CN100515496C - Immunity regulator and its application - Google Patents

Abstract

The invention discloses a group of immunomodulatory agents, which can be used for the treatment of autoimmune diseases or organ transplantation through immunological inhibitory actions.

Description

Immunomodulator and application thereof

The application is to be on February 14th, 2003 applying date, and application number is 03115396.8, and denomination of invention is divided an application for China of " immunomodulator and application thereof " application.

Technical field

The present invention relates to people CD2 conjugated protein (variant and the monoclonal antibody that comprise its receptor, receptor) and CD11a monoclonal antibody or CTLA4 soluble fragments and variant use in conjunction thereof, by immunosuppressive action treatment autoimmune disease or be applied to clinical disease such as organ transplantation.

Background technology

Antigen presenting cell and T cell have important function aspect the cellular immunization mechanism, have the multiple protein molecule can mediate the two and interact, thereby cause the differentiation and the growth of T cell, finally mediate immunoreation.Three examples of this interaction signalling channel are shown in Fig. 1.

One, CD2:LFA3 approach

In the CD2/LFA3 approach, people CD2 molecule is the memebrane protein of a kind of 50KD, be present in most thymocyte cell and all periphery lymphocyte surfaces, its maturation protein is made up of 332 aminoacid, the signal peptide that its precursor is made up of 19 aminoacid (Genebank protein ID:AAA51738.1).This molecule and LFA3 specificity interact, and mediation T cell is to the adhesion of target cell or antigen-presenting cell, thus the functional response of initial T cell.

LFA3 claims CD58 again, is that antigen presenting cell is a kind of transmembrane protein, and its precursor protein has 28 amino acid whose signal peptides, and maturation protein is formed (Genebank protein IDNP001770.1) by 233 aminoacid.LFA3 is distributed widely in various cell surfaces, comprises erythrocyte, endotheliocyte, epithelial cell, fibroblast etc.CD2 is its part.

It is T cell-antigen presenting cell a kind of of multiple signalling channel that interact that CD2/LFA3 interacts, can strengthen the adhesion between T cell and antigen presenting cell or the target cell after the two combination, promote of the conduction of T cell antigenic identification and the signal that CD2 mediated.

The antibody of anti-CD2 or anti-LFA3 can stop the signal conduction of CD2/LFA3, and the immunologic function of T cell is suppressed.

Two .B7:CD28/CTLA4 approach

T-cell, antigen presenting cell surface participate in producing altogether, and the molecule of stimulation is referred to as costimulatory molecules.Generally acknowledge that at present most important stimulation channels altogether is B7-CD27 path (Fig. 1).B7 is positioned at antigen presenting cell, combines with the CD28 molecule of T cell surface and can produce common stimulation.Behind t cell activation, its cell surface can occur another can with the bonded molecule CTLA4 of B7 cell (Cytotoxic TLymphocyte associate Antigen-4).The affinity of CTLA4 and B7 is very high, this molecule with can induce the accent of B7 cell to die after the B7 cell combines.Therefore think that at present it has down regulation to t cell activation, the balance of immune state plays an important role in the human body for keeping.

CTLA4 (being the cytolytic T lymphocyte antigen 4) claims CD152 again, the dimer of being made up of a kind of transmembrane glycoprotein, it can combine with CD80 and CD86, its affinity is than obviously high with CD28, and its function is opposite with CD28, and the T cell activation is had down regulation.Its receptor is B7-1 and B7-2.This molecule is with after B7 combines, can the inducing T cell apoptosis, think at present that therefore it is regulated the t cell activation negative feedback, and the balance of keeping immune state in the human body is played an important role.

CTLA-4 is one of significant molecule that is expressed on the activating T cell surface.Studies show that this composition is a negative regulatory factor of keeping T cell stability in the body, have the value-added function that suppresses activation T-cell, constituted a pair of crucial immunoregulatory factor with CD28.At present, it is used as the immunosuppressant receptor and has carried out extensive studies, might use as the preparation that suppresses immunological rejection clinically.

Three, CD11a:ICAM-1 approach

The CD11/CD18 complex belongs to integrin family, comprises that lymphocyte function associated antigen (LFA) is CD11a/CD18 (this complex also is called LFA-1), and the phagocyte 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 platelet and the lymphocyte combines, the identification of mediated leucocytes, adhesion.The expression of CD11/CD18 is apparently higher than the normal control group on neutrophilic granulocyte in the Mezzane discovery unstable angina patient arteria coronaria hole blood and the mononuclear cell, and the prompting leukocyte is activated through coronary circulation the time, and adheres to increase.

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.

CD11a claims to integrate plain aL, LFA-1a chain, p180 etc. again, is a member in the cellular integration element.This amyloid protein precursor is by 26 signal peptides that aminoacid is formed, and maturation protein is made up of 1144 aminoacid.

ICAM-1 claims CD54 again, is a kind of transmembrane molecule of the APC of being positioned at cell surface, and its precursor protein is by 27 signal peptides that aminoacid is formed, and maturation protein is made up of 505 aminoacid.The CD11a subunit of the LFA-1 molecule of this albumen and CD8 CTL T cell surface interacts, and can strengthen the function of T cell.

Above-mentioned three kinds of signal transduction paths between T cell and the antigen presenting cell are having a very important role aspect the immunologic function adjusting of T cell.The blocking-up of any signal transduction path all might cause the immunologic function downward modulation.Based on this principle, can develop the medicine of treatment immunologic derangement disease such as autoimmune disease.From the genetic engineering angle, the strategy of developing this class medicine mainly contains three kinds.A kind of is the monoclonal antibody of development signalling channel associated protein, and a kind of is the receptor protein of development signalling channel associated protein, and a kind of is the antagonist that utilizes the signalling channel associated protein.These three kinds of strategies all are to utilize a kind of (or multiple) to come the disabling signal conductive process with the bonded albumen of signalling channel protein-specific.

The principal diagram of this strategy is in Fig. 2.The monoclonal antibody of CD11a can combine with the CD11a specificity, thereby blocked combining of CD11a and ICAM1, makes the LFA1-ICAM1 signal transduction path interrupt.CTLA4 is the receptor of B7, and the outer soluble fragments of its born of the same parents can combine with B7 competitively with CD28, because CTLA4 is than the binding ability height of CD28 and B7, therefore, when CTLA4 concentration acquired a certain degree, CD28 promptly capable of blocking combined with B7's, thereby the B7:CD28 signal transduction path is interrupted.In the LFA3:CD2 approach, if exist not with the APC cell complete molecule of bonded free LFA3 or have the imperfect molecule of its CD2 land, then this molecule also can combine with CD2, thereby hindered on the APC cell combining of CD2 molecule on the LFA3 and T cell, made the intercellular signal transduction path of APC and T be blocked.

Discover, adopt one or more of above-mentioned three kinds of approach, can effectively block the intercellular signal conduction of APC and T, T cell development, differentiation are obstructed or cause the apoptosis of T cell, thereby the immunologic function of T cell can not normally be brought into play, and the result causes immunologic function degression.

Up to the present, succeed in developing the panimmunity depressant of functions according to above-mentioned principle, these immunosuppressant can make immunologic function significantly reduce, thereby make autoimmune disease obtain significant treatment.The immunologic function inhibitor of above-mentioned principle development can also be applied in organ or cell transplantation aspect, and immunologic rejection is significantly reduced.

Yet,, go back neither one curative effect medicines clear and definite and significant chemosynthesis or other approach manufacturings both at home and abroad and be used for the treatment of autoimmune and immunologic rejection up at present.Therefore, this area presses for the inhibitor of the new treatment autoimmune disease of exploitation.

Summary of the invention

Purpose of the present invention just provides inhibitor and the method for making thereof that can be used for controlling autoimmune disease.

Another object of the present invention provides a kind of pharmaceutical composition that contains described inhibitor.

In a first aspect of the present invention, a kind of immunomodulator is provided, it is selected from down group:

(a) anti-people CD11a monoclonal antibody, described anti-people CD11a monoclonal antibody has the sequence of heavy chain shown in SEQ ID NO:1 or 3, and/or the sequence of light chain shown in SEQ ID NO:2 or 4;

(b) FLA3Ig fusion rotein, described fusion rotein have the Fc fragment of the human IgG1 shown in the 121-347 position among the CD2 land of the FLA3 shown in the 1-120 position among the SEQ ID NO:25 and the SEQ ID NO:26;

(c) CTLA4Ig fusion rotein, described fusion rotein have the Fc fragment of the human IgG1 shown in the CTLA-4R ectodomain shown in the 37-161 position and SEQ ID NO:17 among the SEQ ID NO:5.

In a preference, described immunomodulator is anti-people CD11a monoclonal antibody, and has human IgG1's constant region.

In another preference, described immunomodulator is the FLA3Ig fusion rotein, and has the aminoacid sequence shown in the SEQ IDNO:26.

In another preference, described immunomodulator is the CTLA4Ig fusion rotein, and also has connection peptides between CTLA-4R ectodomain and human IgG1's Fc fragment, for example the connection peptides shown in the SEQ ID NO:35.

In a second aspect of the present invention, provide the dna molecular that separates the above-mentioned immunomodulator of code book invention.

In a preference, described dna molecular has the nucleotide sequence shown in SEQ ID NO:27 or 28.

In a third aspect of the present invention, a kind of pharmaceutical composition is provided, it contains pharmaceutically acceptable carrier and immunomodulator of the present invention.

Preferably, described pharmaceutical composition contains one or more immunomodulators, for example (a) and (b) and (c) in 2-3 kind immunomodulator.

In another preference, described pharmaceutical composition, contain following two kinds of immunomodulators:

(a) anti-people CD11a monoclonal antibody, described anti-people CD11a monoclonal antibody has the sequence of heavy chain shown in SEQ ID NO:1 or 3, and/or the sequence of light chain shown in SEQ ID NO:2 or 4; With

(b) FLA3Ig fusion rotein, described fusion rotein have the Fc fragment of the human IgG1 shown in the 121-347 position among the CD2 land of the FLA3 shown in the 1-120 position among the SEQ ID NO:26 and the SEQ ID NO:26.

In a fourth aspect of the present invention, a kind of purposes of immunomodulator of the present invention is provided, it is used to prepare the medicine of treatment autoimmune disease and anti-immunologic rejection.

Description of drawings

Fig. 1 has shown several signalling channels of APC cell and T cell interaction.

Fig. 2 has shown the blocking-up approach of signal transduction path.

Fig. 3 is the structure collection of illustrative plates of pMG18 expression vector, and wherein pA is SV40 virus " poly A " tailing signal; HCMV pro is the promoter (containing enhancer sequence) of human cytomegalic inclusion disease virus; AmpR is an ampicillin resistance gene.

Fig. 4 has shown the molecular structure of CTLA4Ig.

Fig. 5 has shown the structure collection of illustrative plates of expression vector pMSG.

Fig. 6 has shown the molecular structure of the people FLA3 extracellular region-Ig fusion rotein of reorganization.

The specific embodiment

The inventor is through deeply and extensive studies has made up a para-immunity regulator, and they can treat autoimmune disease or be applied to clinical disease such as organ transplantation by immunosuppressive action.Particularly, this immunomodulator comprises (a) anti-people CD11a monoclonal antibody; (b) FLA3Ig fusion rotein; (c) CTLA4Ig fusion rotein.On this basis for this reason the present invention.

As used herein, term " autoimmune disease " is meant the rheumatoid arthritis that often runs into clinically, psoriasis, lupus erythematosus, multiple sclerosis etc.

Immunomodulator of the present invention is a polypeptides matter.Except immunomodulator was provided, the present invention also provided the dna molecular of coding immunomodulator.Nucleotide coding sequence of the present invention 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 fragments, and then connect and to obtain the very long fragment of sequence.

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 conventional method.

At present, can be fully obtain the DNA sequence of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This DNA sequence 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 and suitable promoter 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, as bacterial cell; Or eukaryotic cell such as low, as yeast cells; Or higher eucaryotic cells, as mammalian cell.Representative example has: escherichia coli; Fungal cell such as yeast; 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 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 conventional method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used culture medium can be selected from various conventional culture medium 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 promoter of selection with suitable method (as temperature transition or chemical induction), cell is cultivated a period of time again.

In the reconstitution cell culture technique that relates among the present invention, adaptable culture medium has many kinds.Wherein, Ham ' s101, MEM, RPMI-1640, commercial Zooblast culture medium such as DMEM all can use.In these culture medium, can add auxiliary elements such as suitable hormone, somatomedin, nucleic acid degradation product, trace element, to improve cell activity.If necessary, can add any material, as energy factor or the like.The control of pH, dissolved oxygen etc. is operated according to general procedure in the incubation.

The component of any adding ingredient beyond the minimal medium all can be optimized by Orthogonal Experiment and Design or method of least square.The equipment of cell culture can use and roll bottle, shake bottle, zooblast bioreactor etc. and carry out.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cell membrane.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 chroma-tography (HPLC) and other various liquid chromatography (LC) technology and these methods.

In preferred recombinant protein purification technology, because the purification of recombiant protein of the present invention has some common features.Because they all have the Fc fragment, therefore, when elementary purification, can adopt protein A filler to carry out affinity chromatograph and catch.The affinitive layer purification that carries out with protein A has outstanding advantages such as yield height, purification effect be good, but link coupled protein A albumen comes off from carrier easily, and easy inactivation in use can not clean with NaOH etc. after filler pollutes, and is difficult to life-time service.

The present invention also provides a kind of pharmaceutical composition for the treatment of autoimmune disease and anti-immunologic rejection, and it contains above-mentioned immunomodulator and pharmaceutically acceptable carrier.Usually, can these materials are formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can be with being changed to some extent by preparation Substance Properties and disease to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intraperitoneal, intravenous or topical.

Pharmaceutical composition of the present invention can be directly used in treatment autoimmune disease and anti-immunologic rejection.

Pharmaceutical composition of the present invention contains above-mentioned immunomodulator and the pharmaceutically acceptable carrier or the excipient of the present invention of safe and effective amount.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, 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 conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.Pharmaceutical composition such as injection, solution should be made under aseptic condition.The dosage of active component is the treatment effective dose, 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 immunomodulator with safe and effective amount is applied to mammal, 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.

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 normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

The structure of the anti-people CD11a of embodiment 1 recombination human source (change) monoclonal anti body expression vector

1.1 the antibody gene variable region of screening CD11a from antibody library

The building process of human antibody library is as follows:

1). extract peripheral blood from 1125 normal adults, everyone 5 milliliters, mix after the citric acid anticoagulant.

2). extract total RNA with Trizol from above-mentioned mixing peripheral blood, carry out reverse transcription, obtain cDNA with Oligo-dT16.

3). press document Marks et al.By-passing immunization:human antibodiesfrom V-gene libraries displayed on phage.J.Mol.Biol., 222,581-597 and Hoogenboom and Winter, By-passing immunisation:human antibodies fromsynthetic repertoires of germline VH gene segments rearranged in vitro.J.Mol.Biol., 227, the described method of 381-388, with the performing PCR that is combined into of each primer, obtain length respectively and be about the variable region of heavy chain of 330bp and the variable region of light chain amplified production about 310bp.PCR primer sets compound is that the final concentration of every primer is 20pmol/L.Amplification condition: pre-degeneration 94C 1min, major cycle 94C 30sec, 54C 30sec, 72C 30sec, 25 circulations, 72C 3min is extended in the back.PCR product 1.2% sepharose electrophoresis purification rear clone is to pCantab 5E carrier (Pharmacia product).

4). transform: with above-mentioned plasmid DNA of being cloned into Vector for Phage Display pCantabe 5E, to coli strain TG1 (Promega company) conversion of shocking by electricity.The electric shock cell preparation: TG1 cells frozen storing liquid 100ul is inoculated in 10 milliliters of LB fluid mediums that contain the 100ug/ml ampicillin, 37 degree overnight incubation.Get the above-mentioned overnight culture of 1ml, be inoculated in the above-mentioned same culture medium of 2L, 37 degree are cultivated and to be reached 0.4 to OD600 in 8 hours.With behind the 10% ice-cold glycerite washed cell 2 times cell is resuspended in 10ml 10% glycerol behind the centrifugal collection thalline of 5000RPM.Electric shock: above-mentioned cell 100ul joins in advance in the 100ul electric shock cup that is chilled to-20 degree, adds above-mentioned pCantab 5E carrier 2 little lifes, fully shocks by electricity behind the mixing.Electric shock condition: 2500 ohm of resistance, 10 kilovolts of voltages.Above-mentioned electric shock is carried out repeatedly continuously, until all carrier DNAs are used up.

After above-mentioned electric shock is finished, add LB fluid medium (not containing ampicillin) 1 milliliter immediately in electric shock cup, 37 degree are cultivated and are added ampicillin after 10 hours to 100ug/L, and 37 degree cultivate that 5000rpm collects thalline after 6 hours.The thalline of collecting is resuspended in the LB culture medium that contains 7% glycerol, and-70 ℃ of preservations are standby.

Antibody library to preparation, screen antibody according to the following steps:

1). the antibody library bacterial strain of recovery adds 14 milliliters of fresh LB culture medium for 1 milliliter, and 37 degree were cultivated 16 hours in 50 milliliters of triangular flasks.

2) the .12000rpm high speed centrifugation is 10 minutes, shifts in supernatant to one 50 milliliters of aseptic centrifuge tubes, preserves standby.Its titre should be 2 * 10 ¹¹More than.

3). the CD11a albumen (available from Shenzhen brilliant U.S. company) with purification is antigen, and the conventional method bag is by 25 ml cells culture bottles.

4). add in the cell bottle behind the bag quilt and be no less than 3 * 10 ¹⁰Phage particle, 37 degree incubations 1 hour.

5). outwell the liquid in the bottle, wash culture bottle 10 times with 10 milliliters of PBS that added 1%Tween-20.

6). add the TG1 cell of 1 milliliter of logarithmic (log) phase in culture bottle, 37 degree incubation concussions were cultivated 16 hours.

7). repeated for 2～6 steps, carry out 4 repetitive cycling altogether.

8). will on 1.5% agar plate that adds 0.1% ampicillin, cultivate to obtain monoclonal behind cell dilution to the 100000 cell/ml of above-mentioned acquisition.

9). 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.

10). 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 centrifuges, be preserved in after sealing 4 the degree standby.

11). get 10 of 96 orifice plates, add in every hole the conventional bag of CD11a (10ug/ml) 10 microlitres by after, add supernatant 10 microlitres of above-mentioned preservation respectively, 37 degree incubations after 1 hour with the PBS washing that contains 1%Tween-20 20 times.

12). add the goat-anti M13 monoclonal antibody of 1 microlitre HRP labelling, 37 degree incubations use the PBS of 1%Tween-20 to wash 10 times after 30 minutes.

13). add and contain PBS 200 microlitres of 0.025%DAB developer and the H2O2 of 1 microlitre 1%, 37 degree incubation colour developings were read 595 nanometers after 20 minutes on plate reading machine light absorption.

14). determine the hole that chromogenic reaction is strong according to the light absorption reading, the corresponding clone in these holes is the stronger antibody variable region clone of affinity.

15). by said process, filtered out 387 positives of positive colony of anti-people CD11a, determined wherein 5 the strongest clones of affinity according to reading, choosing wherein two the highest be used for follow-up research.These two clone's difference called after pCD11-1, pCD11-2.

16). sequencing analysis, the result is as follows:

Table 1

Aminoacid sequence	Length	SEQ ID NO：
			The pCD11-1 heavy chain	121Aa		1
The pCD11-1 light chain	108Aa						2
			The pCD11-2 heavy chain	120Aa		3
The pCD11-2 light chain	109Aa						4

1.2 the clone of the expression vector of antibody variable region coded sequence

1). above-mentioned 2 clones' bacterial strain is increased in 100ml LB culture medium, with the plasmid DNA extracting and purifying test kit plasmid DNA purification of Promega company.

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

3). separate endonuclease bamhi with after HindIII and the above-mentioned plasmid DNA of Bsi WI enzyme action on 1.5% agarose gel electrophoresis, get band about 320bp and carry out glue and reclaim, the gained fragment is variable region of light chain.At first variable region of heavy chain is inserted into the XbaI/NheI site of expression vector pMG18 (Fig. 3) then, reuse HindIII and Bsi WI are inserted into the HindIII/Bsi WI site of the pMG18 that has inserted variable region of heavy chain with antibody chain variable region, thereby have constituted the expression vector of the anti-CD11a antibody gene of humanization.

1.3CHO the transfection of cell and the screening of recombinant clone

1). the expression vector that has antibody gene of above-mentioned structure increases in 100 milliliters of LB culture medium in the escherichia coli DH5a inoculation, with the Ultrapure Plasmid DNAPurification Kit extracting and purifying plasmid DNA of Qiagen company.The plasmid DNA of above-mentioned purification is adopted the liposome method test kit transfection CHO cell of Invitrogen company, and operation is carried out with reference to the description of producer.

2). the Chinese hamster ovary celI of conversion carries out the extreme dilution at last and cultivates in the selection of selecting to carry out on the culture medium continuous 9 weeks on 96 orifice plates, carry out continuously 3 times, carry out monoclonalization.

3). the monoclonal cell of choosing ties up on the RPM1641 culture medium and cultivates, and supernatant is carried out Western Blotting experiment, judges expression intensity according to staining reaction, picks out and expresses strong clone as the candidate cell strain.

4). Purification of Monoclonal Antibodies: the purification of above-mentioned monoclonal antibody adopts the directly separation and purification from cells and supernatant of Protein A affinity column, and proves that with the SDA-PAGE electrophoresis products therefrom purity is greater than 90%.

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

Result:, filter out above-mentioned two kinds of 13 and 9 of candidate clones (seeing Table 2) that anti-people CD11a antibody gene expression intensity is higher by above operation.

1.4 the research of anti-CD11a antibody gene expression intensity in Chinese hamster ovary celI

The high expressed candidate clone (seeing Table 2) that above-mentioned screening is obtained is incubated at tissue culture's ware of 10cm, adopt the ELISA method to measure the expression of antibody: goat anti-human igg (Fc) bag quilt is in elisa plate, 4 ℃ are spent the night, 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, use H at last in 37 ℃ of effects 10 minutes ₂SO ₄Cessation reaction is surveyed A ₄₅₀Value.(ug/ml) is as shown in table 2 below for the expression of the above-mentioned candidate clone that records:

Table 2

As can be seen from the above table, the expression of 8C9 and 4D8 has very high expression.(18.2ug/ml) compare with the data of abroad having delivered (Kunkel, PNAS, 82:488,1995), expression has improved 17 times and 20.7 times.

Choose 8C9 and 4D8 as further object of study, the cell strain note is made CD11-1 and CD11-2, respectively corresponding pCD11-1 and two kinds of carrier molecule structures of pCD11-2.

Embodiment 2

The cell strain of recombinant human CTLA 4-Ig makes up and expresses

2.1.CTLA-4R the basic document of coded sequence

According to the document of having delivered [GenBank Accession Number L15006], the aminoacid sequence of CTLA-4R (223 aminoacid) is shown in SEQ ID NO:5, wherein the 37-161 position is the extracellular region domain, this fragment has completely and the bonded ability of B7, can be used as the CD28 antagonist.Note is made CTLAs among the present invention.

The cDNA sequence of CTLA-4R gene (5 ' → 3 ', 672b) shown in SEQ ID NO:6, wherein the 109-483 position is the coded sequence of ectodomain CTLAs.

In order in Chinese hamster ovary celI, to obtain high expressed,, utilize DNA/ protein analysis software DNAstar and GCG that the coded sequence that is fit to expressing cho cell is designed according to the segmental aminoacid sequence of above-mentioned CTLAs.

2.2. the CTAs gene of artificial design is synthetic

The CTLAs gene DNA sequence of artificial design is shown in SEQ IDNO:7, and wherein preceding 9 bases are the initiation codons of 3 protections base, a Nhe I restriction enzyme site and addings of interpolation.

Then, by the synthetic artificial total length CTLAs gene that designs of following program:

Utilization GCG software resolves into the fragment that is about 85b with above-mentioned sequence and complementary series thereof, amounts to 25 polynucleotide passages, wherein normal chain first fragment be:

5′-CAG gctagcATGgccatgcacgtggccca gcccgccgtggtgctggcctcctcccgcg-3′(SEQ ID NO：8)

Its front three base is the protection base, and the 4th～9 base is the restriction enzyme site (Nhe I) when inserting expression vector, and first ATG is the start codon that adds.This fragment also is the forward primer when synthesizing the total length fusion gene, and note is made CTLAs-M in this research.Design principle is the complementation district that 18～20 bases are arranged between the complementary adjacent product fragment.Reference literature Chrisostomos Prodromou and Laurenee H, the method for Pearl (1992) is carried out the synthetic of gene.The polynucleotide passage of design entrusts Shanghai to give birth to worker's biological engineering company limited on behalf of synthetic and carry out the PAGE purification.

Synthetic fragment well is dissolved in the tri-distilled water of sterilization according to the concentration of 2 micrograms/microlitre, carries out PCR in accordance with the following methods:

The composition of reaction system is:

Reaction condition is as follows:

Pre-degeneration: 94 degree 2 minutes

Major cycle: 94 degree 1 minute, 55 degree 1 minute, 72 degree 3 minutes.

Period: 20

Extend the back: 72 degree 5 minutes

The PCR process is carried out on PROGENE Genium thermal cycler.

Carry out PCR according to above-mentioned condition.After finishing product is identified on 0.8% agarose gel electrophoresis, found to have in the product band on unique molecular weight 350～400bp right side, reclaim test kit with the glue of Promega company and reclaim this fragment, operate and carry out according to the description of producer.Altogether about 2.5 micrograms of DNA.

Above-mentioned dna fragmentation is cloned on the pUC18 carrier according to conventional method, behind the transformed into escherichia coli DH5a cell at the dull and stereotyped enterprising row filter of IPTG/X-gal, get 18 white bacterial plaques and be inoculated in the liquid LB culture medium that contains ampicillin and increase, with plasmid extraction purification kit (Promega) extracting plasmid DNA and carried out selecting after enzyme action is identified the correct clone of 15 sizes and carried out determined dna sequence.According to sequencing result, 1 the right-on clone of sequence, i.e. CTLAs-M have been confirmed.

2.3. the pcr amplification of natural CTLAs gene, clone and order-checking

(1). primer design

According to the DNA sequence of aforementioned CTLAs gene, carry out design of primers with DNAstar software, the result is as follows:

Forward primer CTLAs-N:5 '-CAG GctagcATGgcaatgcacgtggcccagcc-3 ' (SEQ IDNO:9), wherein, CAG is protection base, the point of contact (Nhe I) when underscore partly is expression vector pMSG (Pharmacia company) clone.This fragment also is the forward primer when making up natural CTLAs/Fc full-length gene, and note is made CTLAs-N.

Downstream primer: 5 '-gtcagaatctgggcacggttc-3 ' (SEQ ID NO:10).

Above-mentioned primer is entrusted Shanghai living worker company to synthesize and is carried out being used for PCR behind the PAGE purification.

(2). the preparation of template DNA

With Trizol reagent extracted total RNA from people MT cell of GIBCO company, operating process is carried out according to the explanation of producer.Extractive total RNA reverse transcription test kit with INVITROGENE company after confirming integrity on 1% sepharose electrophoresis carries out reverse transcription according to producer's explanation, obtains cDNA.Reaction finishes, behind the reaction system DNA of Promega company extracting and purifying test kit purification as the template of PCR.

(3) .PCR amplification

Utilize above-mentioned primer and template DNA to carry out PCR, its reaction system is as follows:

The PCR process is carried out on PROGENE GENIUS thermal cycler, and cycling condition is as follows:

Pre-degeneration: 94 degree 2 minutes.

Major cycle: 94 degree 1 minute, 59 degree 1 minute, 72 degree 2 minutes, totally 20 circulations.

Extend the back: 72 degree 5 minutes.

(4) evaluation of PCR product and glue reclaim

Carry out PCR according to above-mentioned condition.After finishing product is identified on 0.8% agarose gel electrophoresis that finding has the single band of a molecular weight about 375bp in the product, consistent with the 375bp size of expection.Glue recovery test kit with Promega company reclaims this fragment, and operation is carried out according to the description of producer.Altogether about 3.2 micrograms of DNA.

(5). the clone of natural CTLAs gene, evaluation and order-checking

Above-mentioned dna fragmentation is cloned on the pUC18 carrier according to conventional method, behind the transformed into escherichia coli DH5a cell at the dull and stereotyped enterprising row filter of IPTG/X-gal, get 5 white bacterial plaques and be inoculated in the liquid LB culture medium that contains ampicillin and increase, with plasmid extraction purification kit (Promega) extracting plasmid DNA and carried out selecting after enzyme action is identified the correct clone of 5 sizes and carried out determined dna sequence.According to sequencing result, 1 the right-on clone of sequence, i.e. CTLAs-N have been confirmed.

2.4. the design of joint sequence is with synthetic

When two genes or genetic fragment being reassembled into a coding region and constitute the coded sequence of fusion rotein, often need to add a catenation sequence between two target fragment, the selection of this catenation sequence has very strong technical.We are according to for many years experience, and the principle of following when designing this fragment is: 1).Use its encoded polypeptides can not have the ability that forms secondary structure; 2).Its encoded polypeptides can not be by the glycosylation signal; 3).Too much hydrophobic amino acid can not be arranged.In view of the above, the aminoacid sequence of design is (Ala ₃Ser ₂) ₅Its coded DNA sequence is:

5′-ACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCA-3’(SEQ ID NO：11)

According to above sequence, the needs when considering by PCR, these segmental head and the tail with CTLAs coding region and Fc fragment assembly respectively add one respectively with CTLAs coding region 3 '-end and Fc coding region 5 '-hold complementary sequence, each about 20 base length.Therefore, the Linker-M total order of synthetic CTLAs is classified as:

5’-taaccatttccatccataattaaACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAacatgcccaccgtgcccagcac-3’(SEQ ID NO：12)

The Linker-N total order of natural CTLAs is classified as:

5’-cttattttattcccatcaattgaACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAacatgcccaccgtgcccagcac-3’(SEQ ID NO：13)

Above-mentioned two kinds of Linker are synthetic and carry out purification by the living worker in Shanghai company band.

2.5. the acquisition of human IgG1 Fc fragment coding sequence

The sequence of natural total length IgG1 coding region (1416bp, 471 aminoacid+termination codon, Genbank accessionnumber:BC024289) is shown in SEQ ID NO:14, and wherein, the 1-426 position is the VDJ district; The 427-1416 position is human IgG1 Heng Qu, comprises CH1+ hinge region+CH2+CH3; The 718-1416 position is the coding region in hinge region in human IgG1's constant region+CH2+CH3 district.

Obtain human IgG1's coded sequence by following program reverse transcription, and be cloned on the carrier pGEM-T, gained carrier note is pIGG-1.Specific operation process: 100 milliliters of human peripherals, separate the acquisition leukocyte with lymphocyte separation medium (Sigma product), mRNA extracting and purifying test kit extracting mRNA with Qiagen, RT-PCR test kit with GIBCO company after identifying its integrity on 1.2% agarose gel electrophoresis carries out reverse transcription and PCR, and primer is:

Forward primer: (enzyme-added point of contact) atggaactggggctccgctg (SEQ ID NO:15)

Reverse primer: (enzyme-added point of contact) tcatttacccggagacag (SEQ ID NO:16)

(because utilized the A/T clone, this two primer does not need to add the enzyme action point)

PCR condition: pre-degeneration 94C 2 minutes; Major cycle: 94C 1 minute, 56C 45 seconds, 72C 90 seconds, 30 circulations; Extend the back: 72C 4 minutes.

Amplification finishes, on 1% agarose gel electrophoresis, identify the DNA that separates amplification, use the DNA glue recovery test kit of Qiagen company to reclaim the fragment rear clone of 14Kb to pGEM-T carrier (Promega company), after the sequence verification sequence, obtain a right-on clone, note is made pIGG-1.

Be used for the human IgG1 Fc that soluble fragments merges (the segmental aminoacid sequence of hinge region+CH2+CH3) shown in SEQ ID NO:17, totally 232 aminoacid.

The human IgG1 Fc coded sequence of the suitable expressing cho cell of artificial design is shown in SEQ ID NO:18.

The natural coded sequence of human IgG1 Fc is total to 699bp shown in SEQ ID NO:19.In order to obtain the segmental coded sequence of IgG1 Fc, design and synthetic following primer:

3 '-end primers F c-3:5 '-gca CtcgagTtttacccggagacagggagag-3 ' (SEQ ID NO:20), wherein, gca is the protection base, underscore partly is the point of contact (Xho I) when above-mentioned expression vector is cloned.This primer also is the downstream primer that obtains total length CTLAs/Fc fusion gene.

5 '-end primers F c-5:5 '-gagcccaaatcttgtgacaaaac-3 ' (SEQ ID NO:21)

Is that template is carried out PCR with above-mentioned primer with the plasmid pIGG-1 that contains human IgG1's complete sequence, and having obtained length is the product of 708 bases.Electrophoresis evaluation, clone and sequence measurement are the same.The result obtains pIGG1Fc.

2.6. the acquisition of total length CTLAs-Fc fusion gene

(1) acquisition of total length CTLAs-N/Linker/Fc fusion gene

Its aminoacid sequence of the joint sequence that designs in the present embodiment (linker) is:

(Ala ₃Ser ₂) ₅(SEQ ID NO：35)；

One of its DNA sequence is:

5’-ACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCA-3’(SEQ ID NO：36)

This sequence behind the Fc fragment assembly among PCR method and the aforementioned pIGG1Fc, forms the Linker-N/Fc fragment after entrusting Shanghai living worker company synthetic.

DNA with aforesaid Linker-N/Fc and CTLAs-N is a template, is that primer carries out PCR with CTLAs-N and IgG1Fc-3 ' (SEQ ID NO:20), and reaction condition is:

Reaction system is as follows:

The PCR process is carried out on PROGENE GENIUS thermal cycler, and cycling condition is as follows:

Pre-degeneration: 94 degree 2 minutes.

Major cycle: 94 degree 1 minute, 60 degree 1 minute, 72 degree 3 minutes, totally 20 circulations.

Extend the back: 72 degree 5 minutes.

Carry out PCR according to above-mentioned condition.After finishing product is identified on 0.8% agarose gel electrophoresis that finding has the single band of a molecular weight about 1500bp in the product, consistent with the 1540bp size of expection.Glue recovery test kit with Promega company reclaims this fragment, and operation is carried out according to the description of producer.Altogether about 3 micrograms of DNA.

Above-mentioned dna fragmentation is cloned on the commercial pUC18 carrier according to conventional method, behind the transformed into escherichia coli DH5a cell at the dull and stereotyped enterprising row filter of IPTG/X-gal, get 7 white bacterial plaques and be inoculated in the liquid LB culture medium that contains ampicillin and increase, with plasmid extraction purification kit (Promega) extracting plasmid DNA and carried out selecting after enzyme action is identified the correct clone of 3 sizes and carried out determined dna sequence.According to sequencing result, confirmed 1 right-on clone of sequence, note is made CTLAs-N/Linker-N/Fc.

(2) acquisition of total length CTLAs-M/Linker/Fc fusion gene

Its aminoacid sequence of the joint sequence that designs in the present embodiment (linker) is:

(Ala ₃Ser ₂) ₅(SEQ ID NO：35)；

One of its DNA sequence is:

5’-

cttattttattcccatcaattgaACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAACCACAACCTCGTCAacatgcccaccgtgcccagcac-3’(SEQ IDNO：37)

This sequence behind the Fc fragment assembly among PCR method and the aforementioned pIGG1Fc, forms the Linker-M/Fc fragment after entrusting Shanghai living worker company synthetic.

DNA with aforesaid Linker-M/Fc and CTLAs-M (SEQ ID NO:7) is a template, CTLAs-M and IgG1Fc-3 ' SEQ ID NO:20) for primer carries out PCR, reaction system is:

The PCR process is carried out on PROGENE GENIUS thermal cycler, and cycling condition is as follows:

Pre-degeneration: 94 degree 2 minutes.

Major cycle: 94 degree 1 minute, 60 degree 1 minute, 72 degree 4 minutes, totally 25 circulations.

Extend the back: 72 degree 5 minutes.

Carry out PCR according to above-mentioned condition.After finishing product is identified on 0.8% agarose gel electrophoresis that finding has the single band of a molecular weight about 1500bp in the product, consistent with the 1540bp size of expection.Glue recovery test kit with Promega company reclaims this fragment, and operation is carried out according to the description of producer.Altogether about 2.7 micrograms of DNA.

Above-mentioned dna fragmentation is cloned on the pUC18 carrier according to conventional method, behind the transformed into escherichia coli DH5a cell at the dull and stereotyped enterprising row filter of IPTG/X-gal, get 9 white bacterial plaques and be inoculated in the liquid LB culture medium that contains ampicillin and increase, with plasmid extraction purification kit (Promega) extracting plasmid DNA and carried out selecting after enzyme action is identified the correct clone of 6 sizes and carried out determined dna sequence.According to sequencing result, confirmed 1 right-on clone of sequence, note is made CTLAs-M/Linker-N/Fc.

(3) result

By above operation, obtained CTLAs gene and the Linker and the segmental fusion gene CTLAs-M/Linker/Fc of Fc of segmental fusion gene CTLAs-N/Linker/Fc of natural CTLAs gene and Linker and Fc and artificial design.The difference of these two kinds of fusion rotein is that the former CTLAs coding region is natural, and the latter's respective regions is artificial design.Fig. 4 has shown the molecular structure of total length fusion protein expression products.

2.7. the acquisition of total length CTLAs-Fc fusion gene

(1) preparation total length CTLAs-Fc fusion gene

For the detection tabs fragment to improving the effect of this fusion rotein affinity, in following operation, obtained the direct fusion fragment of Fc fragment and CTLAs-N and CTLAs-M, do not have the Linker fragment in this kind structure.If to these two kinds of fusion genes in Chinese hamster ovary celI expression product and the affinity of the expression product of aforementioned CTLAs-N/Linker/Fc and CTLAs-M/Linker/Fc compare, just can confirm of the influence of Linker fragment to affinity.The building process of these two kinds of structures is as follows:

According to the DNA sequences encoding of Fc and CTLA-M, CTLAs-N, design following two kinds of primers:

Primer M：5′- taaccatttccatccataattaatctgaggttgtctggaacccag-3′(SEQ ID NO：22)

Primer N：5′- cttattttattcccatcaattgaacatgcccaccgtgcccagcac-3′(SEQ ID NO：23)

Is template with aforesaid CTLAs-N with the plasmid DNA that has total length IgG1 gene, and CTLAs-N, IgG1Fc-3 ' and Primer N are that primer carries out PCR, carries out glue and reclaim after the acquisition molecular weight is about the single band of 1049bp on the agarose gel electrophoresis.The DNA that obtains is cloned on the pUC18 carrier back transformed into escherichia coli and is carried out blue white macula screening according to conventional method, identifies and a right-on clone of sequence has been confirmed in order-checking that by enzyme action note is made CTLAs-N/Fc.

Repeat said procedure, difference is to replace Primer N with Primer M, replaces CTLAs-N with CTLAs-M.The result has obtained the fusion gene of CTLAs-M and Fc, and note is made CTLAs-M/Fc.

(2) clone of gene and sequencing

At first the natural CTLA of above-mentioned amplification, the CTLA fragment and the pUC18 of artificial design are carried out enzyme action by following operation,

Reaction system is:

Above-mentioned system is spent the night in 37 degree reactions, carry out isolation identification on 0.8% sepharose electrophoresis, with the correct fragment of glue recovery test kit recovery size of Promega company, natural CTLA obtains about 3 micrograms of DNA, about 4 micrograms of CTLA of artificial design, pUC18 obtains about 3.3 micrograms of DNA.

Getting above-mentioned pUC18 DNA is connected according to following program with CTLA DNA:

Reaction system:

Above-mentioned system, and is carried out blue white macula and is screened with the above-mentioned recombinant DNA transformed into escherichia coli DH5a bacterial strain that connects according to conventional method after 16 degree reactions are spent the night on the IPTG/X-Gal plating medium.6 of white clones that get 28 of the white maculas of the CTLAs-M of artificial design, natural CTLAs-N are inoculated in the liquid LB culture medium that contains ampicillin, utilize the plasmid extraction purification kit extraction plasmid of Promega company to carry out the enzyme action evaluation after the 37 degree overnight incubation, finding wherein manually has 16 clones to have correct size among the clone of CTLAs-M, has 4 sizes to conform to expection among the clone of natural CTLAs.These clones are as the candidate clone of order-checking.

Select 10 from 16 clones of above-mentioned artificial design CTLAs-M, choose 2 and carry out dna sequencing from natural CTLAs-N clone, the examining order trust is gone up sea base Kanggong department and is carried out.Sequencing result shows, has obtained correct CTLAs-M/Fc and the CTLAs-N/Fc clone of sequence.

2.8.CTLA expression carrier clone

(1). the preparation of fusion gene DNA and carrier DNA

Aforementioned work has made up CTLAs-N/Linker/Fc, CTLAs-M/Linker/Fc, four kinds of antigen-4 fusion protein genes of CTLAs-M/Fc, CTLAs-N/Fc.The correct clone of above-mentioned affirmation is inoculated in the 20 milliliters of liquid LB culture medium that contain ampicillin, and 37 degree overnight incubation with the Midi plasmid DNA extracting and purifying test kit extracting plasmid DNA of Promega company, are each got about 30 micrograms of plasmid DNA.

Carry out enzyme action and carry out glue and reclaim according to following condition, obtain the dna fragmentation of each fusion gene and ready pMSG carrier (Pharmacia company) (Fig. 5).

Reaction system:

Above-mentioned system is spent the night in 37 degree reactions, on 0.8% sepharose electrophoresis, carry out isolation identification, glue with Promega company reclaims the fragment that test kit recovery size is about 1500b, CTLAs-N/Linker/Fc, CTLAs-M/Linker/Fc, CTLAs-M/Fc, CTLAs-N/Fc respectively obtain about 1.2 micrograms, 2.7 micrograms, 1.6 micrograms, 1.8 micrograms of DNA, and pMSG obtains about 3.4 micrograms.

(2). with the fusion gene fragment pMSG expression vector of recombinating:

Be connected to CTLAs-N/Linker/Fc, CTLAs-M/Linker/Fc, CTLAs-M/Fc, CTLAs-N/Fc gene on the pMSG carrier and transform and blue white macula screening by following reaction:

Reaction system:

Above-mentioned reaction system is reacted spend the night (more than 10 hours) at 16 degree.

(3). the conversion of antibacterial, clone's evaluation and order-checking:

Above-mentioned system, and is carried out blue white macula and is screened with the above-mentioned recombinant DNA transformed into escherichia coli DH5a bacterial strain that connects according to conventional method after 16 degree reactions are spent the night on the IPTG/X-Gal plating medium.Respectively getting 12 of white maculas is inoculated in LB fluid medium 37 degree and cultivates the back and extract plasmids and carry out enzyme action and identify that every kind of fusion gene obtains 4 segmental clones of insertion with correct size at least, as candidate clone.

Carry out dna sequencing with two in the above-mentioned candidate clone, examining order is entrusted and is gone up sea base Kanggong department and carry out.According to sequencing result, confirm that respectively CTLAs-N/Linker/Fc, CTLAs-M/Linker/Fc, CTLAs-M/Fc, CTLAs-N/Fc have the in full accord of 3,2,1 and 1 clones' sequence and design respectively.

The correct clone who obtains in the above-mentioned research is referred to as respectively: pMSG[CTLAs-N/Linker/Fc], pMSG[CTLAs-M/Linker/Fc], pMSG[CTLAs-M/Fc], pMSG[CTLAs-N/Fc].(the N representative is natural, M representative sudden change).

The protein-bonded cell strain of embodiment 3 recombined human CD2 makes up

3.1.FLA3 the analysis of extracellular region encoding gene and design

Shown in SEQ ID NO:24, wherein the 10-762 position is the full length protein coding region to the natural DNA sequence of FLA3 (being also referred to as CD58) (GenBank Accession NumberNM 001779), and the 10-369 position is the coding region of CD2 calmodulin binding domain CaM.Shown in the corresponding amino acid sequence SEQ ID NO:25, total length is 250 aminoacid, and the 1-120 position is the CD2 calmodulin binding domain CaM.

In view of the above, can design primer, utilize PCR method that this district is increased out, and constitute fusion protein F LA3Ig with human IgG1 Fc fragment.Its aminoacid sequence is shown in SEQ ID NO:26, and wherein the 1-120 position is the CD2 calmodulin binding domain CaM, and 121-347 is an IgG1 Fc fragment.

With the reorganization of the Fc coding region of natural CD2 land and IgG1, the dna encoding sequence of formed fusion gene shown in SEQ ID NO:27, the coded sequence of CD2 land, 1-360 position wherein.

As aforementioned, in order to obtain the high expressed in Chinese hamster ovary celI,, designed the FLA3-Ig antigen-4 fusion protein gene of suitable expressing cho cell according to the preferences of mammalian cell to codon, shown in SEQ IDNO:28, the coded sequence of CD2 land, 1-360 position wherein.

The expression product of the fusion gene of above-mentioned structure is FLA3-Ig, and the structure of its maturation protein as shown in Figure 6.

3.2FLA3 the acquisition of extracellular region gene

According to the sequence of the cDNA sequence of aforesaid FLA3 extracellular region and artificial design, it is synthetic to carry out the amplification and the total length of respective area segment DNA with PCR method respectively, obtains the natural encode fragment in this district and the encode fragment of artificial design respectively.Operate as follows:

The structure of natural encode fragment recombinant expression carrier

The acquisition of the natural encode fragment of FLA3

According to the sequence of its cDNA, synthetic following primer:

Forward primer: 5 '-ATGGTTGCTGGGAGCGACGC-3 ' (SEQ ID NO:29)

Reverse primer: 5 '-tttacccggagacagggagag-3 ' (SEQ ID NO:30)

Synthetic primer dissolves according to the ratio that 1.0OD is dissolved in 100 microlitre sterilized water, uses after diluting 10 times.The PCR condition is:

Template DNA (100ng/ul) 1ul, 10mM dNTPs 1.5ul, 2ul 10X PCR, 1.5ul 25mM MgCl ₂, each 1ul of primer adds water to the final volume of 20ul.Cycling condition: pre-degeneration 94C 2 minutes, major cycle is 94C30 second, 56C30 second, 72C 1 minute carries out extending after 3 minutes after 30 circulations.

PCR finishes, and analyzes the PCR product on 1.2% agarose gel electrophoresis, and finding has an only band at 350～400bp near the 350bp place.This band size conforms to theoretical size, is object tape.Reclaim test kit with glue and this band is carried out DNA reclaim, conventional method is cloned on the T-carrier (available from Promega company), carries out sequence verification after selecting positive colony.The in full accord of two sequences and expection arranged in the clone of 5 order-checkings, and getting wherein, pFLA3-N preserves standby.

3.3 total length FLA3Ig antigen-4 fusion protein gene is synthetic

The upstream forward primer: 5 '-gctagcATGGTTGCTGGGAGCGACGC-3 ' (SEQ IDNO:31) (containing restriction enzyme site Nhe I)

The splicing forward primer:

5′-GAAGTTCTTTCTTTATGTCGACAAAACTCACACATGCCCA-3′(SEQ IDNO：32)

The splicing reverse primer:

5′-TGGGCATGTGTGAGTTTTGTCGACATAAAGAAAGAACTTC-3′(SEQ IDNO：33)

The downstream reverse primer: 5 '-TTTACCCGGAGACAGGGAGAGGCctcgag-3 ' (SEQ IDNO:34) (containing restriction enzyme site XhoI)

With above-mentioned primer and pFLA3-N and pIGG-1 is template, with above-mentioned 4 kinds of primers PCR that overlaps, the synthetic total length FLA3-N/Ig gene of amplification.Obtain the full-length gene rear clone to pGEM-T carrier (Promega company product), get a right-on clone of sequence after the sequence verification, preserve standby.This clone's note is made pFLA3Ig-N.

3.4.FLA3Ig artificial gene is complete synthesis

Artificial gene sequence according to design designs 12 fragments, the about 85bp of each length.Add restriction enzyme site in the both sides of complete sequence during the design primer.Entrust Shanghai to give birth to the synthetic back of worker company obtains the 1041bp of this genetic fragment with PCR method total length amplified production.Then, use it is identified, clones and checks order, finally find 1 clone to have right-on sequence, preserve standby.This clone's note is made pFLA3Ig-M.

3.5. the protein-bonded cell strain of recombined human CD2 makes up

With make up the same of CTLA4Ig expression vector among the embodiment 2, the structural gene of above-mentioned FLA3Ig-N and FLA3Ig-M is inserted on the pMSG expression vector (Fig. 5), after screening obtains positive colony, carry out dna sequencing to verify the correctness of gained recon.

The acquisition of embodiment 4 recombinaant CHO cells

1). the extraction and purification of plasmid DNA

Get the coli strain that has recombiant plasmid for preparing among the embodiment 1-3, be inoculated in 500ml respectively and added the 2xYT fluid medium of ampicillin, the Ultrapure Plasmid Purification Kit extracting plasmid DNA of using Qiagen company after 16 hours is cultivated in 37 degree 260rpm concussions, and extractive process is carried out according to the description that producer provides.

2) transfection of .CHO cell and screening

Adopt liposome method that Chinese hamster ovary celI is carried out transfection, the transfection reagent box is got plasmid DNA 100 micrograms of above-mentioned purification respectively and as sample Chinese hamster ovary celI is carried out transfection available from Invitrogene company during transfection, and the transfection operation sequence is carried out according to the description of producer.

Chinese hamster ovary celI after the transfection is through continuous 3 months methotrexate (MTX) screening, and its concentration is from 0.05uM to 10uM, and per two weeks increase a concentration, and the consumption of each MTX is about previous 2 times, concrete visual cell growing state and deciding.Cell culture carries out according to routine, and culture medium is: 15% hyclone (Gibco)+RPM1640/DEME.In 37 degree 5%CO ₂Cultivate in the incubator.Carry out monoclonalization according to routine extreme dilution method then.Then, detect the expression of respectively cloning target recombinant protein respectively, and every kind of fusion gene is selected the highest clone of several expressions in view of the above, confirm that one of them expression is high and stable clone is used for further research with the ELISA method.

The result

1.CTLA4Ig fusion rotein: obtain pMSG[CTLAs-N/Linker-N/Fc altogether] clone 110, pMSG[CTLAs-M/Linker-N/Fc] clone 87, pMSG[CTLAs-M/Fc] clone 93, pMSG[CTLAs-N/Fc] clone 112.Obtain 5 of high-expression clones respectively, 1,2,2.

2. anti-CD11a monoclonal antibody: obtain 1850 clones, wherein CD11-1960, CD11-2890.The former obtains 2 high-expression clones, and the latter obtains 5 high-expression clones.

3.FLA3Ig：

Obtain 428 high-expression clones altogether, wherein FLA3Ig-N is 4,2 of FLA3Ig-M.

3) research of fusion gene expression

With the ELISA method above-mentioned part clone's the expression intensity of CTLAs-Fc fusion gene in Chinese hamster ovary celI studied, concrete data are as follows:

Table 3

Gene type	PMSG [CTLAs-N/Linker/Fc]	PMSG [CTLAs-M/Linker/Fc]	pMSG [CTLAs-N/Fc]	PMSG [CTLAs-M/Fc]
					Tested clone's quantity	47	44	38	48
Average expression (mg/litre supernatant)	0.37±0.21	2.68±0.28	0.39±0.34	2.89±0.52

Above data declaration, the expression intensity of CTLAs-M all have obvious enhanced expression in the fusion structure of Linker Linker being arranged and do not have.This explanation, the artificial design of gene has obviously improved its expression, and the amplitude of raising is up to 14.6 times.The expression that the CTLAs-M gene of the artificial design of this explanation has been significantly improved in Chinese hamster ovary celI has important economic value to this proteinic large-scale production.

The purification of embodiment 5 recombiant proteins

5.1 the purification of anti-people CD11a monoclonal antibody

A kind of purifying process flow process of anti-people CD11a monoclonal antibody can be: protein A post → reversed-phase column → molecular sieve.In this technology, because recombinant monoclonal antibody is secretion type expression, so culture supernatant is through simply centrifugal, can directly carry out affinity chromatograph after removing the cell relic.During with protein A affinity chromatograph, a kind of in can making with the following method:

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.In the Tris-H2SO4 of 100mM pH8.2 buffer after the rinsing 4 times, with carrying out the ammonium sulfate fractional precipitation after the same buffer solution elution that contains 250mM sodium chloride, initial concentration is 35% saturation, stops concentration 63% saturation then.4 degree are handled high speed centrifugation after 12 hours, get precipitation, be dissolved in again in the buffer of above-mentioned not sodium chloride-containing, and concentrate 10 times after same buffer dialysed overnight, and are stand-by.

Above-mentioned sample can be through SDS-PAGE or HPLC purity checking, ACD method or Follin phenol standard measure protein content.Through the recombiant protein of above-mentioned purification, its purity can reach more than 90%, and has notable biological activity.

Second method is that protein A is dressed up pillar, with manual or go up automatically on the sample equipment behind the sample, after the buffer washing limbs of process 100mM Tris-HCl pH8.5 are long-pending,, can obtain the monoclonal antibody of catching through protein A with the eluent eluting that contains 100～400mM sodium chloride.By eluting, the gained sample can be again be further purified through oppositely chromatography and molecular sieve eluting peak at 233～260mMNaCl place, can obtain purity and reach recombiant protein more than 93%.But this method is operated automatization, and connects online monitoring equipment, can understand operating conditions at any time.

Monoclonal antibody also can directly use the ammonium sulfate step-by-step precipitation method to carry out purification without protein A affinity chromatograph.Its yield can reach 12%, and purity can reach more than 90%.

5.2 the purification of recombinant human CTLA 4 Ig fusion rotein

A kind of purifying process flow process of recombinant human CTLA 4 Ig fusion rotein can be: protein A post → molecular sieve → ion exchange column → hydrophobic chromatography.

The same with the CD11a monoclonal antibody, CTLA4Ig also has same Fc fragment, and therefore also available protein A carries out affinity chromatograph.The condition of affinity chromatograph and CD11a basically identical just need reduce the consumption of NaCl when eluting, the consumption during gradient elution drops to 190～210mM, can use 210mM during eluting in batches.

In the CTLA4Ig purifying process, the recombiant protein that affinity chromatograph obtains need be removed part macromole and small molecular weight impurity through the G75 molecular sieve, exchange buffering liquid is to the 100mM phosphate buffer of pH7.5, go up on the Q-Sepharose post the 4 volumes washing back 50mM Tris-HCl eluant solution that contains 250mM NH4SO4 then.Eluent can directly be gone up the Octyle-O post and carry out hydrophobic chromatography, can preserve standby behind the 50mM Tris pH7.5 eluting.

The sample purity that this method obtains can reach more than 95%, and total yield is a kind of more satisfactory purification process more than 75%.This method is amplified easily, suitable industrial-scale production.

5.3 the purification of recombined human FLA3Ig fusion rotein

A kind of purifying process flow process of recombined human FLA3Ig fusion rotein can be: protein A post → ion exchange column → hydrophobic chromatography → molecular sieve.

The same protein A affinity chromatograph that all comprised of the purification of FLA3Ig fusion rotein with the purge process of above-mentioned CTLA4Ig, and it is consistent with elution requirement to go up sample.Eluate is carried out purification once more through the QA-post, and eluant is the Tris-HCl buffer that contains 250mM NH2SO4, and eluent is directly gone up the hydrophobic chromatography post, and eluent is a 50mM Tris-HCl buffer.After eluent further separated through Sephadex G200 SF, its purity can reach 98%, and total yield is about 11.7%.

This method has easy and simple to handle, and the characteristics of amplifying are carried out plant-scale production easily easily.

The experimental verification of embodiment 6 immunologic functions downward modulation

6.1. anti-people CD11a monoclonal antibody physiologically active and affinity research

(1) physiologically active: mixed leukocyte culture test

To the monoclonal antibody that cell strain produced that embodiment 1 obtains, carried out the biologic activity detection.Concrete grammar and result are as follows:

Conventional separation of human peripheral blood lymphocyte, adjusting cell density is 2 * 10 ⁶Cells/ml mixes the back with 2 * 10 with two human PBMCs (the two-way MLR of allogeneic (mixed lymphocyte responses)) equal-volume ⁵Cells/ hole inoculation " U " type 96 well culture plates, the purified fusion protein of adding different volumes is provided with equal-volume corresponding empty carrier transfection supernatant or culture medium and compares, every group 3 multiple hole, 37 ℃, 5%CO ₂Cultivate 5d, 16h adds before stopping cultivating ³H-TdR (final concentration 5 μ Ci/ml), collecting cell are dried on 0.45 μ m microporous filter membrane, and liquid scintillation counter is surveyed the DPM value.Statistical procedures result represents that with x ± s the MicrosoftExcel statistical procedure carries out mean difference significance analysis.

In the MLR reaction system, add 1ul, 5ul and 10ul Protein A purification anti-CD11a monoclonal anti body expression vector or empty carrier transfection CHO cell supernatant respectively, the result shows, express to such an extent that the monoclonal anti physical ability significantly suppresses human peripheral MLR even add 1ul ProteinA purification, suppression ratio 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 factor analysis of variance, its difference reaches utmost point significant level, the results are shown in following table 4.Equally, we also once directly carried 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 stimulation and the proteic inhibitory action of micro-monoclonal antibody of heterologous protein in the purifying cells transfection supernatant not.

The influence that table 4 purification CD11a monoclonal antibody is bred people MLR medium-sized lymphocyte (DPM, n=3)

The MLR experiment shows that this purified fusion protein has the activity that suppresses immune response.Above result shows that embodiment 1 successfully clones and expressed the anti-CD11a monoclonal antibody that biologic activity is arranged in Chinese hamster ovary celI.

The above results shows that also CD11-2 has the inhibitory action stronger than CD11-1.

(2) affinity is identified

Affinity mensuration employing Scatchard analytic process (Munson et al, 1980, Anal.BioChem. 107:220) carries out.

The result shows that the affinity of CD11-1 and two kinds of monoclonal antibodies of CD11-2 reaches 3.2 * 10 respectively ^-9With 8 * 10 ^-6The above results shows, the affinity of CD11-1 and CD11-2 two strain monoclonal antibodies has reached the level of 1nM and 0.1nM respectively, CD11-1 has reached the level of having delivered abroad, and CD11-2 has then improved an order of magnitude than the best result of having delivered abroad.

6.2 the bioactivity research of recombinant human CTLA 4 Ig fusion rotein

(1) CTLAs suppresses the lymphopoiesis Research on ability

With above-mentioned pMSG[CTLAs-N/Linker/Fc], pMSG[CTLAs-M/Linker/Fc], pMSG[CTLAs-M/Fc], pMSG[CTLAs-N/Fc] expression cell line cultivate the back and carry out affinity chromatograph with Protein A-Sepharose, acquisition purity reaches the fusion rotein more than 90%.

Conventional separation of human peripheral blood lymphocyte, adjusting cell density is 2 * 10 ⁶Cells/ml mixes the back with 2 * 10 with two human PBMCs (the two-way MLR of allogeneic) equal-volume ⁵Cells/ hole inoculation " U " type 96 well culture plates add not commensurability purified fusion protein, equal-volume corresponding empty carrier transfection supernatant or culture medium are set compare, every group 3 multiple hole, 37 ℃, 5%CO ₂Cultivate 5d, 16h adds before stopping cultivating ³H-TdR (final concentration 5 μ Ci/ml), collecting cell are dried on 0.45 μ m microporous filter membrane, and liquid scintillation counter is surveyed the DPM value.The result represents that with x ± s Microsoft Excel statistical procedure carries out mean difference significance analysis.

In the MLR reaction system, CTLAs-N/Linker/Fc, the CTLAs-M/Linker/Fc, CTLAs-M/Fc, CTLAs-N/Fc or the empty carrier transfection CHO cell supernatant that add 1 microgram, 5 micrograms and 10 microgram Protein A purification purification respectively, the result shows, even add the fusion rotein of 1 microgram Protein A purification, also can significantly suppress human peripheral MLR, suppression ratio is 60%～70%; And add the empty carrier transfection CHO cell supernatant of same volume, and then there is not this effect, through one factor analysis of variance, the result has statistical significance (seeing Table 5).Equally, also once directly carried out the MLR experiment with the supernatant of unpurified transfectional cell, and failed to observe significant inhibitory effect, this may cancel out each other relevant with the stimulation of heterologous protein in the purifying cells transfection supernatant not and the inhibitory action of micro-fusion rotein.

The influence that table 5 purification CTLA-4/Ig breeds people MLR medium-sized lymphocyte (DPM, n=3)

*：P＜0.01 vs control；△：P＜0.01 vs 0

From above data as can be seen, the fusion rotein that adds Linker has improved about 1.5 times than the fusion rotein that does not have Linker to the inhibitory action of human peripheral MLR.This explanation, the adding of Linker can obviously improve fusion rotein to lymphocytic inhibition ability.

6.3 the bioactivity research of recombined human FLA3Ig fusion rotein

(1) the Jurkat cell is in conjunction with test

The Jurkat cell surface has CD2, with this kind cell of cultivating, is prepared into 5 * 10 with after the RPMI washing that contains 10% hyclone three times ⁶The suspension of cell/ml adds 1ml cell suspension in one 6 each hole of porocyte plate.The FLA3Ig that adds 10 microgram purification in 3 holes respectively gets involved TNFRIg (negative control) respectively in 3 holes.Behind the 0C incubation 4 hours, wash 2 times gently with the RPMI culture medium after, add goat anti-human igg's monoclonal antibody of HRP labelling, add DAB developer and H2O2,37 degree incubations 10 minutes after the RPMI washing 4 times.The situation that observation of cell color reaction and cell mutually combine under 40X and 100X phase contrast microscope.

Found that negative control does not have chromogenic reaction, and the cell in the FLA3Ig hole is brown chromogenic reaction.Microscopic examination shows that the Jurkat cell has tangible agglutination phenomenon.This phenomenon is that two arms of part molecule combine a cell respectively, thereby have caused the coagulation of cell because FLA3Ig is a bivalent molecule.The situation of this kind reaction and agglutinin coagulation erythrocyte is quite similar.

(2) FACS measures

In the Eppendorf pipe, add 1 * 10 ⁵The Jurkat cell, the 1000RPM centrifugal collecting cell, being resuspended to 100ul concentration is in the LFA3Ig solution of 10ug/ml, the buffer composition is: the PBS that contains 0.5%BSA, pH7.2. ice bath is 30 minutes after mixing fully, wash goat anti-human igg's monoclonal antibody ice bath of the FITC labelling that dilutes at 1: 50 with 100ul after 2 times with above-mentioned buffer and use the PBS washed twice after 30 minutes, be resuspended in again among the 300ul PBS.Above-mentioned test is with the negative contrast of TNFRIg.Above-mentioned resuspended cell is analyzed on the FACS instrument.

The result as shown in Figure 7.Fluorescence does not appear in negative control TNFRIg when carrying out FACS.As can be seen from Figure 7, FLA3Ig has significantly and the bonded ability of Jurkat cell.

(3) MLR reaction

Respectively extract 20 milliliters of fresh bloods from the donor of two affinity-less relations, Ficoll gradient separations leukocyte.One kind of cell carries out the r ray with 2000RADs to be handled.Above-mentioned two kinds of cells all transfer to 3 * 10 ⁶Cell/ml.

Two human PBMCs (the two-way MLR of allogeneic) equal-volume is mixed the back with 2 * 10 ⁵Cells/ hole inoculation " U " type 96 well culture plates add not commensurability purified fusion protein, equal-volume corresponding empty carrier transfection supernatant or culture medium are set compare, every group 3 multiple hole, 37 ℃, 5%CO ₂Cultivate 5d, 16h adds before stopping cultivating ³H-TdR (final concentration 5uCi/ml), collecting cell are dried on the 0.45um microporous filter membrane, and liquid scintillation counter is surveyed the DPM value.The result represents that with x ± s Microsoft Excel statistical procedure carries out mean difference significance analysis.

In the MLR reaction system, the FLA3Ig-N that adds 1 microgram, 5 micrograms and 10 microgram Protein A purification respectively, FLA3Ig-M or empty carrier transfection CHO cell supernatant (negative control), the result shows, even add the fusion rotein of 1 microgram Protein A purification, also can significantly suppress human peripheral MLR, suppression ratio is 60%～70%; And add the empty carrier transfection CHO cell supernatant of same volume, and then there is not this effect, through one factor analysis of variance, the result has statistical significance (seeing Table 6).Equally, also once directly carried out the MLR experiment with the supernatant of unpurified transfectional cell, and failed to observe significant inhibitory effect, this may cancel out each other relevant with the stimulation of heterologous protein in the purifying cells transfection supernatant not and the inhibitory action of micro-fusion rotein.

The influence that table 6 purification FLA3Ig breeds people MLR medium-sized lymphocyte (DPM, n=3)

*：P＜0.01 vs control；△：P＜0.01 vs 0

From above-mentioned data as can be seen, the relative activity of FLA3Ig-M is obviously than FLA3Ig-N height.Consider that the two proteinic aminoacid sequence is identical, obvious this difference is owing to the expression difference causes.This illustrates that also the dna encoding sequence of FLA3Ig-M is more suitable for expressing in the Chinese hamster ovary celI than the DNA sequence of FLA3Ig-N.

The animal model checking of embodiment 7 clinical practices

To 6 kinds of above-mentioned three classes [2 kinds of antibody (CD11-1 and CD11-2) wherein, 2 kinds of FLA3Ig (FLA3Ig-N and FLA3Ig-M), CTLA4/Fc 2 kinds (CTLAs-M/Fc and CTLAs-M/Fc)] function of material carried out animal model test, and main result of study is as follows:

7.1. therapeutical effect to the rheumatoid arthritis model

(1) the treatment function of single medicine

Above-mentioned 6 kinds of materials use separately separately, have carried out following research.

100.mu.gII Collagen Type VI (CII) adds complete Freund's adjuvant, intradermal injection B10.RIII mice is repeated once after 14 days again, at this moment occurs collagen-induced rheumatoid arthritis (CIA) symptom in the mice body, and 92% mice shows serious CIA symptom after 28 days.Mouse peritoneum is injected anti-CD11a monoclonal antibody of the present invention, observe its influence in the mice body the symptom of CIA.Detect after 12 weeks.Experimental mice 3 times weekly, 6 kinds of materials of the aforementioned preparation of per injection 10.mu.g were from the 1st day to 35 days; Inject PBS as negative control to control group mice.

Observe to find that symptoms such as redness and swelling of joints, ankylosis all take place control group mice, then have only slight swelling in the administration group, but serious symptoms such as ankylosis does not take place.This shows that 6 kinds of materials compare with matched group, all can significantly reduce the rheumatoid order of severity.Therefore, monoclonal antibody of the present invention might be developed the medicine that becomes treatment rheumatoid arthritis or other autoimmune diseases.

(2). the treatment function of composite reagent

Respectively select from 6 kinds of medicines of aforementioned 3 classes a kind (CD11-1, FLA3Ig-N and CTLAs-N/Fc), the research that above-mentioned same model suppresses rheumatoid arthritis has been carried out in combination in twos, and the result is as shown in table 7 below.

Table 7

Combination	CD11-1+ FLA3Ig-N	CD11-1+ CTLAs-N/Fc	FLA3Ig-N+ CTLAs-N/Fc	CD11-1	FLA3Ig- N	CTLAs- N
							Therapeutic effect	+++++	++++	++++	++	+++	+

From The above results as can be seen, when two kinds of medication combined application, better than the effect of independent application of treatment rheumatoid arthritis.

7.2 anti-immunologic rejection test-mice organ transplantation test

(1). the anti-repulsion test of single medicine

When above-mentioned antibody is used for the mouse heart inhibition test, can obviously improve the survival rate and the average survival natural law of animal subject.Following table 8 has shown the result of this test.For the reagent thing is anti-people CD11a monoclonal antibody, FLA3Ig and CTLA4Ig.

Normal Kunming kind white mice, male and female half and half, 12 every group.Wherein 6 is receptor, and 6 are carried out heart transplantation for donor.

Table 8

The blank supernatant of 0=negative control; A=CD11-1, B=CTLAs-N/Fc, C=FLA3Ig, D=CD11-1+FLA3Ig-N, E=CD11-1+CTLAs-N/Fc, F=FLA3Ig-N+CTLAs-N/Fc.

As can be seen, Mus source and humanized anti-people CD11a monoclonal antibody, FLA3Ig and CTLA4Ig can both significantly improve the survival rates on the 30th of organ transplantation receptor and the natural law of on average surviving from above-mentioned experiment.The medicine that shows these immunologic function downward modulations has certain function that overcomes immunosuppressive effect, might be used for the drug development of anti-immunologic rejection.

(2) the anti-repulsion test of composite reagent

In the composition of medicine test in the last table, also shown three kinds of effects that medicine has tangible anti-immunologic rejection, wherein the effect of CD11 obviously is better than other drug.Under the situation of composition of medicine, shown that equally effect obviously improves when containing the CD11-1 component, and usually showed tangible additive effect between medicine.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition 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

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gacatcgccg tggagtggga gtccaacggc cagcccgaga acaactacaa gaccaccccc 540

cccgtgctgg actccgacgg ctccttcttc ctgtactcca agctgaccgt ggacaagtcc 600

cgctggcagc agggcaacgt gttctcctgc tccgtgatgc acgaggccct gcacaaccac 660

tacacccaga agtccctgtc cctgtccccc ggcaag 696

<210>19

<211>699

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>19

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 60

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240

tacaacagca cgtaccgggt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 360

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660

tacacgcaga agagcctctc cctgtctccg ggtaaatga 699

<210>20

<211>31

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(31)

＜223〉primer

<400>20

gcactcgagt tttacccgga gacagggaga g 31

<210>21

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(23)

＜223〉primer

<400>21

gagcccaaat cttgtgacaa aac 23

<210>22

<211>45

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(45)

＜223〉primer

<400>22

taaccatttc catccataat taatctgagg ttgtctggaa cccag 45

<210>23

<211>45

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(45)

＜223〉primer

<400>23

cttattttat tcccatcaat tgaacatgcc caccgtgccc agcac 45

<210>24

<211>900

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>24

cgacgagcca tggttgctgg gagcgacgcg gggcgggccc tgggggtcct cagcgtggtc 60

tgcctgctgc actgctttgg tttcatcagc tgtttttccc aacaaatata tggtgttgtg 120

tatgggaatg taactttcca tgtaccaagc aatgtgcctt taaaagaggt cctatggaaa 180

aaacaaaagg ataaagttgc agaactggaa aattctgaat tcagagcttt ctcatctttt 240

aaaaataggg tttatttaga cactgtgtca ggtagcctca ctatctacaa cttaacatca 300

tcagatgaag atgagtatga aatggaatcg ccaaatatta ctgataccat gaagttcttt 360

ctttatgtgc ttgagtctct tccatctccc acactaactt gtgcattgac taatggaagc 420

attgaagtcc aatgcatgat accagagcat tacaacagcc atcgaggact tataatgtac 480

tcatgggatt gtcctatgga gcaatgtaaa cgtaactcaa ccagtatata ttttaagatg 540

gaaaatgatc ttccacaaaa aatacagtgt actcttagca atccattatt taatacaaca 600

tcatcaatca ttttgacaac ctgtatccca agcagcggtc attcaagaca cagatatgca 660

cttataccca taccattagc agtaattaca acatgtattg tgctgtatat gaatggtatt 720

ctgaaatgtg acagaaaacc agacagaacc aactccaatt gattggtaac agaagatgaa 780

gacaacagca taactaaatt attttaaaaa ctaaaaagcc atctgatttc tcatttgagt 840

attacaattt ttgaacaact gttggaaatg taacttgaag cagctgcttt aagaagaaat 900

<210>25

<211>250

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>25

Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val

1 5 10 15

Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln

20 25 30

Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn

35 40 45

Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala

50 55 60

Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg

65 70 75 80

Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr

85 90 95

Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp

100 105 110

Thr Met Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr

115 120 125

Leu Thr Cys Ala Leu Thr Asn Gly Ser Ile Glu Val Gln Cys Met Ile

130 135 140

Pro Glu His Tyr Asn Ser His Arg Gly Leu Ile Met Tyr Ser Trp Asp

145 150 155 160

Cys Pro Met Glu Gln Cys Lys Arg Asn Ser Thr Ser Ile Tyr Phe Lys

165 170 175

Met Glu Asn Asp Leu Pro Gln Lys Ile Gln Cys Thr Leu Ser Asn Pro

180 185 190

Leu Phe Asn Thr Thr Ser Ser Ile Ile Leu Thr Thr Cys Ile Pro Ser

195 200 205

Ser Gly His Ser Arg His Arg Tyr Ala Leu Ile Pro Ile Pro Leu Ala

210 215 220

Val Ile Thr Thr Cys Ile Val Leu Tyr Met Asn Gly Ile Leu Lys Cys

225 230 235 240

Asp Arg Lys Pro Asp Arg Thr Asn Ser Asn

245 250

<210>26

<211>347

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(347)

＜223〉fusion protein F LA3Ig

<400>26

Met Val Ala Gly Ser Asp Ala Gly Arg Ala Leu Gly Val Leu Ser Val

1 5 10 15

Val Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gln Gln

20 25 30

Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn

35 40 45

Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala

50 55 60

Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg

65 70 75 80

Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr

85 90 95

Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp

100 105 110

Thr Met Lys Phe Phe Leu Tyr Val Asp Lys Thr His Thr Cys Pro Pro

115 120 125

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

130 135 140

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

145 150 155 160

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

165 170 175

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

180 185 190

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

195 200 205

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

210 215 220

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

225 230 235 240

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

245 250 255

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

260 265 270

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

275 280 285

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

290 295 300

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

305 310 315 320

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

325 330 335

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345

<210>27

<211>1041

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(1041)

＜223〉coded sequence of fusion protein F LA3Ig

<400>27

atggttgctg ggagcgacgc ggggcgggcc ctgggggtcc tcagcgtggt ctgcctgctg 60

cactgctttg gtttcatcag ctgtttttcc caacaaatat atggtgttgt gtatgggaat 120

gtaactttcc atgtaccaag caatgtgcct ttaaaagagg tcctatggaa aaaacaaaag 180

gataaagttg cagaactgga aaattctgaa ttcagagctt tctcatcttt taaaaatagg 240

gtttatttag acactgtgtc aggtagcctc actatctaca acttaacatc atcagatgaa 300

gatgagtatg aaatggaatc gccaaatatt actgatacca tgaagttctt tctttatgtc 360

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 420

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 480

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 540

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 600

cgggtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 660

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 720

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 780

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 840

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 900

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 960

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1020

ctctccctgt ctccgggtaa a 1041

<210>28

<211>1041

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(1041)

＜223〉codon optimized FLA3-Ig antigen-4 fusion protein gene

<400>28

atggtggccg gctccgacgc cggccgcgcc ctgggcgtgc tgtccgtggt gtgcctgctg 60

cactgcttcg gcttcatctc ctgcttctcc cagcagatct acggcgtggt gtacggcaac 120

gtgaccttcc acgtgccctc caacgtgccc ctgaaggagg tgctgtggaa gaagcagaag 180

gacaaggtgg ccgagctgga gaactccgag ttccgcgcct tctcctcctt caagaaccgc 240

gtgtacctgg acaccgtgtc cggctccctg accatctaca acctgacctc ctccgacgag 300

gacgagtacg agatggagtc ccccaacatc accgacacca tgaagttctt cctgtacgtg 360

gacaagaccc acacctgccc cccctgcccc gcccccgagc tgctgggcgg cccctccgtg 420

ttcctgttcc cccccaagcc caaggacacc ctgatgatct cccgcacccc cgaggtgacc 480

tgcgtggtgg tggacgtgtc ccacgaggac cccgaggtga agttcaactg gtacgtggac 540

ggcgtggagg tgcacaacgc caagaccaag ccccgcgagg agcagtacaa ctccacctac 600

cgcgtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 660

tgcaaggtgt ccaacaaggc cctgcccgcc cccatcgaga agaccatctc caaggccaag 720

ggccagcccc gcgagcccca ggtgtacacc ctgcccccct cccgcgacga gctgaccaag 780

aaccaggtgt ccctgacctg cctggtgaag ggcttctacc cctccgacat cgccgtggag 840

tgggagtcca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggactcc 900

gacggctcct tcttcctgta ctccaagctg accgtggaca agtcccgctg gcagcagggc 960

aacgtgttct cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1020

ctgtccctgt cccccggcaa g 1041

<210>29

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉primer

<400>29

atggttgctg ggagcgacgc 20

<210>30

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(21)

＜223〉primer

<400>30

tttacccgga gacagggaga g 21

<210>31

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(26)

＜223〉primer

<400>31

gctagcatgg ttgctgggag cgacgc 26

<210>32

<211>40

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(40)

＜223〉primer

<400>32

gaagttcttt ctttatgtcg acaaaactca cacatgccca 40

<210>33

<211>40

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(40)

＜223〉primer

<400>33

tgggcatgtg tgagttttgt cgacataaag aaagaacttc 40

<210>34

<211>29

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(29)

＜223〉primer

<400>34

tttacccgga gacagggaga ggcctcgag 29

<210>35

<211>25

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(25)

＜223〉joint sequence

<400>35

Ala Ala Ala Ser Ser Ala Ala Ala Ser Ser Ala Ala Ala Ser Ser Ala

1 5 10 15

Ala Ala Ser Ser Ala Ala Ala Ser Ser

20 25

<210>36

<211>75

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(75)

＜223〉coded sequence of joint sequence

<400>36

accacaacct cgtcaaccac aacctcgtca accacaacct cgtcaaccac aacctcgtca 60

accacaacct cgtca 75

<210>37

<211>120

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(120)

＜223〉coded sequence of joint peptide and both sides sequence

<400>37

cttattttat tcccatcaat tgaaccacaa cctcgtcaac cacaacctcg tcaaccacaa 60

cctcgtcaac cacaacctcg tcaaccacaa cctcgtcaac atgcccaccg tgcccagcac 120

Claims (8)

1. immunomodulator, it is characterized in that, described immunomodulator is the CTLA4Ig fusion rotein, and described fusion rotein has the Fc fragment of the human IgG1 shown in the CTLA-4R ectodomain shown in the 37-161 position and SEQ ID NO:17 among the SEQ ID NO:5;

And described CTLA4Ig fusion rotein also has the connection peptides shown in the SEQ ID NO:35 between CTLA-4R ectodomain and human IgG1's Fc fragment.

2. immunomodulator as claimed in claim 1 is characterized in that, the CTLA-4R ectodomain of described CTLA4Ig fusion rotein is coded by the nucleotide sequence shown in SEQ ID NO:6 or 7.

3. dna molecular that separates coding claim 1 described immunomodulator.

4. dna molecular as claimed in claim 3, it is characterized in that, the part of described CTLA-4R ectodomain of encoding has the nucleotide sequence of 109-483 position among the SEQ ID NO:6 or the nucleotide sequence shown in the SEQ ID NO:7, and the segmental part of the described human IgG1's that encodes Fc has the nucleotide sequence shown in the 718-1416 position among the SEQ ID NO:14.

5. a pharmaceutical composition is characterized in that, it contains pharmaceutically acceptable carrier and the described immunomodulator of claim 1.

6. pharmaceutical composition as claimed in claim 5 is characterized in that, it also contains the immunomodulator that is selected from down group:

(a) anti-people CD11a monoclonal antibody, described anti-people CD11a monoclonal antibody has the sequence of light chain shown in sequence of heavy chain shown in the SEQ ID NO:3 and the SEQ ID NO:4; Or

(b) FLA3Ig fusion rotein, described fusion rotein have the Fc fragment of the human IgG1 shown in the 121-347 position among the CD2 land of the FLA3 shown in the 1-120 position among the SEQ ID NO:25 and the SEQ ID NO:26.

7. pharmaceutical composition as claimed in claim 6 is characterized in that, described pharmaceutical composition also contains following two kinds of immunomodulators simultaneously:

(a) anti-people CD11a monoclonal antibody, described anti-people CD11a monoclonal antibody has the sequence of light chain shown in sequence of heavy chain shown in the SEQ ID NO:3 and the SEQ ID NO:4; With

(b) FLA3Ig fusion rotein, described fusion rotein have the Fc fragment of the human IgG1 shown in the 121-347 position among the CD2 land of the FLA3 shown in the 1-120 position among the SEQ ID NO:25 and the SEQ ID NO:26.

8. the purposes of immunomodulator as claimed in claim 1 is characterized in that, is used to prepare the medicine of treatment autoimmune disease and anti-immunologic rejection.

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