CN1189484C

CN1189484C - Recombined human CD11a monoclone antibody and its preparation and medicinal composition

Info

Publication number: CN1189484C
Application number: CNB021108668A
Authority: CN
Inventors: 王皓; 王俊林
Original assignee: ZHONGXIN GUOJIAN PHARMACEUTICAL CO Ltd SHANGHAI
Current assignee: Antibodies National Engineering Research Center
Priority date: 2002-02-20
Filing date: 2002-02-20
Publication date: 2005-02-16
Anticipated expiration: 2022-02-20
Also published as: CN1439651A

Abstract

The present invention provides an anti-CD11a monoclone antibody. The variable region of a light chain has an amino acid sequence shown in the SEQ ID NO : 1 or the SEQ ID NO : 1, and the variable region of a heavy chain has an amino acid sequence shown in the SEQ ID NO : 2 or the SEQ ID NO : 6. The bioactivity of the monoclonal antibody and the expression amount of the monoclonal antibody in a host cell are obviously enhanced. The present invention also provides DNA molecules for encoding the monoclonal antibody, a preparation method of the monoclonal antibody and a medical composition containing the monoclonal antibody.

Description

Recombinant anti human CD11a monoclonal antibody and method for making thereof and pharmaceutical composition

Invention field

The present invention relates to biological technical field.Specifically, the present invention relates to a kind of new recombinant anti human CD11a monoclonal antibody.In addition, the pharmaceutical composition that the invention still further relates to this MONOCLONAL ANTIBODIES SPECIFIC FOR method and contain this monoclonal antibody.

Background of invention

The CD11/CD18 mixture belongs to integrin family, comprises that LFA is CD11a/CD18, LFA-1; The scavenger cell differentiation antigen is CD11b/CD18, and Mac-1 and glycoprotein are P150/95, CD11c/CD18.They respectively be distributed in endotheliocyte, the respective ligand on thrombocyte and the lymphocyte combines, the identification of mediated leucocytes, adhesion.The expression of CD11/CD18 is apparently higher than the normal control group on neutrophil leucocyte in the Mezzane discovery unstable angina patient coronary artery hole blood and the monocyte, the prompting white corpuscle is activated through coronary circulation the time, and adhere to increase [people .Science such as Cybul S, 1991,251:788-789].

CD11a is relevant with the T cells whose development, and the inhibition of its function can cause the reduction of T cellular immune function.Therefore, it is relevant with autoimmune disease.Studies show that the immunologic function that the monoclonal antibody clonal antibody of CD11a can suppressor T cell, its mechanism of action are to suppress combining of T-cell and other cell types, thereby reduce the immunologic function of T cell.There are some researches prove that the expression of downward modulation CD11a can obviously improve the symptom of psoriatic (psoriasis cascade), its main mechanism is relevant with psoriatic three critical process.They are:

1) by doing mutually to combine with the T cell with the adhesion molecule of endothelial cell surface;

2) the T cell is transported to skin;

3) activated T cell.

Above-mentioned these effects are all relevant with the skin injury that causes after the misgrowth of skin cells and psoriatic are attacked.The CD11a monoclonal antibody might become the medicine of treatment relative disease.

In sum, therefore the effect that the monoclonal antibody of CD11a can suppressor T cell can be used for the treatment of the immunological rejection that occurs in inflammation (especially skin inflammation), autoimmune disease and tissue (cell) migration process clinically.

Yet the biological activity and the expression amount of existing anti-CD11a monoclonal antibody are lower at present.Therefore, need the recombinant anti human CD11a monoclonal antibody that new biological activity increases in this area.

Goal of the invention

The anti-people CD11a monoclonal antibody that the purpose of this invention is to provide a kind of reorganization.

Another object of the present invention provides the dna molecular of the above-mentioned anti-CD11a monoclonal antibody of coding.

Another object of the present invention provides a kind of pharmaceutical composition that contains said monoclonal antibody.

A further object of the invention provides the method for preparing said monoclonal antibody.

To achieve the above object of the invention, one aspect of the present invention provides a kind of anti-people CD11a monoclonal antibody of reorganization, this antibody contains variable region of heavy chain and variable region of light chain, it is characterized in that, variable region of light chain has the aminoacid sequence shown in SEQ ID NO:1 or the SEQ ID NO:5, and variable region of heavy chain has the aminoacid sequence shown in SEQ ID NO:2 or the SEQ ID NO:6.

Preferably, the variable region of light chain in this monoclonal antibody has the aminoacid sequence shown in the SEQ ID NO:1, and variable region of heavy chain has the aminoacid sequence shown in the SEQ ID NO:2.

The present invention provides the dna molecular of coding said monoclonal antibody on the other hand.

In a preferable example, this dna molecular contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in SEQ ID NO:3 or the SEQ ID NO:7, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in SEQ ID NO:4 or the SEQ ID NO:8.

Third aspect present invention provides a kind of expression vector, the expression regulation sequence that this expression vector contains above-mentioned dna sequence dna and links to each other with this series of operations.

Fourth aspect present invention provides a kind of host cell, it is characterized in that, it is transformed by above-mentioned expression vector.In a preferable example, this host cell is a Chinese hamster ovary celI.

Fifth aspect present invention provides a kind of pharmaceutical composition for the treatment of inflammation, it is characterized in that, it contains the pharmaceutically above-mentioned human monoclonal antibodies and the pharmaceutically acceptable carrier of significant quantity.In a preferable embodiment, the inflammation of being treated is a skin inflammation.

Sixth aspect present invention provides a kind of method for preparing said monoclonal antibody, it is characterized in that, this method comprises:

A) provide an expression vector, the expression regulation sequence that this expression vector contains above-mentioned dna sequence dna and links to each other with this series of operations;

B) with the described expression vector transformed host cell of step a);

C) host cell of gained culturing step b under the condition that is fit to described monoclonal antibody expression); With

D) separation and purification obtains described monoclonal antibody.

The avidity and the expression amount in host cell (as the COS cell) that the invention has the advantages that monoclonal antibody of the present invention are significantly increased than existing monoclonal antibody.Other purpose of the present invention and advantage can be learnt by following detailed.

Description of drawings

Fig. 1 has shown the restriction enzyme mapping of the used pMG18 carrier of the present invention.Ck represents the constant region gene of antibody kappa light chain among the figure; The IgG1 constant region is represented the weight chain constant area gene of people's IgG antibody 1; PA represents that SV40 adds poly A site.

Detailed Description Of The Invention

The present invention relates to a kind of anti-human CD11a monoclonal antibody of restructuring, this antibody comprises variable region of heavy chain and variable region of light chain, it is characterized in that, variable region of light chain has the amino acid sequence shown in SEQ ID NO:1 or the SEQ ID NO:5, and variable region of heavy chain has the amino acid sequence shown in SEQ ID NO:2 or the SEQ ID NO:6.

Term used herein " monoclonal antibody (monoclonal antibody) " refers to the antibody that obtains from the colony of the basic homogeneous of a class, and the single antibody that namely comprises in this colony is identical, the sudden change of the natural generation that may exist except minority. Monoclonal antibody is with high specificity for single antigen site. And from conventional Anti-TNF-α body preparation (normally having the different antibodies for different determinants) difference, each monoclonal antibody is for the single determinant on the antigen. Except their specificity, the benefit of monoclonal antibody is that also they are next synthetic by the hybridoma cultivation, can not polluted by other immunoglobulin (Ig). Modifier " monoclonal " has represented the characteristic of antibody, is to obtain from the antibody population of basic homogeneous, and this should not be interpreted into and need to produce antibody with any specific process.

Term used herein " antibody " and " immunoglobulin (Ig) " are the about 150000 daltonian different four glycan albumen that the same structure feature is arranged, and it is comprised of with two identical heavy chains (H) two identical light chains (L). Every light chain links to each other with heavy chain by a covalent disulfide bonds, and the disulfide bond number between the heavy chain of different Immunoglobulin Isotypes is different. Every heavy chain and light chain be the intrachain disulfide bond at regular interval also. One end of every heavy chain has variable region (VH), is thereafter a plurality of constant regions. One end of every light chain has variable region (VL), and the other end has constant region; The constant region of light chain is relative with first constant region of heavy chain, and the variable region of light chain is relative with the variable region of heavy chain. Special amino acid residue forms the interface between the variable region of light chain and heavy chain.

Some part of variable region is different on sequence in term used herein " variable " the expression antibody, and it has formed various specific antibodies to combination and the specificity of its specific antigen. Yet changeability is not evenly distributed in the whole antibody variable region. It concentrates in three fragments that are called in light chain and the variable region of heavy chain in complementary determining region (CDR) or the hypervariable region. Part conservative in the variable region is called framework region (FR). Each self-contained four FR district in the variable region of natural heavy chain and light chain, they are the beta sheet configuration haply, are linked to each other by three CDR that form connecting ring, but forming section β-pleated sheet structure in some cases. CDR in every chain closely is close together by the FR district and has formed the antigen-binding site (referring to Kabat etc., NIH Publ.No.91-3242, volume I, 647-669 page or leaf (1991)) of antibody with the CDR of another chain. Constant region is not participated in the combination of antibody and antigen directly, but they show different effector functions, for example participates in the cytotoxicity that depends on antibody of antibody.

" light chain " of vertebrate antibody (immunoglobulin (Ig)) can be classified as according to the amino acid sequence of its constant region the class in visibly different two classes (being called κ and λ). According to the amino acid sequence of its CH, immunoglobulin (Ig) can be divided into different kinds. Mainly contain 5 immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM, some of them also can further be divided into subclass (isotype), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. CH corresponding to the inhomogeneity immunoglobulin (Ig) is called α, δ, ε, γ and μ. The subunit structure of inhomogeneity immunoglobulin (Ig) and 3-d modelling are well-known.

Monoclonal antibody can make with the whole bag of tricks well known to those skilled in the art. For example, monoclonal antibody can use hybridoma method (by Kohler etc., Nature, 256:495 (1975) at first proposes) to make, or makes with recombinant DNA method (U.S. Patent No. 4,816,567). Monoclonal antibody is also available such as Clackson etc., Nature, and 352:624-628 (1991) and Marks etc., J.Mol.Biol., the described technology of 222:581-597 (1991) is separated acquisition from phage antibody library.

The present invention also provides the dna molecular of code book invention recombinant anti human CD11a monoclonal antibody. In a better example, this dna molecular contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in SEQ ID NO:3 or the SEQ ID NO:7, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in SEQ ID NO:4 or the SEQ ID NO:8.

Behind the nucleotide sequence that obtains code book invention recombinant anti human CD11a monoclonal antibody variable region of heavy chain and variable region of light chain, usually can prepare by the following method monoclonal antibody of the present invention.

At first, provide the nucleotide sequence that contains code book invention monoclonal antibody and the expression vector of the expression regulation sequence that links to each other with this series of operations.

Term used herein " expression regulation sequence " is often referred to and participates in the sequence that the control nucleotide sequence is expressed. Expression regulation sequence comprises promoter and the termination signal that links to each other with target nucleotide sequence operability. They also comprise the suitably required sequence of translation of nucleotide sequence usually. " operability links to each other " refers to that some part of linear DNA sequence can affect the activity of same other parts of linear DNA sequence. For example, if promoter or enhancer have increased transcribing of coded sequence, then it is that operability links to each other with coded sequence.

The dna sequence dna of code book invention monoclonal antibody can make with conventional means well known to those skilled in the art. For example, can be manually synthetic or increase with the PCR method and to obtain encoding the nucleotide sequence of this monoclonal antibody variable region of heavy chain and variable region of light chain according to sequence disclosed by the invention. Then, these nucleotide sequences are inserted in the suitable expression vector by the suitable restriction enzyme site of selection with the whole bag of tricks well known in the art, make them respectively before expression vector entrained CH coded sequence and constant region of light chain coded sequence, and make them in same frame. Used expression vector is various commercially available expression vector well known by persons skilled in the art among the present invention, for example available from the expression vector of Qiagen and Promega company, and commercially available expression vector pMG18 (" carrying out the exploitation of the instrument of environmental monitoring according to the INCP-9 plasmid sequence " (DEVELOPMENT OF TOOLS FOR ENVIRONMENTAL MONITORING BASED ON INCP-9 PLASMIDS SEQUENCES), A.Greated, R.Krasowiak, M.Titok, C.M.Thomas School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK and Faculty of Biology, Dept of Microbiology, Belarus State University Scorina Av.4, Minsk 220080 Belarus).

Subsequently, the expression vector with above-mentioned acquisition transforms suitable host cell. " host cell " generally comprises prokaryotic and eukaryotic. The example of prokaryotic host cell commonly used comprises Escherichia coli, hay bacillus etc. Eukaryotic host cell commonly used comprises yeast cells, insect cell and mammalian cell. In the present invention, better host cell is Chinese hamster ovary celI. Method with the expression vector transformed host cell has a variety of, used Transformation Program to depend on host to be transformed. The method that heterologous polynucleotide is imported in the mammalian cell is known in the art, it comprises transfection, calcium phosphate precipitation, the Polybrene (1 of glucan mediation, 5-dimethyl-1,5-phenodiazine 11 methylene gather Methobromide) mediation transfection, protoplast fusion, electroporation, liposome-mediated transfection and with the direct microinjection of DNA in karyon. In the present invention, better method is electroporation or liposome mediated-method etc. For example can adopt the liposome method kit of Invitrogen company to come transfection CHO cell.

Then, under the condition that is fit to monoclonal antibody expression of the present invention, cultivate the host cell that transforms gained. Then use conventional immunoglobulin purification step, obtain recombinant anti human CD11a monoclonal antibody of the present invention such as conventional separation and purification means purifying well known to those skilled in the art such as albumin A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion-exchange chromatography, hydrophobic chromatography, sieve chromatography or affinity chromatographys.

The gained monoclonal antibody can be identified with conventional means. The binding specificity of monoclonal antibody can be measured with immunoprecipitation or external combination test (such as radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA) (ELISA)). The binding affinity of monoclonal antibody such as available Munson etc., Anal.Biochem., the Scatchard of 107:220 (1980) analyzes to measure.

The present invention also provides a kind of pharmaceutical composition that is used for the treatment of inflammation, especially scytitis. Said composition contains pharmaceutically monoclonal antibody of the present invention and the pharmaceutically acceptable carrier of effective dose. Term used herein " pharmaceutically acceptable " refers to when molecule body and composition suitably give the animal or human, and they can not produce disadvantageous, irritated or other bad reaction. " pharmaceutically acceptable carrier " used herein should be compatible with mutain of the present invention, can be with its blend the effect of decrease pharmaceutical composition under normal conditions not. The object lesson that can be used as some materials of pharmaceutically acceptable carrier or its component is carbohydrate, such as lactose, dextrose plus saccharose; Starch is such as cornstarch and potato starch; Cellulose and derivative thereof are such as sodium carboxymethylcellulose, ethyl cellulose and methylcellulose; The tragacanth powder; Fructus Hordei Germinatus; Gelatin; Talcum; Kollag is such as stearic acid and dolomol; Calcium sulfate; Vegetable oil is such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cupu oil; Polyalcohol is such as propane diols, glycerine, D-sorbite, mannitol and polyethylene glycol; Alginic acid; Emulsifying agent is such as Tween ; Wetting agent is such as NaLS; Colouring agent; Flavor enhancement; Tablet agent, stabilizing agent; Antioxidant; Anticorrisive agent; Apirogen water; Deng oozing salting liquid; With phosphate buffer etc.

Pharmaceutical composition of the present invention can be made various formulations as required, and can by the doctor according to patient's kind, age, body weight and roughly the factor such as disease condition, administering mode determine the useful dosage of patient is used.

Below in conjunction with embodiment the present invention is described in further detail.Yet should be appreciated that and enumerate these embodiment, and be not to be used for limiting the present invention just for an illustration.

Embodiment 1 screens the antibody gene variable region of CD11a from antibody library

1) structure of human antibody library

According to people J.Mol.Biol. such as Marks, 222,581-597, Hoogenboom and Winter, J.Mol.Biol., people J Immunol Methods.2001 Nov 1 such as 227.381-388, Haidaris CG; 257 (1-2): 185-202, Griffiths, people EMBO J. such as A.D., 13,3245-3260 (1994), Nissim, people EMBO J. such as A., 13, the method for describing among the 692-698 (1994) makes up human antibody library.

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 is transferred to supernatant liquor in 50 milliliters of aseptic centrifuge tubes, preserves standby.Guarantee that its titre should be 2 * 10 ¹¹More than.With the CD11a albumen of purifying (available from Shenzhen brilliant U.S. company) as antigen, with the ordinary method bag by 25 ml cells culturing bottles.Add in the cell bottle behind the bag quilt and be no less than 3 * 10 ¹⁰Phage, 37 ℃ of incubations 1 hour.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 the described step 4 of epimere time.

Cell dilution to 10 with above-mentioned acquisition ⁵Cultivate being added with on 1.5% agar plate of 0.1% penbritin after the cells/ml, obtain mono-clonal.

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.

Above-mentioned deep-well plates after centrifugal 20 minutes, is transferred to new aseptic deep-well plates with supernatant at 5000RPM on the 96 orifice plate whizzers, be preserved in after sealing 4 the degree standby.

Get 10 of 96 orifice plates, add in every hole the conventional bag of CD11a (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 (available from Pharmacia company) 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.This test-results filters out 387 positive colonies altogether, determines wherein 5 the strongest clones of avidity according to its reading, is used for next step research.

3) the antibody variable region encoding sequence is to the clone of expression vector

Above-mentioned 5 clones' bacterial strain is increased, then according to the plasmid DNA extracting and purifying test kit plasmid DNA purification of manufacturer's specification sheets in 100 milliliters of LB substratum with Promega company.

With XbaI with on 1.5% agarose gel electrophoresis, separate endonuclease bamhi after NheI Restriction Enzyme enzyme is cut above-mentioned plasmid DNA, get band about 350bp and carry out glue and reclaim, the gained fragment is variable region of heavy chain.With Hind III with on 1.5% agarose gel electrophoresis, separate endonuclease bamhi after BsiWI Restriction Enzyme enzyme is cut above-mentioned plasmid DNA, get band about 320bp and carry out glue and reclaim, the gained fragment is variable region of light chain.

Cut expression vector pMG18 with XbaI and NheI Restriction Enzyme enzyme.The restriction enzyme mapping of this expression vector as shown in Figure 1.Above-mentioned variable region of heavy chain is inserted in the XbaI/NheI site of this expression vector then.Equally, utilize HindIII and Bsi WI Restriction Enzyme that antibody chain variable region is inserted in the HindIII/Bsi WI site of the pMG18 carrier that inserts variable region of heavy chain, obtain containing the expression vector of anti-CD11a antibody gene of recombinating thereby make up.

4) screening of the transfection of Chinese hamster ovary celI and recombinant clone

The expression vector that has antibody gene of above-mentioned structure is inoculated in the escherichia coli DH5a bacterial strain in 100 milliliters of LB substratum and increases, with ultrapure plasmid DNA purification kit (Ultrapure PlasmidDNA Purification Kit) the extracting and purifying plasmid DNA of Qiagen company.Adopt the liposome method test kit of Invitrogen company, with the plasmid DNA transfection Chinese hamster ovary celI of above-mentioned purifying, operation is carried out with reference to the specification sheets of producer.

The Chinese hamster ovary celI that transforms carries out the selection in continuous 9 weeks on HAT selection substratum, cultivate in the enterprising limit by row dilution of 96 orifice plates at last, carries out continuously 3 times, carry out mono-clonalization.

The monoclonal cell of choosing ties up on the RPM1641 substratum and cultivates, and supernatant is carried out the test of Western trace, judges expression intensity according to staining reaction, picks out 12 and expresses strong clone as the candidate cell strain.

5) Purification of Monoclonal Antibodies

Go out said monoclonal antibody with the direct separation and purification from cells and supernatant of Protein A affinity column, prove that through the SDA-PAGE electrophoresis products therefrom purity is greater than 90%.The product of affinity chromatography passes through sieve chromatography once more, has obtained the sample of purity＞98%.These samples are used for following further analysis and research.

The expression intensity of embodiment 2 antibody genes in Chinese hamster ovary celI

12 high expression level candidate clones that above-mentioned screening is obtained are incubated in the tissue culture ware of 10cm, the expression amount of measuring antibody with the ELISA method as described below.Goat anti-human igg (Fc) is wrapped quilt in elisa plate, 4 ℃ are spent the night, sealed 2 hours in 37 ℃ through 2%BSA, add culture supernatant to be measured and standard substance (human IgG1), 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 H at last ₂SO ₄Termination reaction is surveyed A ₄₅₀Value.The expression amount that has shown 12 candidate clones that above-mentioned screening obtains in the following table 1.

The expression amount of table 1 candidate clone in Chinese hamster ovary celI

The cell strain numbering	1B3	2D4	3D7	5E3	8C9	6D2	3E9	2H8	3H4	2F1	3G6	7G8	1A7
The cell strain numbering	1B3	2D4	3D7	5E3	8C9	6D2	3E9	2H8	3H4	2F1	3G6	7G8	1A7	Expression amount (mcg/ml)	121.1	247.2	212.0	98.6	309.1	278.2	211.7	289.4	227.8	145.6	165.7	141.2	146.6

As can be seen from Table 1,8C9,6D2 and 2H8 have very high expression level.Compare with the data of abroad having delivered (Kunkel, PNAS, 82:488,1995) (18.2 mcg/ml), its expression level has improved 17 times, 15.3 times and 15.9 times respectively.

The dna sequencing of embodiment 3 anti-CD11a monoclonal antibody genes

According to pedigree, the 2H8 of above-mentioned acquisition and the anti-CD11a monoclonal antibody gene of 6D2 cell strain are carried out dna sequencing.The result is as follows: SEQ ID NO:3 shown 2H8 chain variable region gene sequence (5 ' to 3 ', 327bp), the aminoacid sequence of its supposition is presented among the SEQ ID NO:1; SEQ ID NO:4 shown 2H8 heavy chain variable region gene sequence (5 ' to 3 ', 366bp), the aminoacid sequence of its supposition is presented among the SEQ ID NO:2; SEQ ID NO:7 shown 6D2 chain variable region gene sequence (5 ' to 3 ', 324bp), the aminoacid sequence of its supposition is presented among the SEQ IDNO:5; SEQ ID NO:8 shown 6D2 heavy chain variable region gene sequence (5 ' to 3 ', 363bp), the aminoacid sequence of its supposition is presented among the SEQ ID NO:5.

Embodiment 4 monoclonal antibody physiologically actives and avidity research

1) mixed leukocyte culture test

The biologic activity of the monoclonal antibody that higher cell strain 8C9,6D2 and the 2H8 of above-mentioned three expression levels produced detects.Concrete test method is as follows:

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 (mixed lymphocyte responses)) 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 ℃, 5%CO ₂Cultivated 5 days, and stopped cultivating preceding 16 hours adding 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 the MicrosoftExcel statistics program.

In the MLR reaction system, add 1 microlitre, 5 microlitres and 10 microlitres Chinese hamster ovary celI supernatant respectively through anti-CD11a 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 utmost point conspicuous level, the results are shown in following table 2.Equally, directly carry out the MLR experiment with the supernatant of unpurified transfectional cell, fail to observe significant inhibitory effect, this may cancel out each other relevant with the hormesis and the proteic restraining effect of micro-monoclonal antibody of heterologous protein in the purifying cells transfection supernatant not.

The influence that table 2 purifying CD11a monoclonal antibody is bred people MLR medium size lymphocyte (DPM, n=3)

Test grouping (microlitre/hole)	0	1	5	10
Test grouping (microlitre/hole)	0	1	5	10	The monoclonal antibody 8C9 of purifying	5629±563	2987±198 ^*	2237±240 ^*	1054±224 ^*
The monoclonal antibody 6D2 of purifying	5576±487	3017±206	2144±197	803±217	The monoclonal antibody 8C9 of purifying	5629±563	2987±198 ^*	2237±240 ^*	1054±224 ^*
The monoclonal antibody 6D2 of purifying	5576±487	3017±206	2144±197	803±217	The monoclonal antibody 2H8 of purifying	5467±354	2631±187	1876±114	462±173
Contrast	5579±367	5367±313	5627±309	5324±306	The monoclonal antibody 2H8 of purifying	5467±354	2631±187	1876±114	462±173

The MLR experiment shows that the albumen of this purifying has the activity that suppresses immune response, and 2H8 has the strongest restraining effect, and 6D2 takes second place, and the restraining effect of 8C9 is minimum.Above result also shown successfully the clone and expressed the anti-CD11a monoclonal antibody that biologic activity is arranged in Chinese hamster ovary celI, thereby lays a good foundation for the anti-CD11a monoclonal antibody of further great expression purifying and Immunocompetence Study thereof and clinical application.

2) evaluation of avidity

Avidity measure to adopt the Scatchard analytical method (people such as Munson, 1980, Anal.BioChem. 107:220) carries out.The result shows that the avidity of 2H8,6D2 and three kinds of monoclonal antibodies of 8C9 reaches 3.2 * 10 respectively ^-9, 1.18 * 10 ^-8With 8 * 10 ^-6, wherein avidity (Kunkel, PNAS, 82:488,1995,2 * 10 of 2H8 and 6D2 two strain monoclonal antibodies ^-8) have greatly improved.

Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.

Sequence table

＜110〉Shanghai CP Guojian Pharmaceutical Co.,Ltd

＜120〉recombinant anti human CD11a monoclonal antibody and method for making thereof and pharmaceutical composition

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<211>366

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>4

gaggtgcagc tggtgaagtc cggcatgggc ctggtgcagc ccggcgtgtc cctgcgcctg 60

tcctgcaccg cctccggcta ctcccgcacc ggccactgga tgaactgggt gcgcaccgcc 120

cccggccgcg gcctggagtg ggtgggcaac gaccacccct ccgactccga gacccgctac 180

aaccagaagt tcaaggactg cttcaccatc tccgtggaca cccgctccaa gaacaccctg 240

tacctgcaga tgaactccct gcgcgccaag gacaccaacg tgtactactg cgccaccggc 300

atctacttcc gcggcaccac ctacaccgac tactggggcc agtgcaccct ggtgaccgtg 360

tcctcc 366

<210>5

<211>108

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>5

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 Arg Ala Ser Lys Thr Ile Ser Lys Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Leu

85 90 95

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

100 105

<210>6

<211>121

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>6

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly His

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Gly Ile Tyr Phe Tyr Gly Thr Thr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>7

<211>324

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>7

gacattcaga tgacccagag cccttcctca ctctctgcct ctgtgggcga ccgggtgacc 60

atcacctgtc gcgcctccaa aaccatttct aaatacctcg cttggtacca gcagaagccc 120

ggcaaggctc caaagctgct catttacagc ggctctactt tgcagtctgg agtcccatct 180

cgtttcagcg ggtctggttc agggactgac tttacattga caatttcaag cctgcagcct 240

gaagattttg ctacctatta ttgccaacag cacaacgagt acccactgac ctttggccaa 300

ggcaccaagg ttgaaatcaa acgt 324

<210>8

<211>363

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>8

gaggtccagc tggttgagtc tgggggcgga cttgtgcagc cagggggcag ccttagactt 60

tcatgcgctg cctcgggtta tagcttcacc ggccactgga tgaactgggt ccgccaggcc 120

cctgggaagg gcctggagtg ggtcgggatg attcaccctt ctgacagcga gaccagatat 180

aaccagaaat tcaaagacag gttcacaatt tctgttgata aatcaaagaa taccctgtat 240

ctgcaaatga attccctcag ggccgaagac actgccgttt attactgcgc ccgcggcatt 300

tatttttacg gcacgaccta ctttgactac tgggggcagg ggactctcgt aactgtctct 360

tcc 363

Claims

1. recombinant anti human CD11a monoclonal antibody, it comprises variable region of heavy chain and variable region of light chain, it is characterized in that, variable region of light chain has the aminoacid sequence shown in SEQ ID NO:1 or the SEQ ID NO:5, and variable region of heavy chain has the aminoacid sequence shown in SEQ ID NO:2 or the SEQ ID NO:6.

2. monoclonal antibody according to claim 1 is characterized in that variable region of light chain has the aminoacid sequence shown in the SEQ IDNO:1, and variable region of heavy chain has the aminoacid sequence shown in the SEQ ID NO:2.

3. a dna molecular is characterized in that, the described monoclonal antibody of its coding claim 1.

4. dna molecular according to claim 3, it is characterized in that, this dna molecular contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in SEQ ID NO:3 or the SEQ ID NO:7, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in SEQ ID NO:4 or the SEQ ID NO:8.

5. an expression vector is characterized in that, the expression regulation sequence that it contains the described dna sequence dna of claim 3 and links to each other with this series of operations.

6. an eukaryotic host cell is characterized in that, it is transformed by the described expression vector of claim 5.

7. eukaryotic host cell according to claim 6 is characterized in that it is a Chinese hamster ovary celI.

8. treatment and the pharmaceutical composition that CD11a expresses relevant inflammation is characterized in that it contains the pharmaceutically described recombinant human monoclonal antibody of claim 1 and the pharmaceutically acceptable carrier of significant quantity.

9. the pharmaceutical composition of treatment inflammation according to claim 8 is characterized in that, the described inflammation relevant with the CD11a expression is skin inflammation.

10. one kind prepares the described monoclonal antibody method of claim 1, it is characterized in that this method comprises:

A) provide an expression vector, the expression regulation sequence that this expression vector contains the described dna sequence dna of claim 3 and links to each other with this series of operations;

B) transform eukaryotic host cell with the described expression vector of step a);

C) eukaryotic host cell of gained culturing step b under the condition that is fit to described monoclonal antibody expression); With

D) separation and purification obtains described monoclonal antibody.