cell (preferred CHO), expressing.Finished the present invention on this basis.

The invention provides the anti-CD52 Humanized monoclonal antibodies of a kind of reorganization, it comprises people source Heng Qu (as permanent district, people source IgG1-Fc), has unique prior art constructions that is different from through variable region of heavy chain after the artificial design and variable region of light chain.

The present invention also provides aminoacid sequence and its variable region chain thereof of anti-CD52 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 identical or at least 90% homology of hypervariable region of this hypervariable region and light chain of the present invention and heavy chain, preferably at least 95% homology.

The antigen binding characteristic of antibody can be described by 3 specific zones that are positioned at heavy chain and variable region of light chain, be called hypermutation zone (CDR), should intersegmentally be divided into 4 frame areas (FR), the aminoacid sequence of 4 FR is relatively conservative, does not participate in association reaction directly.These CDR form ring texturees, and the βZhe Die that the FR by therebetween forms is close mutually on space structure, and the CDR on CDR on the heavy chain and the corresponding light chain has constituted the antigen binding site of antibody.Can determine which amino acid has constituted FR or CDR zone by the aminoacid sequence of antibody more of the same type.

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

Herein the hypervariable region of the V chain of Jian Dinging or complementary determining region (complementarity determiningregion, CDR) interesting especially because relate to conjugated antigen to small part in them.Therefore, the present invention includes those the monoclonal antibody light chains and the molecule of weight chain variable chain, as long as its CDR has the homology of (preferably more than 95%) more than 90% with the CDR that identifies herein with band CDR.

The present invention not only comprises complete monoclonal antibody, also comprises having immunocompetent antibody fragment, as Fab or (Fab ') ₂Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody, as have the murine antibody binding specificity but keep antibody from people's antibody moiety.

The present invention also provides coding said monoclonal antibody or its segmental dna molecular.The Nucleotide full length sequence of monoclonal antibody of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.A kind of feasible method is to synthesize relevant sequence, especially fragment length more in short-term with artificial synthetic method.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.

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

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

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 to transform appropriate host cell, so that it can marking protein.

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS7,293 cells etc.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, 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 used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

The present invention also provides a kind of pharmaceutical composition for the treatment of transplant rejection, and it contains above-mentioned monoclonal antibody or immune conjugate, and pharmaceutically acceptable carrier.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties 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.

Pharmaceutical composition of the present invention can be directly used in prevention and treatment transplant rejection.In addition, also can use the other treatment agent simultaneously.

Pharmaceutical composition of the present invention contains above-mentioned monoclonal antibody and the pharmaceutically acceptable carrier or the vehicle of the present invention of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the 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.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that immune conjugate with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

Outstanding advantage of the present invention is: the physiologically active of monoclonal antibody of the present invention and the expression amount in host cell (as Chinese hamster ovary celI) increase significantly than existing monoclonal antibody.And monoclonal antibody of the present invention is humanized, and its avidity has reached the level of 0.1nM.The monoclonal antibody of this high-affinity has significant values clinically.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described 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 people such as normal condition such as Sambrook, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The variable region gene of screening anti-CD 52 antibody from antibody library

1) structure of human antibody library

According to people J.Mol.Biol. such as Marks, 222,581-597; Hoogenboom and Winte, J.Mol.Biol., 227,381-388; Haidaris CG etc., J Immunol Methods.2001 Nov 1; 257 (1-2): 185-202; Griffiths, EMBO J. such as A.D., 13,3245-3260 (1994); Nissim, EMBO J. such as A., 13, the method for describing among the 692-698 (1994) makes up human antibody library.In brief, the gene for preparing heavy chain immunoglobulin and light chain from peripheral blood lymphocyte, carry out amplification in vitro with PCR method, and it is cloned into phage vector, the characteristics of utilizing the antibody molecule fragment to present at phage surface, (available from Shenzhen brilliant U.S. company) is antigen with CD52 albumen, screens corresponding specific antibody.

2) screening

The antibody library bacterial strain of recovery is added 14 milliliters of fresh LB substratum for 1 milliliter, in 50 milliliters of triangular flasks, cultivated 16 hours for 37 ℃.

12000rpm high speed centrifugation 10 minutes shifts in supernatant to one 50 milliliters of aseptic centrifuge tubes, preserves standby.Its titre should be 2 * 10 ¹¹More than.CD52 albumen (available from Shenzhen brilliant U.S. company) with purifying is antigen, and bag is by 25 ml cells culturing bottles.Add in the cell bottle behind the bag quilt and be no less than 3 * 10 ¹⁰Phage particle, 37 ℃ of incubations 1 hour.

Then, outwell the liquid in the bottle, wash culturing bottle 10 times with 10 milliliters of PBS that added 1%Tween-20.The TG1 cell that adds 1 milliliter of logarithmic phase in culturing bottle, 37 ℃ of incubation concussions were cultivated 16 hours.Repeat 4 circulations.

With cell dilution to 100000 cells/ml of above-mentioned acquisition, on 1.5% agar plate that adds 0.1% penbritin, cultivate to obtain mono-clonal then.Get being cloned on the 96 hole depth orifice plates on the above-mentioned flat board and cultivate, the clone in every hole does 960 clones (10 96 orifice plates) altogether.With above-mentioned deep-well plates centrifugal 20 minutes of 5000rpm on 96 orifice plate whizzers, supernatant is transferred to new aseptic deep-well plates, be preserved in after sealing 4 ℃ standby.

Get 10 of 96 orifice plates, add in every hole the conventional bag of CD52 (10 mcg/ml) 10 microlitres by after, add supernatant 10 microlitres of above-mentioned preservation respectively, 37 ℃ of incubations after 1 hour with the PBS washing that contains 1%Tween-20 20 times.The goat-anti M13 monoclonal antibody that adds 1 microlitre HRP mark, 37 ℃ of incubations use the PBS of 1%Tween-20 to wash 10 times after 30 minutes.

Add and contain PBS 200 microlitres of 0.025%DAB developer and the H of 1 microlitre 1% ₂O ₂, the 37 ℃ of incubation colour developing was read 595 nanometers after 20 minutes on plate reading machine photoabsorption.Determine the hole that color reaction is strong according to the photoabsorption reading, the corresponding clone in these holes is the stronger antibody variable region clone of avidity.

Filtered out 296 positive colonies by said process, the strongest clone's avidity measurement result sees Table 1.Determine wherein 1 clone 5E6 that avidity is the strongest according to reading, be used for following research.

Each clone's of table 1 avidity

	2A3	5D1	4E3	6F2	5E6	4F9
	2A3	5D1	4E3	6F2	5E6	4F9	Photoabsorption	16.73±1.18	12.3±1.32	15.45±1.27	19.8±0.52	23.59±1.46	18.04±0.87

Embodiment 2

The clone of the expression vector of antibody variable region encoding sequence

The clone's that embodiment 1 is obtained bacterial strain increases in 100 milliliters of LB substratum, with the plasmid DNA extracting and purifying test kit plasmid DNA purification of Promega company.

With XbaI with on 1.5% agarose gel electrophoresis, separate endonuclease bamhi after the NheI enzyme is cut above-mentioned plasmid DNA, get band about 350bp and carry out glue and reclaim, the gained fragment is the variable region of heavy chain encoding sequence.

With HindIII with on 1.5% agarose gel electrophoresis, separate endonuclease bamhi after Bsi WI enzyme is cut above-mentioned plasmid DNA, get band about 320bp and carry out glue and reclaim, the gained fragment is the variable region of light chain encoding sequence.

At first above-mentioned variable region of heavy chain encoding sequence is inserted into expression vector pMG18-3K then (referring to books DEVELOPMENT OF TOOLS FOR ENVIRONMENTAL MONITORINGBASED ON INCP-9PLASMIDS SEQUENCES.A.Greated, R.Krasowiak, M.Titok, C.M.Thomas School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B 152TT, UK and Faculty of Biology, Dept ofMicrobiology, Belarus State University Scorina Av.4, Minsk 220080Belarus1992 publishes.In addition, this carrier can be available from Invitrogen company) the XbaI/NheI site in, with Hind III and Bsi WI above-mentioned antibody chain variable region encoding sequence is inserted in the HindIII/Bsi WI site of the pMG18-3K that is inserted with the variable region of heavy chain encoding sequence again, is built into the humanized anti-CD 52 antibody expression carrier.

Embodiment 3

The transfection of Chinese hamster ovary celI and the screening of recombinant clone

The expression vector that has antibody gene that makes up in the foregoing description 2 changes at e.colistraindh5, be inoculated in then in 100 milliliters of LB substratum and increase, with ultrapure plasmid DNA purification kit (Ultrapure Plasmid DNA Purification Kit) the extracting and purifying plasmid DNA of Qiagen company.The plasmid DNA of above-mentioned purifying is adopted the liposome method test kit transfection CHO cell of Invitrogen company, and operation is carried out with reference to the specification sheets of producer.

The Chinese hamster ovary celI that transforms carries out the extreme dilution at last and cultivates in the selection of selecting to carry out on the substratum continuous 9 weeks on 96 orifice plates, carry out continuously 3 times, carry out mono-clonalization.

The monoclonal cell of choosing ties up on the RPM1641 substratum and cultivates, supernatant is carried out Western Blotting experiment, judge expression intensity according to staining reaction, pick out and express strong clone as the candidate cell strain, obtain 9 of the higher candidate clones of expression intensity, i.e. 1B4,2D4,3F7,5E3,7C9,6D2,4D9,2H7,3H6.

Embodiment 4

Purification of Monoclonal Antibodies

Present embodiment comes monoclonal antibody purification with ammonium sulfate precipitation method.The purifying of above-mentioned monoclonal antibody is adopted the directly separation and purification from cells and supernatant of Protein A affinity column.Culture supernatant is through simply centrifugal, can directly carry out protein A affinity chromatography after removing the cell relic.

Behind the ratio adding filler of protein A filler, stirred at a slow speed 24 hours at 4 degree according to 1 milliliter of per 100 milliliters of supernatant.Then at the Tris-H of 100mM pH8.2 ₂SO ₄After the rinsing 4 times, with carrying out the ammonium sulfate fractional precipitation after the same buffer solution elution that contains 250mM sodium-chlor, initial concentration is 35% saturation ratio, stops concentration 63% saturation ratio in the damping fluid.4 degree are handled high speed centrifugation after 12 hours, get precipitation, be dissolved in again in the damping fluid of above-mentioned not sodium chloride-containing, and concentrate 10 times after same damping fluid dialysed overnight, and are stand-by.

Above-mentioned sample found that purified recombinant protein through HPLC purity checking and Follin phenol standard measure protein content, and its purity can reach more than 90%, and has notable biological activity.

The product of above affinity chromatography passes through sieve chromatography once more, has obtained the sample of purity＞98%.These samples can be used for following further analysis and research.

Embodiment 5

Expression intensity and the avidity of antibody gene in Chinese hamster ovary celI is measured

(a) expression intensity

The high expression level candidate clone that above-mentioned screening is obtained is incubated at the tissue culture ware of 10cm, adopt the ELISA method to measure the expression amount of antibody: goat anti-human igg (Fc) bag quilt is in elisa plate, 4 ℃ are spent the night, and in 37 ℃ of sealings 2 hours, add culture supernatant to be measured and standard substance (human IgG1) through 2%BSA, hatched 2 hours for 37 ℃, add HRP-goat anti-human igg (κ) and carry out association reaction, hatched 1 hour for 37 ℃, add TMB in 37 ℃ of effects 10 minutes, use the H2S04 termination reaction at last, survey the A450 value.The expression amount that records above-mentioned 9 candidate clones is as follows:

Table 2 antibody gene expression intensity in Chinese hamster ovary celI

The cell strain numbering	1B4	2D4	3F7	5E3	7C9	6D2	4D9	2H7	3H6
The cell strain numbering	1B4	2D4	3F7	5E3	7C9	6D2	4D9	2H7	3H6	Expression amount (ug/ milliliter)	49.1	61.2	82.0	128.6	69.1	78.2	99.7	59.4	55.8

As can be seen from Table 2, the expression level that is numbered the cell strain of 5E3 and 4D9 has very high expression level (about 100ug/ milliliter or higher), has exceeded the expression level (generally being 10-80ug/ml now) of domestic and international monoclonal antibody class abroad.

(b) avidity is measured

Cell culture fluid to each clone carries out purifying by the following method.Centrifugal cell and the cell debris removed of 10000rpm, filter membrane ultrafiltration and concentration to 1/10 volume of 100Kd molecular weight cut-off, the ultrafiltration damping fluid is 100mMTris-HCl, pH7.5.Cross the SPA-sepharose affinity column, sample solution is 100mM Tris-HCl, and pH7.5, elutriant are 100mM Tris-HCl, pH7.5,100mM NaCl.Molecular sieve (SephadexG200) chromatography.Elutriant is 100mM Tris-HCl, pH7.5.Get pure product.

Avidity mensuration employing Scatchard analytical method (Munson et al, 1980, Anal.BioChem. 107:220) carries out, and the result shows that the avidity of 5E3 and 4D9 two strain monoclonal antibodies reaches 4.5 * 10 respectively ^-9With 1.58 * 10 ^-8

The above results shows that the avidity of 5E3 and 4D9 two strain monoclonal antibodies has reached the level of 0.1nM and 1nM respectively, has very high avidity.

Embodiment 6

The sequence of CD52 monoclonal antibody gene is determined

Anti-CD52 monoclonal antibody gene to above-mentioned cell strain 5E3 and 4D9 carries out dna sequencing.The source antibody variable region universal primer of promptly choosing carries out pcr amplification, and the light chain of acquisition and variable region of heavy chain entrust Shanghai to give birth to worker company to carry out the PCR order-checking.

The result is presented in the sequence table.For monoclonal antibody 5E3, SEQ ID NO:1 and SEQID NO:3 are respectively the variable region of heavy chain of the high reactivity high expression level CD52 monoclonal antibody 5E3 that obtains of the present invention and the dna encoding sequence of variable region of light chain; SEQ ID NO:2 and SEQ ID NO:4 are respectively according to the variable region of heavy chain of above-mentioned dna encoding sequence supposition and the aminoacid sequence of variable region of light chain.

For monoclonal antibody 4D9, SEQ ID NO:11 and SEQ ID NO:13 are respectively the variable region of heavy chain of the high reactivity high expression level CD52 monoclonal antibody 5E3 that obtains of the present invention and the dna encoding sequence of variable region of light chain; SEQ ID NO:12 and SEQ ID NO:14 are respectively according to the variable region of heavy chain of above-mentioned dna encoding sequence supposition and the aminoacid sequence of variable region of light chain.

5E3 heavy chain variable region gene sequence (3 ', 363bp)	SEQ ID NO：1
5E3 heavy chain variable region gene sequence (3 ', 363bp)	SEQ ID NO：1	5E3 weight chain variable region amino acid sequence (121aa)	SEQ ID NO：2
5E3 chain variable region gene sequence (5 ' 3 ', 324bp)	SEQ ID NO：3		SEQ ID NO：2
5E3 chain variable region gene sequence (5 ' 3 ', 324bp)	SEQ ID NO：3	The aminoacid sequence of 5E3 variable region of light chain (108aa)	SEQ ID NO：4
4D9 heavy chain variable region gene sequence (5 ' 3 ', 363bp)	SEQ ID NO：11		SEQ ID NO：4
	SEQ ID NO：11	4D9 weight chain variable region amino acid sequence (121aa)	SEQ ID NO：12
4D9 chain variable region gene sequence (5 ' 3 ', 324bp)	SEQ ID NO：13		SEQ ID NO：12
4D9 chain variable region gene sequence (5 ' 3 ', 324bp)	SEQ ID NO：13	4D9 light chain variable region amino acid sequence (108aa)	SEQ ID NO：14

The variable region	CDR1 sequence SEQ ID NO:		CDR2 sequence SEQ ID NO:		CDR3 sequence SEQ ID NO:
The variable region	CDR1 sequence SEQ ID NO:		CDR2 sequence SEQ ID NO:		CDR3 sequence SEQ ID NO:		The 5E3 variable region of heavy chain	DFYMN	5	FIRDKAKGYTTEYNPSV KG	6	EGHTAAPFDY	7
The 5E3 variable region of light chain	KASQNIDKYLN	8	NTNNLQT	9	LQHISRPRT	10	The 5E3 variable region of heavy chain	DFYMN	5	FIRDKAKGYTTEYNPSV KG	6	EGHTAAPFDY	7
The 5E3 variable region of light chain	KASQNIDKYLN	8	NTNNLQT	9	LQHISRPRT	10	The 4D9 variable region of heavy chain	DFYMN	15	FIRDKGKGYTSEYNPSV KG	16	EGHNLAPFDY	17
The 4D9 variable region of light chain	KLSQNIDKYIN	18	NTNNAQT	19	LQHITRPRT	20	The 4D9 variable region of heavy chain	DFYMN	15	FIRDKGKGYTSEYNPSV KG	16	EGHNLAPFDY	17

Annotate: SEQ ID NO:5 is identical with the sequence of SEQ ID NO:15.

Embodiment 7

The biologic activity of the monoclonal antibody that 5E3 and 4D9 produced detects

With ordinary method separation of human peripheral blood lymphocyte, adjusting cell density is 2 * 10 ⁶Cells/ml.Two human PBMCs (the two-way MLR of allogeneic) equal-volume is mixed, with 2 * 10 ⁵Density inoculation " U " type 96 well culture plates of cells/well.Add the purified fusion protein of different volumes, equal-volume corresponding empty carrier transfection supernatant or substratum are set compare, every group of 3 holes, 37 Fei, 5%CO ₂Cultivated 5 days, and stopped cultivating adding in preceding 16 hours ³H-TdR (final concentration 5 μ Ci/ml).Collecting cell is on 0.45 micron millipore filtration, and oven dry is surveyed the DPM value with liquid scintillation counter.Statistical procedures result represents with mean value ± standard deviation, carries out mean difference significance analysis with Microsoft Excel statistics program.

In the MLR reaction system, add 1 microlitre, 5 microlitres and 10 microlitres Chinese hamster ovary celI supernatant respectively through anti-CD52 monoclonal anti body expression vector or empty carrier transfection, Protein A purifying.The result shows, also can significantly suppress human peripheral MLR even add the cell conditioned medium of 1 microlitre Protein A purifying, and inhibiting rate is 66%～75%; And add the Chinese hamster ovary celI supernatant of the empty carrier transfection of same volume, then there is not this effect.Through one-way analysis of variance, its difference reaches the level of highly significant, the results are shown in Table 3.

The influence that table 3 purifying CD52 monoclonal antibody is bred people MLR medium size lymphocyte (DPM, n=3)

Test grouping (μ l/ hole)	0	1	5	10
Test grouping (μ l/ hole)	0	1	5	10	The monoclonal antibody 4D9 of purifying	4570±420	2355±174	1854±210	854±214
The monoclonal antibody 5E3 of purifying	4432±240	2417±206	1744±194	702±227	The monoclonal antibody 4D9 of purifying	4570±420	2355±174	1854±210	854±214
The monoclonal antibody 5E3 of purifying	4432±240	2417±206	1744±194	702±227	Contrast	4579±362	4367±313	4627 scholars 309	4324±296

The MLR experiment shows that this albumen has the activity that suppresses immune response, and 5E3 has stronger restraining effect, and 4D9 takes second place.Above result shows expressed the anti-CD52 monoclonal antibody that biologic activity is arranged in Chinese hamster ovary celI.In addition, anti-CD 52 antibody of the present invention can be used for treating chronic lymphocytic leukemia.

Should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉horse, cyanines

＜120〉anti-CD52 monoclonal antibody, its encoding sequence and application

<130>027427

<160>20

<170>PatentIn version 3.1

<210>1

<211>363

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(363)

＜223〉encoding sequence of anti-CD 52 antibody heavy chain

caggtccaac tgcaggagag cggtccaggt cttgtgagac ctagccagac cctgagcctg 60

acctgcaccg tgtctggcag caccttcagc gatttctaca tgaactgggt gagacagcca 120

cctggacgag gtcttgagtg gattggattt attagagaca aagctaaagg ttacacaaca 180

gagtacaatc catctgtgaa ggggagagtg acaatgctgg tagacaccag caagaaccag 240

ttcagcctga gactcagcag cgtgacagcc gccgacaccg cggtctatta ttgtgcaaga 300

gagggccaca ctgctgctcc ttttgattac tggggtcaag gcagcctcgt cacagtctcc 360

tca 363

<210>2

<211>121

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(121)

＜223〉aminoacid sequence of anti-CD 52 antibody heavy chain

<400>2

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Phe Ile Arg Asp Lys Ala Lys Gly Tyr Thr Thr Glu Tyr Asn Pro

50 55 60

Ser Val Lys Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln

65 70 75 80

Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Glu Gly His Thr Ala Ala Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Ser Leu Val Thr Val Ser Ser

115 120

<210>3

<211>324

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(324)

＜223〉encoding sequence of anti-CD 52 antibody light chain

<400>3

gacatccaga tgacccagag cccaagcagc ctgagcgcca gcgtgggtga cagagtgacc 60

atcacctgta aagcaagtca gaatattgac aaatacttaa actggtacca gcagaagcca 120

ggtaaggctc caaagctgct gatctacaat acaaacaatt tgcaaacggg tgtgccaagc 180

agattcagcg gtagcggtag cggtaccgac ttcaccttca ccatcagcag cctccagcca 240

gaggacatcg ccacctacta ctgcttgcag catataagta ggccgcgcac gttcggccaa 300

gggaccaagg tggaaatcaa acgt 324

<210>4

<211>108

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(108)

＜223〉aminoacid sequence of anti-CD 52 antibody light chain

<400>4

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile Asp Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Thr Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln His Ile Ser Arg Pro Arg

85 90 95

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

100 105

<210>5

<211>5

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(5)

＜223〉heavy chain CDR1

<400>5

Asp Phe Tyr Met Asn

1 5

<210>6

<211>19

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(19)

＜223〉heavy chain CDR2

<400>6

Phe Ile Arg Asp Lys Ala Lys Gly Tyr Thr Thr Glu Tyr Asn Pro Ser

1 5 10 15

Val Lys Gly

<210>7

<211>10

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(10)

＜223〉heavy chain CDR3

<400>7

Glu Gly His Thr Ala Ala Pro Phe Asp Tyr

1 5 10

<210>8

<211>11

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(11)

＜223〉light chain CDR1

<400>8

Lys Ala Ser Gln Asn Ile Asp Lys Tyr Leu Asn

1 5 10

<210>9

<211>7

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(7)

＜223〉light chain CDR2

<400>9

Asn Thr Asn Asn Leu Gln Thr

1 5

<210>10

<211>9

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(9)

＜223〉light chain CDR3

<400>10

Leu Gln His Ile Ser Arg Pro Arg Thr

1 5

<210>11

<211>363

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(363)

＜223〉encoding sequence of anti-CD 52 antibody heavy chain

<400>11

caggtgcagc tgcaggagtc cggccccggc ctggtgcgcc cctcccagac cctgttcctg 60

acctgcaccg tgtccggctc caccttctcc gacttctaca tgaactgggt gcgcttcccc 120

cccggccgcg gcgccgagtg gatcggcttc atccgcgaca agggcaaggg ctacacctcc 180

gagtacaacc cctccgtgaa gggccgcgtg accatgtccc gcgacaactc caagaactcc 240

ttctccctgc gcctgtccac ccgcaccgcc cccgacaccg ccgtgtacta ctgcgcccgc 300

gagggccaca acctggcccc cttcgactac tggggcatcg gctccctggt gaccgtgtcc 360

tcc 363

<210>12

<211>121

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(121)

＜223〉aminoacid sequence of anti-CD 52 antibody heavy chain

<400>12

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln

1 5 10 15

Thr Leu Phe Leu Thr Cys Thr Val Ser Gly Ser Thr Phe Ser Asp Phe

20 25 30

Tyr Met Asn Trp Val Arg Phe Pro Pro Gly Arg Gly Ala Glu Trp Ile

35 40 45

Gly Phe Ile Arg Asp Lys Gly Lys Gly Tyr Thr Ser Glu Tyr Asn Pro

50 55 60

Ser Val Lys Gly Arg Val Thr Met Ser Arg Asp Asn Ser Lys Asn Ser

65 70 75 80

Phe Ser Leu Arg Leu Ser Thr Arg Thr Ala Pro Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Glu Gly His Asn Leu Ala Pro Phe Asp Tyr Trp Gly

100 105 110

Ile Gly Ser Leu Val Thr Val Ser Ser

115 120

<210>13

<211>324

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(324)

＜223〉encoding sequence of anti-CD 52 antibody light chain

<400>13

gacatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga ccgcgtgacc 60

atctcctgca agctgtccca gaacatcgac aagtacatca actggtacca gcagaagccc 120

ggcgagtccc ccaagctgct gatctacaac accaacaacg cccagaccgg cgtgcccttc 180

cgcttctccg gctccggcac cggcaccgac tccaccctga ccatctccac cctgcagccc 240

gaggacgtgg ccaccttcta ctgcctgcag cacatcaccc gcccccgcac ctccggccag 300

ggcaccaagg tggagatcaa gcgc 324

<210>14

<211>108

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(108)

＜223〉aminoacid sequence of anti-CD 52 antibody light chain

<400>14

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Lys Leu Ser Gln Asn Ile Asp Lys Tyr

20 25 30

Ile Asn Trp Tyr Gln Gln Lys Pro Gly Glu Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Thr Asn Asn Ala Gln Thr Gly Val Pro Phe Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Phe Tyr Cys Leu Gln His Ile Thr Arg Pro Arg

85 90 95

Thr Ser Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210>15

<211>5

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(5)

＜223〉heavy chain CDR1

<400>15

Asp Phe Tyr Met Asn

1 5

<210>16

<211>19

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(19)

＜223〉heavy chain CDR2

<400>16

Phe Ile Arg Asp Lys Gly Lys Gly Tyr Thr Ser Glu Tyr Asn Pro Ser

1 5 10 15

Val Lys Gly

<210>17

<211>10

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(10)

＜223〉heavy chain CDR3

<400>17

Glu Gly His Asn Leu Ala Pro Phe Asp Tyr

1 5 10

<210>18

<211>11

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(11)

＜223〉light chain CDR1

<400>18

Lys Leu Ser Gln Asn Ile Asp Lys Tyr Ile Asn

1 5 10

<210>19

<211>7

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(7)

＜223〉light chain CDR2

<400>19

Asn Thr Asn Asn Ala Gln Thr

1 5

<210>20

<211>9

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(9)

＜223〉light chain CDR3

<400>20

Leu Gln His Ile Thr Arg Pro Arg Thr

1 5

Claims

1. monoclonal antibody V _HChain is characterized in that, its complementary determining region CDR has the aminoacid sequence of the CDR of the group of being selected from down:

CDR1 shown in the SEQ ID NO:5,

CDR2 shown in the SEQ ID NO:6 or 16,

CDR3 shown in the SEQ ID NO:7 or 17,

And, described monoclonal antibody V _HChain has the aminoacid sequence shown in SEQ ID NO:2 or 12.

2. monoclonal antibody V _LChain is characterized in that, its complementary determining region CDR has the aminoacid sequence of the CDR of the group of being selected from down:

CDR1 shown in the SEQ ID NO:8 or 18,

CDR2 shown in the SEQ ID NO:9 or 19,

CDR3 shown in the SEQ ID NO:10 or 20,

And, described monoclonal antibody V _LChain has the aminoacid sequence shown in SEQ ID NO:4 or 14.

3. a monoclonal antibody is characterized in that, its V _HChain has the aminoacid sequence shown in the SEQ ID NO:2 and its V _LChain has the aminoacid sequence shown in the SEQ ID NO:4; Perhaps its V _HChain has the aminoacid sequence shown in the SEQ ID NO:12 and its V _LChain has the aminoacid sequence shown in the SEQ ID NO:14.

4. monoclonal antibody as claimed in claim 3 is characterized in that, it is humanized monoclonal antibody.

5. a dna molecular is characterized in that, its coding is selected from down the protein of group:

The described monoclonal antibody V of claim 1 _HChain;

The described monoclonal antibody V of claim 2 _LChain;

The described monoclonal antibody of claim 3.

6. dna molecular as claimed in claim 5 is characterized in that, it has the dna sequence dna of the group of being selected from down: SEQ ID NO:1,3,11 or 13.

7. a pharmaceutical composition is characterized in that, it contains monoclonal antibody and pharmaceutically acceptable carrier, the V of described monoclonal antibody _HChain and V _LChain has the complementary determining region shown in SEQ ID NO:5-7 and the SEQ ID NO:8-10 respectively,

And the V of described monoclonal antibody _HChain has the aminoacid sequence shown in the SEQ ID NO:2 and its V _LChain has the aminoacid sequence shown in the SEQ ID NO:4; The perhaps V of described monoclonal antibody _HChain has the aminoacid sequence shown in the SEQ ID NO:12 and its V _LChain has the aminoacid sequence shown in the SEQ ID NO:14.