CN100429233C

CN100429233C - Recombinant solvent protein, its production and use

Info

Publication number: CN100429233C
Application number: CNB2005100238152A
Authority: CN
Inventors: 沈茜; 王健; 张鹏; 杨佳荟; 张军
Original assignee: Shanghai Changhai Hospital
Current assignee: Shanghai Changhai Hospital
Priority date: 2005-02-03
Filing date: 2005-02-03
Publication date: 2008-10-29
Anticipated expiration: 2025-02-03
Also published as: CN1817907A

Abstract

The present invention relates to recombinant fusion protein for inducing costimulatory molecules, which is recombination protein formed by merging function molecules and the induced costimulatory molecules. The present invention can adopt conventional methods, such as a genetic engineering method, a chemical method, etc. to prepare the recombinant fusion protein according to the basic steps of merging, expressing, purifying, etc. The present invention is used for particularly inhibiting the functions of memory and activated T-cells and has an obvious and particular inhibiting function for inhibiting the ICOS molecular activity of mammals. Thus, the recombinant fusion protein has the advantages of stable induced costimulatory molecule, half life extension and large scale preparation, is used for preparing particular immunosuppression products with high biological safety, safe raw material for treating, diagnosing, detecting and researching autoimmune diseases, graft rejection and relevant disease symptom products and has wide market application and development prospects.

Description

A kind of recombination fusion protein and preparation method thereof and purposes

Technical field

The present invention relates to medicine, biology, food, technical field of beverage, specifically relate to biological technology products and its production and use, more particularly relate to a kind of recombination fusion protein and its production and use.

Background technology

(1) progress of autoimmune disorder

1, overview

The human immune system has the function of resolution " oneself " and " non-own ", and it can not produce immune response to oneself tissue and cell.But, the human immune system occurs when unusual, also may produce immune response to tissue and the cell of oneself, thereby destroy or damage oneself tissue or cell, it is autoimmune disease, one of its important symbol is the autoantibody that forms anti-autologous tissue composition, and the inspection of this autoantibody and control are the important diagnostic means of autoimmune disease and the basis of clinical treatment.

Autoimmune disorder is a class important diseases of serious harm human health.The typical disease of autoimmune disorder is systemic autoimmune disorder, comprise systemic lupus erythematous (being called for short SLE), rheumatoid arthritis (rheumatoidarthritis is called for short RA), scleroderma (being called for short PSS), dermatomyositis, polyarteritis, autoimmune hemolytic anemia, polymyositis, system's (pilosity) property sclerosis, CREST syndrome, ankylosing spondylitis, osteoarthritis, adult onset still disease, Behcet's disease, special syndrome, psoriatic, relapsing polychondritis, the systemic vasculitis etc. of relying.In addition, autoimmune disorder also comprises the organ specificity autoimmune disorder, as endocrine system autoimmune disease, blood system autoimmune disease, cardiovascular systems autoimmune disease, respiratory system autoimmune disease, Digestive tract autoimmune disease, urinary system autoimmune disease, neural system autoimmune disease, autoimmunity illness in eye, autoimmune skin disease etc.

Diseases such as the systemic lupus erythematous of systematicness in the autoimmune disorder, rheumatoid arthritis, scleroderma, dermatomyositis, polyarteritis, ankylosing spondylitis, osteoarthritis, psoriatic, relapsing polychondritis once were named as " connective tissue disease (CTD) ", international afterwards and domesticly all they and rheumatism unification were classified as rheumatosis.

2, pathogeny

Autoimmune disorder is a class important diseases of serious harm human health, and it is that the human immune system attacks self cell and the disease that produces mistakenly as the exotic invasive material.

The reason that autoimmune disease produces is very complicated, mainly is that human immunity recognition function and homeostatic function get muddled; Or the tissue of self and cell be because of the change of infection recurring structure or component, makes immunity system think " non-oneself " by mistake and attacked; Or original hidden or besieged tissue or cell (as the eyeball content, the sperm in the male testical etc.) manifest, and are thought by immunity system " non-oneself "; Also may be that above-mentioned situation exists simultaneously.

That is to say, the pathogenesis of autoimmune disorder be self tolerance destruction so that autoantibody or/and primed lymphocyte damage contain the organ-tissue of corresponding autoantigen and cause clinical symptom; In the pathologic process, the activation of autoreactive T cell is the key of immunopathogenesis damage.

And inquire into wherein important one big class disease---the pathogenesis of rheumatismal complexity, seek method of early diagnosis, determine that curative effect is sure and the little methods of treatment of side effect is the objective of the struggle of whole wind diseases caused by dampness educational circles always.

In a word, difficult point and the focus that autoimmune more profound reason and countermeasure thereof are still medical research takes place.

3, epidemiology situation and clinical manifestation

Autoimmune disorder patient various places all over the world, morbidity is with 100,000/several calculating; With systemic lupus erythematous disease wherein is example, is 70.41/10 ten thousand in Chinese morbidity, women's morbidity be 113.33/10 ten thousand (yellow inscription is new etc. the systemic lupus erythematous epidemiology survey. CHINESE JOURNAL OF INTERNAL MEDICINE, 1985,14:451.).

Such disease clinical manifestation mostly is chronic disease, and any age, sex, race all can fall ill, and general age of onset is many person between twenty and fifty, and the women sees many.For example, RA be a kind of modal serve as the systemic autoimmune disorder of main performance with chronic polyarthritis disease, the course of disease is long, easily repeatedly, causes very big misery to the patient; The sickness rate of this interior syndrome state is about 0.36%.Patient's age of onset is many, and the women was more than the male sex at 20～50 years old, and men and women's ratio is 1: 3; Its outstanding early stage clinical manifestation is a symmetry multi-joint red and swollen heat pain, the little joint of common four limbs, near-end arthroncus between finger, the palm refers to arthralgia and movable difficulties such as (sole of the foot toe), wrist, elbow, ankle even temporo jaw, the daystart ankylosis, in the afternoon alleviate gradually, symptom has 20% patient subcutaneous nodule can occur approximately outside the joint; The permanent symptom in late period that does not heal then has in various degree joint deformity and tetanic, and function of joint forfeiture etc. can damage internal organs, many histoorgans, cause ischemic necrosis of the femoral head, and are big to human consumption, the disability rate height.

4, the present situation of Clinics and Practices and prospect

At present, autoimmune disorder is not still had the radical cure method, and treatment all is to use nonspecific immunosuppressive agent, as glucocorticosteroid, endoxan, azathioprine, cyclosporin A, Chinese medicine etc., its curative effect is not certainly and often influence the normal immunne response of body simultaneously, can suppress marrow after the prolonged application, cause anaemia, leukopenia, reduce the body anti-infection ability, bring out condition invasive organism and parasitic infection, and because tumour is easily suffered from the inhibition of immunosurveillance ability.

The optimal selection of autoimmune disorder control induces body that autoantigen is produced specific immunologic tolerance again exactly, and therefore, people explore the immunosuppressor of tool antigen-specific always.

And rheumatology is the frontier branch of science of a youth, multidisciplinary intersection, and development in recent years is very fast.All have autoantibody in rheumatisant's the body, " significant antibody " wherein can be used for detecting, diagnoses and treatment, judges significant for diagnosis, treatment and the prognosis of rheumatism; Its inspection, methods of treatment are several times improved, and are worth increasingly, so the application of biotechnological formulation in rheumatosis has bright development prospect.

The example that is diagnosed as with rheumatoid arthritis (RA).

Because the cause of disease of RA is not fully aware of so far, and early stage its atypical clinical manifestations, lack specific diagnostic method in addition, bring certain difficulty for disease early diagnosis and treatment.Proved that now RA patient's arthropathy is with fastest developing speed back 1 year of morbidity, falling ill can occur irreversible osteoarthrosis in 1 year and destroy the early stage exhibition of spouting of using the medicine may command disease that changes the state of an illness.Therefore, the treatment key of RA is early treatment, and the prerequisite of early treatment is early diagnosis, so early diagnosis just becomes the hot issue that everybody paid close attention to.

Definition about early stage RA is a rheumatosis educational circles the question in dispute always, EULAR meeting in 2003 has proposed the definition of very early stage RA and early stage RA, the RA that the course of disease was less than 12 weeks is defined as very early stage RA, the RA of the course of disease between 12 weeks and 2 years is defined as early stage RA, and emphasizes no matter very early stage still early stage RA all needs an antirheumatic that promptly gives mitigate the disease after diagnosing (to be called for short: DMARD) treatment.

U.S. sacroiliitis foundation chairman and CEO Klippel think, anti-cyclic citrullinated peptide (cyclic citrullinated peptide, be called for short: CCP) antibody is to the meaning of RA, (be called for short: PSA) meaning to the examination prostate cancer is suitable with prostate specific antigen, this foundation was summarized to the annual major progress of sacroiliitis research field in 2003 first, and the anti-CCP antibody of the new mark of RA just is cited as one of sacroiliitis research ten big progress.The value of anti-CCP antibody in the RA early diagnosis has also been affirmed by rheumatosis educational circles such as Europe, result of study shows that the independent parameter of prediction RA is anti-CCP antibody, arthroncus number, grip and the AKA positive, best prediction index is anti-CCP antibody, help that the several years accurately detects RA before clinical symptom occurs, and list this antibody in conventional sense.

In recent years, many in the world rheumatologists are devoted to the research of RA early diagnosis, comprise and set up early stage sacroiliitis outpatient service, pay attention to early stage clinical manifestation, propose early stage RA the gross diagnosis standard, set up the autoantibody detection method of RA early diagnosis, and the imaging examination means of sensitivities such as CT, MRI and B ultrasonic being applied to the early diagnosis etc. of RA, this is to instruct to begin the important step of effectively treating RA, improving prognosis in early days.

(2) progress of organ transplantation

Organ transplantation is meant by surgical means, gives patient to replace the operation of its sick organ that decreases other people organ transplantation with vigor.Organ transplantation is one of the fastest subject of current clinical medicine scientific development, the researchdevelopment that it produces, uses and develops drive and promoted a series of basic subjects such as immunology, pharmacology, genetics and molecular biology.And the progress of these basic subjects has promoted the more deep comprehensive of organ transplantation and has been effectively applied to clinical treatment to suffer from the whole end property disease of various organs patients conversely.We can say that now except head and spinal cord can not be transplanted, the equal portable displacement of each organ-tissue of whole body, even a plurality of organ combined transplantation are simultaneously effected a radical cure many " incurable diseases " of in the past thinking.Surpass 160,000 person-times to global renal transplantation in 1989 according to statistics, the heart, liver transplantation are all above 4000 person-times, and bone marrow transplantation is advanced with the inferior speed of 2500～3000 examples every year, and pancreas is transplanted and reached 1500 routine times.Can predict, transplant operation can become the conventional treatment means of increasing mortality disease gradually in the near future.

But, the raising of transplantation technological method and improve and fail to guarantee the postoperative transplant organ function survival, the transplanting between twin children only arranged for a long time.Reason is to exist between people's Different Individual the difference of blood group and tissue matching, and this species diversity causes acceptor not accepted being implanted into the intravital organ that supplies, i.e. immunological rejection.

The key of graft long-term surviving is to select with donee's tissue compatibility antigen and other related antigens highly conforming donor is arranged, but in clinical practice is used but extremely difficulty reach, because the complicacy of major histocompatibility antigen and other related antigen systems and diversity, and be used at present that transplanted organ, tissue and cell are mainly derived from corpse of the same race or the relatives' donor of living, so immunological rejection will inevitably take place in post-transplantation.Therefore rejection becomes the major obstacle that organ transplantation is succeedd.In order to prevent and treat rejection, make the graft long-term surviving, must adopt immunosuppressant measure.

(3) progress of immunosuppressor

1, classification

At present, the immunosuppression measure of clinical use comprises immunosuppressor and other immunosuppression measures of use used.Wherein, with being most widely used of immunosuppressor, immunosuppressor comprises that chemicals immunosuppressor, biological immunosuppressant agent are antilymphocyte antibody.

The above-mentioned organ transplantation that is applied as of immunosuppressor obtains long-term good efficacy and has created condition, reaches more than 95% as renal transplantation one annual survival rate, and the heart, liver transplantation survival rate branch reach more than 90% and 80%.Thereby, immunosuppressor be acknowledged as modern clinical organ transplantation success a strong guarantee (Xia Suisheng. clinical transplantation medical science. the .1999 of Zhejiang science tech publishing house, 8～9.).

2, developing history

Four-stage has been experienced in the development of immunosuppressant treatment:

Fs (1908～1967): reach 70 years, adopt radioactive rays or chemicals to destroy the cell of all differentiation indiscriminately, this immunosuppressor causes the incidence height of severe infections death, so be not widely used in clinical.

Second stage (1967～1976): mainly focus on to the research of T cell inhibiting, employing is to the inhibiting polyclonal serum preparation of T cellular immunization, though this immunosuppressor can make multiple T cytoactive lower, improve the graft survival effect, but increased the side effect of receptor's resistibility.

Three phases (1976～so far): be that the research medicine suppresses to participate in immunoreactive cell, adopt chemical immunosuppressant, its representative is a ciclosporin A.Huge pushing effect has been played in the development that appears as organ transplantation over nearly 20 years of ciclosporin A.So far, ciclosporin A is still main immunosuppressor.

Ciclosporin A is that the incidence of infection that the third generation immunosuppressor of representative causes reduces, the tumour incidence is not higher than other immunosuppressor, its shortcoming is drug-induced organ toxicity and complication, for example, liver toxicity, renal toxicity, intestinal complications, internal secretion complication, neurological complication.

The 4th generation immunosuppressor, the period of just preparing the better immunosuppressor studied.

3, clinical application effect and research and development prospect

Immunosuppressor is used in clinical organ transplantation and has been experienced nearly 50 years history, and each new progress has all promoted the development of clinical organ transplantation; Yet, do not reach satisfied effect.Though graft one annual survival rate such as renal transplantation have reached 95%, the incidence of acute rejection is still up to 50%, and the incidence of chronic rejection reaches 10%, and existing immunosuppressor can't be prevented and treated; In case take place, can only transplant once more, but the success ratio of transplanting once more only 50% (old reality. transplantation immunology. the .1998 of Hubei science tech publishing house).

So far, all right and wrong are special to be applied to the immunosuppressor of each clinical prevention and treatment of rejection, they can reduce receptor's general immunity function, thereby lowered acceptor to infecting and the resistivity of tumour and the toxic side effect of medicine in addition; If can not tolerate, often be forced to drug withdrawal, influence the graft long-term surviving.The optimal selection of rejection control induces the host that antigen of donor is produced specific immunologic tolerance again exactly, and therefore, having the antigen specific immune inhibitor is optimal selection.

Although the research of immunosuppressor has had remarkable progress, but, the ideal immunosuppressor should be a T lymphocyte clone that the specific immune that suppresses to be caused by antigen of donor reacts and bone-marrow-derived lymphocyte clone, yet all present immunosuppressor all do not reach this requirement.

Secular from now on target is the life-time service immunosuppressor, reach and can treat disease, does not cause patient's immune deficiency and the complication that causes other again.So, seek novel, specificity inhibit feature and the little immunosuppressor of toxic side effect are arranged is very necessary.From economics point,, and cost an arm and a leg because the range of application of immunosuppressor is wide; Only the output value in the application every year aspect organ transplantation is just in multi-million dollar, thereby the economic benefit of development neotype immunosuppressant also is very considerable.

(4) progress of costimulatory molecules immunosuppressor

Costimulatory molecules is that a class participates in immunoreactive complementary molecule, is present in T lymphocyte, bone-marrow-derived lymphocyte, antigen presenting cell and (is called for short: APC) and the surface of target cell.The costimulatory signal that costimulatory molecules and its receptors bind are produced plays important effect in T cell activation, helper cell differentiation, signal transduction and responsiveness cytokine secretion.If there is not the participation of costimulatory molecules, very fast apoptosis after having discerned antigenic T lymphocyte and can't activating or activate usually and show clonal anerge or immunological tolerance.

The activation of T cells needs the costimulatory signal of B7-CD28, CTLA-4 and CD40-CD154, but the performance of effector T cell function is not rely on these to stimulate approach altogether to a great extent.

Recent findings in the reactivation process of memory T cell, exists a kind of new costimulatory molecules---and (Inducible costimulator is called for short: ICOS) inducible co-stimulator.The ICOS gene is positioned on the human chromosome 2q33, closely links to each other with the gene of CD28 and CTLA-4, and (be called for short: in zone bp), begin with the initiator codon of CD28, the terminator codon of ICOS finishes, and is separated from one another to be arranged in a length and to be 252000 bases; The ICOS gene contains 5 exons, 4 intron (Hutloff A, Dittrich A M, Beier K C et al.ICOS is an inducible T-cell co-stimulatorstructurally and functionally related to CD28.Nature, 1999,397:263-266.).

ICOS is the B7/CD28 superfamily member, predominant expression is on activated T cells, can promote propagation and differentiation, enhancing cytokine secretion and killing activity, the auxiliary lymphocyte subgroup of the anti-T of adjusting of activated T cell (to be called for short: polarization TH1/Th2), the antibody that promotes the T cell to rely on produces (Hutloff, A. (1999) Nature 397:263-266).CD28-CTLA 4-ICOS gene regions links to each other with the autoimmune type of numerous disease and is relevant, and as type i diabetes, autoimmune thyroid disease, multiple sclerosis, celiaca etc., this makes them very important for the research of autoimmune disease.

Present research confirms: the common stimulation of T effector cell's activation and the essential ICOS of function performance is (referring to for example Tafurl, A. (2001) Nature, 409 (6816): 105-109.), ICOS can promote the propagation and the secretion of activated T cell, regulates the polarization of Th1/Th2 cell, the B cell function that enhancing relies on the T cell etc.ICOS provides costimulatory signal for activating memory T cell, and blocking-up ICOS stimulates approach can increase the apoptosis of memory, activating T cell altogether.Experiment in vivo and vitro reports that all blocking-up ICOS costimulatory signal causes cytokine-expressings minimizing, immunoglobulin classes such as IL-4, IL-10, IFN-γ, CC and CXC chemokine to change impaired; ICOS can alleviate the experimental autoimmune cerebrospinal meningitis (to be called for short: clinical symptom EAE); ICOS can prolong the survival time of allotransplantation heart, liver; ICOS can alleviate infections with leishmaniasis inflammatory (referring to for example Rottman, JB. (2001) Nature Immunology (2); 605-611.; Guo, L. (2002) Transplantation, 73; 1027-1032.; Greenwald, RJ. (2002) J Immunol 168 (3): 991-995.).

By literature search etc., up to the present, do not find that as yet with ICOS stimulation approach altogether be the relevant report of the recombination fusion protein of target spot as immunosuppression thing and its production and use aspect.

Summary of the invention

The technical problem that will solve required for the present invention is to disclose a kind of recombination fusion protein and its production and use, to overcome the above-mentioned defective that prior art exists.

That is to say, the invention is intended to study a kind of recombination fusion protein and its production and use, so with it as new immunosuppression thing, diagnostic reagent, detection reagent etc., be used to prepare corresponding product such as medicine, reagent etc.Be that one of goal of the invention provides this recombination fusion protein, promptly use the partial amino-acid fragment of whole amino acid moleculars or the ICOS of ICOS, merge the recombinant protein of formation with functional molecular; Two of purpose provides the preparation method of this recombination fusion protein; Three of purpose provides the purposes of this recombination fusion protein, so that using and using of aspects such as scientific research, disease treatment, medical diagnosis on disease, disease detection to be provided.

(1) technical conceive

Technical conceive of the present invention is such:

1, general introduction

China's medicament research and development has a long history, accumulated rich experience at everyways such as prevention, diagnosis, treatments, the independent development original new drug is a present urgent task of Chinese Medicine circle, and promptly seeks effective activeconstituents or product is a valid approach.Therefore, the present invention passes through the systematic study to costimulatory molecules immunosuppressor and associated viscera, and screens and prove the activity and the purposes of this related products.

On the theoretical investigation of costimulatory molecules immunosuppressor and scientific experiment result, all reflect a kind of valid approach that blocking-up ICOS stimulation approach altogether is expected to become treatment, diagnosis, detection and studies autoimmune disorder and transplant rejection.Be present in the activation or the ICOS on memory T cell surface, combine with the ligand molecular of its ligand expression cell surface form mixture after, its biological function of competence exertion.If, the combining of blocking-up ICOS and its part, the function of the T cell that then can suppress to activate, and do not influence other lymphocytic functions, can not cause the body's immunity defective, thereby be the ideal selection of specific immunosuppressive agent.Therefore, be the immunosuppressor of target spot with the active inhibition of ICOS, might become the desirable immunosuppressive drug of disease treatment, diagnosis, detection and researchs such as autoimmune disorder and transplant rejection immunoregulation.

The bonded method of described blocking-up ICOS and its part, the inventor thinks four kinds of approach; By these four kinds of Basic Ways, the researchist can access has the immunosuppressor of specificity at ICOS, and what that is to say that the contriver researchs and develops has specificity and at the immunosuppressor of ICOS four kinds of base types can be arranged.Described four kinds of Basic Ways are as follows: the one, destroy or destroy ICOS, the 2nd, cover ICOS and go up and part bonded site or zone or functionally active site or zone, the 3rd, destroy or destroy the part of ICOS, the 4th, cover corresponding binding site or zone or functionally active site or zone on the part of ICOS; These four kinds of Basic Ways all can successfully hinder ICOS and combine with its part, can not form the mixture of expection.The thinking of this research and development also has general directive function to other similar antibody or inhibitor research and development in the biological technical field, certainly particularly other relate to as similarly research and development such as antibody in the field of medicaments to the life science field, also has great importance, as the research of antibody chemicals.

2, Yan Jiukaifa means

At present, if to obtain can specificity be to seek or therapeutic antibodies that preparation has the ICOS specific inhibitory effect at the common methods of the immunosuppressor of ICOS.

Up to now, the antibody that is used for the treatment of effect is total to three major types: first kind treatment antibody the earliest, it is the hybridoma excretory mouse monoclonal antibody for preparing by lymphocyte with the corresponding antigens mice immunized, this antibody-like has very high avidity to reach therapeutic purpose in conjunction with corresponding antigens with corresponding antigens, but they give with human body in use, can cause producing anti-mouse antibodies in the human body and causing a series of associated problem, for example serum half-life short, can not produce the function of body effcct thing and cause the immunne response of anti-this mouse antibodies in the deleterious human body.The second class therapeutic antibodies, be to utilize gene engineering method to carry out humanization modified to murine antibody, to overcome the variety of issue that in human body, uses the circle murine antibody to cause, but because these humanized antibodies have still kept some mouse sequences, therefore they still can cause some undesired immune responses, for example anti-humanized antibody reaction of people.The 3rd class therapeutic antibodies is complete human antibodies, this antibody-like is to utilize in the human peripheral blood lymphocyte of the generation antigen-specific autoantibody that exists naturally to produce by people's hybridoma technology, but thinks that at present the mono-clonal autoantibody in these hybridomas sources can't reach therapeutic action to the affinity of corresponding antigens is too low.

Except that seeking or preparation has the therapeutic antibodies of ICOS specific inhibitory effect, can also adopt engineered means or technology to prepare recombination fusion protein with ICOS specific inhibitory effect.

Recombination fusion protein (claims not only: recombinant protein) be meant with certain functional molecular certain protein (but also is claimed: target protein) modify a kind of protein with above-mentioned two-part structure territory newly that obtains then; For example, one of modifying method that carries out on gene level is, by the recombination fusion protein that the gene of target protein is linked to each other and obtains with the gene of modified protein.This class recombination fusion protein that obtains can be effective to aspects such as treatment of diseases, diagnosis, detection and scientific research.Therefore, comprising the characteristic of medicine, reagent etc. to strengthen or to improve target protein as the product of practicality by protein modification, is a kind of shortcut of research and development product innovation.The survey article of describing protein modification comprises Francis, Focus on GrowthFactors, and 3:4-10, in May, 1992, by Mediscript, London, UK publishes.

One of method of protein modification is, merge mutually with certain part of immunoglobulin (Ig) or the structural domain of certain several part by target protein, for example, application function molecule such as immunoglobulin (Ig) (claim again: antibody, be called for short: constant fragment Ig) (claims again: the Fc district, be called for short: Fc), can make recombination fusion protein obtain new characteristic.Antibody comprises two function independent parts, promptly is called the variable domains and the constant domain that is called " Fc " of " Fab ".Variable domains conjugated antigen, and constant domain joint efficiency subfunction thing, described effector function thing comprises complement, Fc acceptor, phagocytic cell etc.The Fc fragment of immunoglobulin (Ig) has long plasma half-life, and Fab lifetime weak point (Capon etc., Nature, 1989,337:525-531).Wherein, the nucleotide sequence of human IgG1 Fc is seen sequence table 1, and its aminoacid sequence is seen sequence table 2.

This protein modification, its advantage is a lot: the binding ability that 1. remains with antigen or ligand/receptor.2. by carrying out affinity chromatography with the constant segmental antibody of anti-immunoglobulin, staphylococcal protein A,SPA or staphylococcal protein G be the Fc fragment of purifying Ig easily, and preparation cost and use cost are obviously lowered.3. utilize anti-Fc section antibody, can make detection of fusion proteins convenient, special, responsive.4. the Fc section can be passed the cytotoxicity that placenta, conjugated complement mediation complement relies on, and (be called for short: ADCC) effect etc. can be used for the targeted therapy of some disease to the cytotoxicity that mediate antibody relies on.5. the Fc of IgG, IgM, IgA can form multimeric molecule, improves the ability of recombinant protein conjugated antigen or part.6. by reduction or elimination proteolysis,, make it have the longer transformation period to strengthen this proteinic protection and to reduce its degraded; In addition, in some cases, can also further increase this proteinic stability, cycling time and biological activity etc., be more suitable in intravital application.7. directly produce, not only more be fit to body and absorb, and avoided acquired immune deficiency syndrome (AIDS) (to be called for short: AIDS), hepatitis virus and other pathogeny microbiological contamination near the situation of natural molecule with the Mammals engineering cell.

Adopt Fc district to prepare curative proteinaceous product, so that transformation period prolongation or mix following function: for example, the Fc receptors bind that the Fc albumen of immunoglobulin (Ig) has, A protein binding, complement combination and placenta forwarding function; Again for example, the Fc district with IgG1 antibody is fused to CD30 part (abbreviation: N-end CD30-L), described CD30-L is the molecule that is combined in the CD30 acceptor of expressing on the following cell: Hokdkin disease tumour cell, degeneration are grown lymphoma (anaplastic lymphoma) cell, T chronic myeloid leukemia cell and other malignant cell type and (are seen: United States Patent (USP) the 5th, 480, No. 981); For example, a kind of antiphlogistic drug and anti-repellents IL-10 have been fused among the mouse Fc γ 2a with transformation period of increasing the short cytokine of circulating half-life (Zheng etc., The Journal of Immunology, 1995,154:5590-5600).The Tumor Necrosis Factor Receptors treatment septic shock patient's that application and human IgG1's Fc albumen is connected research (Fisher etc., N.Engl.J.Med., 1996,334:1697-1702 have also been estimated; Van Zee etc., The Journalof Immunology, 1996,156:2221-2230).Also Fc and CD4 acceptor have been merged with manufacture of therapeutic protein be used for the treatment of AIDS (Capon etc., Nature, 1989,337:525-531).In addition, also with interleukin-2 (be called for short: N-terminal IL-2) is fused to the Fc fragment of IgG1 or IgG3, with overcome short transformation period of IL-2 and system toxicity thereof (Harvill etc., Immunotechnology, 1995,1:95-105).

The present invention is exactly by preparing a kind of recombination fusion protein that can cover binding site corresponding on the part of ICOS or zone or functionally active site or zone, to reach the immunosuppressive effect of expection.The product and the experimental result of the present invention's research and development have also further confirmed the exactness of contriver in the product research developing thought.

3, design process

The contriver is an example with the ICOS in people source on the specific design of this recombination fusion protein research and development of products, the theing contents are as follows of thinking:

The ICOS that derives from the people (is the ICOS in people source, be called for short: hICOS) as deriving from the hICOS of people's activatory peripheral blood lymphocyte, its mRNA sequence encoding is NM-012092, total length 2610bp, the coding region is 26～625, and can translate and produce length is the polypeptide chain of 199aa.ICOS is the heterodimeric protein that is connected by disulfide linkage, and relative molecular weight is 55,000～60,000.(be called for short: kDa) sequence with two peptide chains of 29kDa is consistent, has only produced some difference in the glycosylation of post transcriptional modificaiton process for 27 kilodaltons of ICOS.ICOS and CD28 molecule have 24% sequence identical, and 39% sequence similarity is arranged; ICOS and CTLA-4 molecule have 17% sequence identical, and 39% sequence similarity is arranged.

The amino acid sequence analysis of ICOS shows, the ICOS in people source (aminoacid sequence number: S78540) 199 amino acid of polypeptide chain total length, wherein 1～21 is its signal sequence, 22～199 is the peptide sequence of ICOS, wherein 22～138 is extracellular region, 26～132 immunoglobulin-like zones, 139～164 is its transmembrane domains, 165～199 is its intracellular region.This structure explanation, sophisticated ICOS is that the I type changes membrane molecule, and the peptide chain of ICOS is promptly partly arranged at kytoplasm; ICOS has the strand of an immunoglobulin-like on the express cell surface, this structure is by the conservative halfcystine on 42 and 109 and stablized; Transmembrane domains is made up of 26 amino acid; Cytoplasmic region is the peptide tail of 35 amino-acid residues; This structure is very similar with CTLA-4 to CD28.The cysteine residues that is positioned the 136th of ICOS is that (same structure is 141 at CD28 in the position that disulfide linkage forms between the participation homodimer; On CTLA-4, also have).The part binding motif MYPPY that does not have CD28 and CTLA-4 on the ICOS; The part binding motif of ICOS is FDPPPF.

We know, the ideal immunosuppressor should only suppress the T lymphocyte that specific antigen causes and the clone of bone-marrow-derived lymphocyte, the T lymphocyte that causes as transplantation antigen and the clone of bone-marrow-derived lymphocyte.ICOS does not express on inmature T cell, and it is only specific expressed on memory or activated T cell, thereby blocking-up ICOS stimulates approach altogether, can reach the effect that specificity suppresses memory T cell and activating T cell function, and can not have influence on other lymphocytic functions, this is the desirable target spot of immunosuppressant activity, can play the effect that specific immunity suppresses.

Natural ICOS is that a total length is 199 amino acid whose membrane protein molecules, behind the T lymphocyte activation ICOS is expressed on the cytolemma.Utilize genetic engineering technique ICOS to be carried out computer mould is built technology and deletion mutantion is discovered, (its nucleotide sequence is seen sequence table 3 to 21～140 amino acid fragments of N end of ICOS, its aminoacid sequence is seen sequence table 4), have its part combined function territory, have natural completely ICOS part binding bioactive; If using gene engineering technique synthesizes this fragment, then can compete native ligand in conjunction with ICOS, reach the effect that suppresses the ICOS biologic activity.Using gene engineering technique, in mammalian cell, intestinal bacteria, yeast, insect cell, all can express 21～140 amino acid fragments of N end of preparation ICOS, but have the difficulty that expression amount is not high, subsequent purification is difficult, degrade easily in the inside and outside, the transformation period is short.And if utilize recombination fusion protein as this specific immunosuppressive agent, then not only make it have immunosuppressant function, can also make it have other functional moleculars such as the constant segmental feature of immunoglobulin (Ig), can increase medicine transformation period in vivo, the pollution of having avoided medicine to cause by pathogenic micro-organism in process of production, but also the process that can simplify preparation reaches the advantages such as effect that reduce cost.

So, on this basis, the contriver has designed a kind of novel immunosuppression thing---whole amino acid moleculars of ICOS or the partial amino-acid fragment of ICOS or its variant or fragment at the blocking-up of ICOS stimulation approach altogether, recombinant protein with the functional molecular fusion, unified in this article abbreviating as: recombination fusion protein claims again: the ICOS recombination fusion protein.On this basis, the whole amino acid moleculars of research and development ICOS or partial amino-acid fragment or its variant or the fragment of ICOS are with the constant fragment of immunoglobulin (Ig) or the recombinant protein (Inducible costimulatorimmunoglobulin fusion protein) of its variant or fragment fusion; When this recombination fusion protein has R ₂-R ₁During type, unified in this article abbreviating as: ICOS-Ig.And further research and develop partial amino-acid fragment or its variant or the fragment of whole amino acid moleculars or the ICOS of people ICOS, with the constant fragment of immunoglobulin G while 1 or the recombinant protein of its variant or fragment fusion, unified in this article abbreviating as: ICOS-IgG1, the recombinant protein that the constant fragment of wherein preferred ICOS polypeptide and immunoglobulin G while 1 merges, unified in this article abbreviating as: hICOS-IgG1; And the partial amino-acid fragment of the whole amino acid moleculars of mouse ICOS or mouse ICOS or its variant or fragment, with the constant fragment of mouse immuning ball protein G2a or the recombinant protein of its variant or fragment fusion, unified in this article abbreviating as: mICOS-IgG2a.That is to say that the present invention from many aspects, multi-angle proves and the exactness of having examined this design.

For ease of narration, the N of wherein said people ICOS is held the 21st～140 segmental amino acid (its nucleotide sequence is seen sequence table 3, and its aminoacid sequence is seen sequence table 4), unified in this article abbreviating as: the ICOS polypeptide also claims: the ICOS ligand binding domain.That is to say that for people ICOS, wherein the ICOS polypeptide is the ligand binding domain of people ICOS, can compete the native ligand in conjunction with ICOS, suppress the activity of ICOS.

(2) recombination fusion protein

Recombination fusion protein provided by the invention such as ICOS-Ig, hICOS-IgG1 etc. are a kind of a kind of novel immunosuppression things with the preparation of methods such as gene engineering method (comprising engineered means or technology), chemical process.

Recombination fusion protein of the present invention is that the partial amino-acid fragment of the whole amino acid moleculars of ICOS or ICOS is modified resulting recombinant protein, and promptly functional molecular (is used code R ₁Expression) (uses code R with the partial amino-acid fragment of the whole amino acid moleculars of ICOS or ICOS or its variant or fragment ₂Expression) merges the recombinant protein of formation, the just so-called ICOS recombination fusion protein of this paper.

Described ICOS recombination fusion protein, its structure is as follows:

This recombination fusion protein is to have one or more recombinant proteins that are selected from the following composition form: R ₁-R ₂, R ₂-R ₁, R ₁-L-R ₂And R ₂-L-R ₁Preferred R ₂-L-R ₁, R ₂-R ₁, further preferred R ₂-R ₁Wherein said L is a junction fragment.

Described R ₁Being arbitrary functional molecular, is to comprise in polypeptide, the albumen etc. one or more; Preferred Fc albumen or its variant or fragment, streptavidin, polyhistidine or green fluorescent protein (are called for short: one or more GFP) etc.; Continue in preferred Fc albumen or its variant or the fragment etc. one or more; Continue one or more of Fc albumen among preferred IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or the IgD etc. or its variant or fragment etc. again; One or more of Fc albumen among further preferred IgG1, IgG2, IgG4, IgA or the IgM etc. or its variant or fragment etc., one or more of Fc albumen among continuation further preferred IgG1, IgG2 or the IgM etc. or its variant or fragment etc., further preferably one or more of the Fc albumen among IgG1 or the IgG2 etc. or its variant or fragment etc., most preferably one or more of the Fc albumen among the IgG1 or its variant or fragment etc. again.

Wherein, described Fc albumen or its variant or fragment are selected from following one or more:

(1) Fc aminoacid sequence, the Fc aminoacid sequence shown in the preferred sequence table 1, i.e. the Fc aminoacid sequence of human IgG1's Fc aminoacid sequence, or people IgM, or a kind of in the Fc aminoacid sequence of mouse IgG2 etc., further preferred human IgG1's Fc aminoacid sequence;

(2) Fc aminoacid sequence, the Fc aminoacid sequence shown in the preferred sequence table 1, i.e. the Fc aminoacid sequence of human IgG1's Fc aminoacid sequence, or people IgM, or a kind of in the Fc aminoacid sequence of mouse IgG2 etc., further preferred human IgG1's Fc aminoacid sequence; Have the different aminoacids that replaces or lack in following one or more positions:

1. one or more cysteine residues;

2. one or more tyrosine residuess;

3. disappearance or with the halfcystine of L-Ala the position of substitution 5;

4. disappearance or with the leucine of L-glutamic acid the position of substitution 20;

5. lack or replace the L-glutamic acid of position 103 with L-Ala;

6. disappearance or with the Methionin of L-Ala the position of substitution 105;

7. disappearance or with the Methionin of L-Ala the position of substitution 107;

8. the disappearance or the position of substitution 1,2,3,4 and 5 in one or more amino acid;

9. replace or lack one or more residues to eliminate described Fc receptor binding site;

10. replace or lack one or more residues to eliminate described complement (C1q) binding site; With

(11) above inferior part combination 1.～10.;

(3) the above inferior partly aminoacid sequence of (1) or (2) has a methionyl residue at the N end;

(4) any Fc albumen or its variant, fragment or derivative in above inferior partly (1) or (3) comprises the chemical part that is connected to described protein part;

(5) above inferior partly a kind of derivative of (4), wherein said chemical part is the water-soluble polymers part;

(6) above inferior partly a kind of derivative of (5), wherein said water-soluble polymers partly is a polyoxyethylene glycol; With

(7) above inferior partly a kind of derivative of (5), wherein said water-soluble polymers partly are connected unique N end of described protein part.

Described R ₂Multiple source is arranged, and mainly is to derive from Mammals;

Wherein, described Mammals; be to comprise in people, mouse, rat, sheep, monkey, ox, pig, horse, rabbit, dog, chimpanzee, baboon, marmoset, macaque or the rhesus monkey etc. one or more; in preferred people, mouse, rat, chimpanzee, sheep, monkey, ox or the pig etc. one or more; in further preferred people, mouse, rat or the chimpanzee etc. one or more, optimum is chosen.

Described R ₂Be the whole amino acid moleculars that comprise ICOS, or in the partial amino-acid fragment of ICOS etc. one or more;

Wherein, the partial amino-acid fragment of described ICOS; be to comprise in the partial amino-acid fragment of all mammiferous ICOS one or more; in the partial amino-acid fragment of the ICOS of preferred people, mouse, rat, chimpanzee, sheep, monkey, ox or pig etc. one or more; the partial amino-acid of ICOS in further preferred people, mouse, rat or the chimpanzee etc. segmental one or more; in further preferred again ICOS polypeptide or its variant, fragment or the derivative etc. one or more, most preferably ICOS polypeptide.

Described ICOS polypeptide or its variant, fragment or derivative, preferably below oneself:

(a) the ICOS amino acid sequence of polypeptide (is seen: any residue of the 42nd amino acids displacement gained sequence table 3);

(b) any residue of the displacement of the 83rd amino acids shown in ICOS amino acid sequence of polypeptide gained;

(c) any residue of the displacement of the 63rd amino acids shown in ICOS amino acid sequence of polypeptide gained;

(d) any residue of the displacement of the 109th amino acids shown in ICOS amino acid sequence of polypeptide gained;

(e) any residue of the displacement of the 52nd amino acids shown in ICOS amino acid sequence of polypeptide gained;

(f) any residue of the displacement of the 115th～121 amino acids shown in ICOS amino acid sequence of polypeptide gained;

(g) any residue of the displacement of the 136th amino acids shown in ICOS amino acid sequence of polypeptide gained;

(h) any residue of the displacement of the 137th amino acids shown in ICOS amino acid sequence of polypeptide gained;

(i) any one described ICOS albumen or its variant, fragment or derivative in above inferior partly (a) to (h) comprise the chemical part that is connected to described protein part;

(j) the proteic a kind of derivative of above inferior part (i) described ICOS, wherein said chemical part is the water-soluble polymers part;

(k) the proteic a kind of derivative of above inferior part (j) described ICOS, wherein said water-soluble polymers partly is a polyoxyethylene glycol;

(l) the proteic a kind of derivative of above inferior part (j) described ICOS, wherein said water-soluble polymers partly is the polyamino acid part; With

(m) the proteic a kind of derivative of above inferior part (j) described ICOS, wherein said water-soluble polymers partly are connected unique N end of described protein part.

Described junction fragment is the one or more amino acid that comprise in glycine, l-asparagine, Serine, Threonine or the L-Ala etc.In being preferably as follows one or more:

(a)ala-ala-ala；

(b)ala-ala-ala-ala；

(c)ala-ala-ala-ala-ala；

(d)gly-gly；

(e)gly-gly-gly；

(f)gly-gly-gly-gly-gly；

(g)gly-gly-gly-gly-gly-gly-gly；

(h)gly-pro-gly；

(i)gly-gly-pro-gly-gly；

(j)val；

(k)ser-gly-gly-gly-gly-gly-gly-gly-gly；

(l)gly-gly-ser-gly-ser-ala-gly-ser-gly-ser-gly-gly-gly-ser-gly-ser-gly-gly；

(m) chemical part; With

(n) above any combination of inferior part (a)～(m).

Work as R ₁Be Fc albumen or its variant or fragment, R ₂During for the partial amino-acid fragment of the whole amino acid moleculars of ICOS or ICOS or its variant or fragment, R ₂-R ₁The recombination fusion protein of type is called for short: ICOS-Ig.

Work as R ₁The Fc albumen of the IgG1 in behaviour source or its variant or fragment, R ₂During for ICOS polypeptide or its variant or fragment, R ₂-R ₁The recombination fusion protein of type, be called for short: hICOS-IgG1, its nucleotide sequence is seen sequence table 5, its aminoacid sequence is seen sequence table 6; That is to say, its structure specifically: N end is the 21st～140 segmental amino acid in 199 amino acid of inducible co-stimulator total length, and its C end is immunoglobulin G while 1 a constant segmental amino acid.

Work as R ₁Be Fc albumen or its variant or the fragment of the IgG2a in mouse source, R ₂During for the ICOS in mouse source or its variant or fragment, R ₂-R ₁The recombination fusion protein of type is called for short: mICOS-IgG2a.

In sequence table 1, sequence table 2, sequence table 3, sequence table 4, sequence table 5, sequence table 6:

A represents Ala, Full Name in English: Alanine, Chinese full name: L-Ala;

M represents Met, Full Name in English: Methionine, and Chinese full name: methionine(Met) (or claims: methionine(Met));

B represents Asx, Full Name in English: Asparagine, Chinese full name: l-asparagine; Or Full Name in English: Aspartic acid, Chinese full name: aspartic acid;

N represents Asn, Full Name in English: Asparagine, Chinese full name: l-asparagine;

C represents Cys, Full Name in English: Cysteine, Chinese full name: halfcystine;

P represents Pro, Full Name in English: Proline, Chinese full name: proline(Pro);

D represents Asp, Full Name in English: Aspartic, and Chinese full name: aspartic acid:

Q represents Gin, Full Name in English: Glutamine, Chinese full name: glutamine;

E represents Glu, Full Name in English: Glutamic acid, Chinese full name: L-glutamic acid;

F represents Phe, Full Name in English: Phenylalanine, Chinese full name: phenylalanine;

R represents Arg, Full Name in English: Arginine, Chinese full name: arginine;

S represents Ser, Full Name in English: Serine, Chinese full name: Serine;

G represents Gly, Full Name in English: Glycine, Chinese full name: glycosides propylhomoserin;

T represents Thr, Full Name in English: Threonine, Chinese full name: Threonine;

H represents His, Full Name in English: Histidine, Chinese full name: Histidine;

V represents Val, Full Name in English: Valine, Chinese full name: Xie Ansuan;

I represents Ile, Full Name in English: Isoleucine, Chinese full name: Isoleucine;

W represents Trp, Full Name in English: Tryptophan, and Chinese full name: tryptophane:

K represents Lys, Full Name in English: Lysine, Chinese full name: Methionin;

Y represents Tyr, Full Name in English: Tyrosine, Chinese full name: proline(Pro);

L represents Leu, Full Name in English: Leucme, Chinese full name: leucine;

Z represents Glz, Full Name in English: Glutamine, Chinese full name: glutamine; Or Full Name in English: Glutamic acid, Chinese full name: L-glutamic acid.

Described fusion method is meant by prior art well known in the art, ICOS and functional molecular are carried out the bonded method, comprise in engineered means or technology, the chemical process etc. one or more, the means of preferred gene engineering or technology directly or by connector connect indirectly, and further the means or the technology of preferred gene engineering directly connect.

For example, ICOS polypeptide and functional molecular such as Fc structural domain can be merged with various method, so that the ICOS fusion rotein of generation has as variable biological nature and the variable effect of potential for the treatment of, diagnose, detect and study product such as medicine, reagent etc.Again for example, the aminoterminal (be called for short: N holds) or the carboxyl terminal of Fc structural domain and ICOS polypeptide (can be called for short: the C end) merge, can directly merge or merge by junction fragment, and/or according to one of the modifiable Fc of its natural form or ICOS polypeptide portion or the two.These different ICOS fusion roteins constitute things can be at expression level, be easy to separate and/or the having nothing in common with each other of aspects such as purifying, biological activity.

Recombination fusion protein provided by the invention, as the ICOS ligand binding domain that ICOS-Ig comprises, this zone is playing an important role aspect the specificity/affinity of recombination fusion protein.When the height that keeps this recombination fusion protein suppresses active and part high-affinity, also be possible at other amino-acid substitution of its ligand binding domain, particularly use conservative amino acid replacement." conservative amino acid replacement " used herein is meant that one of them amino-acid residue is had the aminoacid replacement of similar side chain by another.Amino-acid residue with similar side chain comprises basic side chain (Methionin for example, arginine, Histidine), acid side-chain (aspartic acid for example, L-glutamic acid), neutral side chain (glycine for example, aspartic acid, L-glutamic acid, Serine, Threonine, tyrosine, halfcystine,), non-polar sidechain (L-Ala for example, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), branched building block (Threonine for example, Xie Ansuan, Isoleucine) and aromatic side chains (tyrosine for example, phenylalanine, tryptophane, Histidine).

Recombination fusion protein of the present invention can be derived for or be connected to another functional molecular for example another polypeptide or albumen.Therefore, recombination fusion protein of the present invention comprises the form of recombination fusion protein deutero-described herein and other modification.For example, recombination fusion protein of the present invention can functionally connect (comprise that, heredity coupled by chemistry is merged, non-covalent combination or other) to one or more other molecular entities, for example another antibody (comprise two specially property antibody or double antibodies etc.) but detection agent, cell toxicant medicament, medicinal medicament and/or can mediate this recombination fusion protein and another molecule bonded albumen or peptide (for example, streptavidin core area or polyhistidine mark etc.).

But the detection agent of the recombination fusion protein of the present invention that can be used to derive comprises fluorescent chemicals etc.; But typical fluorescence detection agent comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalic sulfonic chloride, phycoerythrin or the like.Can also be with detecting the enzyme recombination fusion protein of deriving, described enzyme (is called for short: HRP), notatin (is called for short: GOD) or the like: when with can detect enzyme and derive recombination fusion protein the time, but detect this antibody by adding the other reagent that this enzyme is used to produce the detection reaction product for comprising alkaline phosphatase (be called for short: AKP or ALP), horseradish peroxidase.For example, but when the detection agent horseradish peroxidase, add hydrogen peroxide and diaminobenzidine and produce detectable colored reaction product.Can also be with the vitamin H recombination fusion protein of deriving, and by indirect measurement avidin or streptavidin in conjunction with detection.

One type the recombination fusion protein of deriving is to prepare by crosslinked two or more antibody (same type or dissimilar is for example created bi-specific antibody etc.).Suitable linking agent, comprise those Heterobifunctionals, have two unique reactive groups that have suitable spacer to separate, disuccinimidyl suberate etc. for example.

(3) preparation method of recombination fusion protein

The preparation method of recombination fusion protein comprises the steps:

1. merge: the synthetic recombinant dna fragment that can transcribe and can translate into recombination fusion protein;

2. express: described recombinant dna fragment is cloned on the expression vector, will carries the recombinant expression vector of recombinant dna fragment then and in host cell, express;

3. purifying: separation and purification can obtain described recombination fusion protein.

Described term " expression vector " is meant the nucleic acid molecule of having the ability to transport connected another nucleic acid, and can instruct the genetic expression of above-mentioned another nucleic acid molecule.One type of expression vector is " plasmid ", is meant the circular double stranded DNA ring that wherein can connect other dna fragmentation.The another kind of type of expression vector is a virus vector, wherein can connect other dna fragmentation in viral genome.Some expression vector can be in the host cell of its importing self-replicating, for example, have the bacterial expression vector and the episome Mammals carrier of bacterium replication orgin.Other expression vector such as non-add body Mammals carrier can be integrated into the genome of host cell when importing this host cell, can duplicate with this host genome thus.In addition, some expression vector can instruct the expression of gene that they are operably connected.This expression vector is referred to herein as " recombinant expression vector " (or abbreviate as " expression vector ").The common form of the expression vector that generally speaking, uses in recombinant DNA technology is plasmid.Comprise the expression vector of other form, for example one or more in Mammals carrier for expression of eukaryon (for example pSEC Tag2/Hygro A, pcDNA4/His Max or pcDNA3 etc.), virus vector (for example replication defect type retrovirus, adenovirus or adeno-associated virus), bacterial expression vector, Yeast expression carrier or the insect expression vector etc. in this explanation with identical functions.

Described term " host cell " is meant the cell that is used to express target protein, comprise Chinese hamster ovary cell (be called for short: Chinese hamster ovary celI), Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary celI (be called for short: the dhfr-CHO cell), the African green monkey kidney fibroblast-like cells (be called for short: the COS cell) or HEKC 293 cells) etc. (be called for short: one or more in.Should understand this term and not only refer to specific object cell, and refer to the offspring of this cell.Because in the follow-up generation, because some modification may take place for sudden change or environmental influence etc., in fact this offspring can be different with parental cell, but within still the be contained in term as used herein scope of " host cell ".In preferred Chinese hamster ovary celI or dhfr-CHO cell and the progeny cell thereof etc. one or more, further preferred Chinese hamster ovary celI and progeny cell thereof.

With ICOS-Ig is example, and its preparation method comprises the steps:

1. merge: the synthetic recombinant dna fragment that can transcribe and can translate into ICOS-Ig;

3. purifying: separation and purification can obtain described ICOS-Ig.

With hICOS-IgG1 is example, and its preparation method comprises the steps:

1. merge: the synthetic recombinant dna fragment that can transcribe and can translate into hICOS-IgG1:

3. purifying: separation and purification can obtain described hICOS-IgG1.

Can be by recombinant expressed to prepare ICOS-Ig of the present invention or hICOS-IgG1 in host cell.Be preparation ICOS-Ig or hICOS-IgG1, recombinant expression vector transfection host cell with the dna fragmentation that carries this recombination fusion protein of coding, make described recombination fusion protein in this host cell, express also preferably secretion and to the substratum of cultivating described host cell, can reclaim described recombination fusion protein from this substratum.Used " molecular cloning: laboratory manual " (third edition such as Sa nurse Brooker, the cold spring port, New York, calendar year 2001) described standard recombinant dna method, to obtain the recombination fusion protein gene, these genes added recombinant expression vector and described carrier is imported host cell.

For expressing ICOS-Ig or hICOS-IgG1, at first obtain the dna fragmentation of described ICOS-Ig of coding or hICOS-IgG1.Can (be called for short: PCR) amplification and modify the ICOS gene order and constant region for immunoglobulin (IgGFc) sequence prepares recombinant DNA by using the polymerase chain reaction.Dna fragmentation for the ligands specific land that obtains coding ICOS-Ig or hICOS-IgG1, build the ligands specific calmodulin binding domain CaM that technology and deletion mutantion technology are determined the ICOS molecule by computer mould, and with the ligands specific calmodulin binding domain CaM of standard pcr amplification ICOS gene.For the dna fragmentation of the constant region for immunoglobulin that obtains coding ICOS-Ig or hICOS-IgG1, by standard pcr amplification constant region for immunoglobulin territory.

In case after obtaining the dna fragmentation of the ligands specific calmodulin binding domain CaM of described ICOS gene and constant region for immunoglobulin, can modify these sequences to obtain encode described ICOS-Ig disclosed herein or hICOS-IgG1 aminoacid sequence.At first will be by the aminoacid sequence utilization homology mould construction method of the dna sequence encoding of the ICOS space conformation of analog IC OS molecule respectively, and build the mixture model of ICOS molecule and its ligand interaction, model presumes ICOS molecule ligand is in conjunction with the territory, to obtain the aminoacid sequence of ICOS molecule ligand in conjunction with the aminoacid sequence or derivatives thereof in territory of encoding in view of the above.By standard method for example PCR mediated mutagenesis (wherein described sudden change nucleic acid is added described PCR primer, make described PCR product contain described sudden change) or site-directed mutagenesis.

In case after obtaining the dna fragmentation of described human normal immunoglobulin constant region, can connect these dna fragmentations, obtain the dna fragmentation of recombination fusion protein by the standard recombinant dna technology.At first will may be operably coupled to the dna fragmentation of coding constant region for immunoglobulin by the ligands specific calmodulin binding domain CaM of ICOS gene.Constant region for immunoglobulin can be one or more in IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or the IgD constant region etc., one or more in IgG1, IgG4, IgA or the IgM constant region etc. preferably, further IgG1 constant region preferably.Term used herein " is operably connected ", is meant to connect these two dna fragmentations, makes to be remained in the framework of protein translation by these two dna fragmentation amino acid sequence coded.

For expressing ICOS-Ig or hICOS-IgG1, the gene fragment DNA insertion expression vector with the above-mentioned ICOS-Ig that obtains makes described gene may be operably coupled to and transcribes and translate control sequence.In this article, the term gene sheet that is meant ICOS-Ig or hICOS-IgG1 etc. that " is operably connected " is connected in the carrier, make this year intravital transcribe and translate the control sequence performance they regulate genetic transcription such as this ICOS-Ig or hICOS-IgG1 and translation promptly decide function.Select this expression vector and expression control sequenc to make it compatible with used expression host cell.By standard method (if the complementary restriction site on the gene that for example connects this ICOS-Ig or hICOS-IgG1 and the carrier or without limits the site be connected with regard to carrying out flush end) gene of described ICOS-Ig or hICOS-IgG1 is inserted this expression vector.In addition, the gene of this ICOS-Ig or hICOS-IgG1 can insert this recombination fusion protein is cloned into the recombinant expression vector of the above-mentioned signal peptide of encoding from the signal peptide gene of secretory host cell or with ICOS-Ig or hICOS-IgG1, makes this signal peptide be connected to the N-terminal of this recombination fusion protein gene in the transcription and translation framework.This signal peptide can be the signal peptide of ICOS molecule self or immunoglobulin (Ig) signal peptide or allos the signal peptide signal peptide of NIg (promptly from).Except that described ICOS-Ig or hICOS-IgG1 gene, recombinant expression vector of the present invention also carries the adjusting sequence that control described ICOS-Ig or hICOS-IgG1 gene are expressed in host cell.Term " adjusting sequence " comprises other expression controlling elements (for example polyadenylation signal) of described ICOS-Ig genetic transcription of promotor, enhanser and control or translation.This adjusting sequence, for example be described in Transcriptional Regulation in Eukaryotes:Concepts such as Michale Carey, Strategies andTechniques355, Cole Spring Harbor Laboratory Press, New York, USA (2000).The selection of this expression vector can be depended on all factors, for example selects the expression level of desire transformed host cells, desirable proteins etc.Preferred mammalian cell expression is regulated the viral element that sequence comprises guidance high-level protein expression in mammalian cell, and for example (be called for short: CMV) (for example CMV promotor/enhanser), simian virus 40 (are called for short: SV40) (for example SV40 promotor/enhanser) or adenovirus (one or more in adenovirus major late promoter's (be called for short: AdMLP) and the promotor and/or the enhanser of polyoma) etc. for example from cytomegalovirus.Further describing of relevant viral regulatory element and sequence thereof, for example No. 4,968,615, the United States Patent (USP) of Schaffner etc.

Except that described recombination fusion protein gene and adjusting sequence, recombinant expression vector of the present invention can also carry other sequence, for example regulates sequence (for example replication orgin) and the selectable marker gene that this carrier duplicates in host cell.This selectable marker gene is convenient to select to import the host cell of this carrier.For example, generally this selectable marker gene is given the host cell that imports this carrier resistance to medicine (for example G418, Totomycin or methotrexate).Preferred selectable marker gene comprises that Tetrahydrofolate dehydrogenase (is called for short: DHFR) gene (be used for dhfr-host cell with methotrexate selections/amplification) and neo gene (being used for the G418 selection).

For expressing described ICOS-Ig or hICOS-IgG1, the recombinant expression vector of described ICOS-Ig of coding or hICOS-IgG1 is transfected into host cell by standard technique.The various forms of term " transfection " has been contained numerous various common technologies that are used for foreign gene is imported protokaryon or eukaryotic host cell.For example electroporation, calcium phosphate precipitation, deae dextran transfection, liposome etc.Although can in prokaryotic host cell or eukaryotic host cell, express ICOS-Ig of the present invention or hICOS-IgG1 in theory, but most preferably in eukaryotic cell, most preferably in mammalian cell, express ICOS-Ig or hICOS-IgG1, because this eukaryotic cell, particularly mammalian cell more likely assemble and secrete the ICOS-Ig or the hICOS-IgG1 of correct folding and tool immunosuppressive activity than prokaryotic cell prokaryocyte.Preferred mammalian host cell of expressing recombination fusion protein of the present invention comprise Chinese hamster ovary cell (be called for short: CHO), Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary celI (be called for short: the dhfr-CHO cell), the African green monkey kidney fibroblast-like cells (be called for short: the COS cell) or HEKC 293 cells) etc. (be called for short: one or more in.When the recombinant expression vector of coding ICOS-Ig or hICOS-IgG1 gene is imported mammalian host cell, by cultivating the enough time of described host cell in described host cell, expressing this recombination fusion protein, or more preferably this recombination fusion protein is secreted in the substratum of described host cell growth and produces described ICOS-Ig or hICOS-IgG1.Use the standard protein purification method can from this substratum, reclaim ICOS-Ig or hICOS-IgG1.

In the commending system of a recombinant expressed ICOS-Ig of the present invention or hICOS-IgG1, import Chinese hamster ovary celI by will both the encode recombinant expression vector of this recombination fusion protein of liposome-mediated transfection.In this recombinant expression vector, described ICOS-Ig or hICOS-IgG1 gene may be operably coupled to the enhancers/promoters regulatory element and (for example transcribe with the high level that drives described gene from SV40, CMV etc., this recombinant expression vector also carries dihydrofolate reductase gene (to be called for short: the DHFR gene), can select/increase to select to have used the Chinese hamster ovary celI of this carrier transfection with methotrexate, cultivate this transformant host cell of selecting expressing described ICOS-Ig or hICOS-IgG1, and from this substratum, reclaim ICOS-Ig or hICOS-IgG1.Use the standard molecular biology method to prepare this recombinant expression vector, the described host cell of transfection, select transformant, cultivate described host cell and from this substratum, reclaim this ICOS-Ig or hICOS-IgG1.

In view of aforementioned, another aspect of the present invention relates to nucleic acid, carrier and the host cell combination that can be used for recombinant expressed ICOS-Ig of the present invention or hICOS-IgG1.

(4) purposes of recombination fusion protein

1, general introduction

Further purpose of the present invention provides a kind of product with immunosuppressive action and treatment autoimmune disorder or transplant rejection and associated conditions thereof, comprises medicine, detection reagent, diagnostic reagent etc., especially a kind of medicine.

Recombination fusion protein of the present invention can be used for animal especially Mammals prevention and treatment " wherein the ICOS activity is deleterious disorder ".This " wherein the ICOS activity is deleterious disorder " can develop because of the existence of ICOS and worsen, and suppresses the ICOS activity and just can treat this disorder.Described Mammals comprises people and non-human mammal, and described non-human mammal comprises one or more in mouse, rat, sheep, monkey, ox, pig, horse, rabbit, dog, chimpanzee, baboon, marmoset, macaque or the rhesus monkey etc.Recombination fusion protein of the present invention can be given and people curee with therapeutic purpose.Recombination fusion protein of the present invention is given and non-human mammal, to reach animal doctor's purpose or as the animal model of human diseases.The relevant latter, this animal model can be used to estimate the treatment effectiveness (for example testing drug effect, dosage and time course) of recombination fusion protein of the present invention.

Term used herein " wherein the ICOS activity is deleterious disorder " comprises disease and other disorder.Wherein, in suffering from this disorderly curee, the existence of ICOS has demonstrated or is under a cloud or this sick physiopathology is responsible for or is enough to the factor that causes this disease to increase the weight of.Therefore, wherein ICOS is deleterious disorder a kind of disorder that comes to this, and wherein the active inhibition expection of ICOS will alleviate this disorderly symptom and/or process.This disorder can confirm by the increase (for example, expressing increase on cell, the spleen cell) of ICOS expression in the organism that for example suffers from this sick curee.It is examples of deleterious disorder that many wherein ICOS are arranged.

Below the purposes of ICOS120 recombination fusion protein of the present invention in preventing, detect, diagnose, treat and study concrete disorderly hole further is discussed.

Disorder as herein described comprises:

Autoimmune disorder: comprise systemic autoimmune disorder and associated conditions thereof (as systemic lupus erythematous, rheumatoid arthritis, scleroderma, ankylosing spondylitis, osteoarthritis, adult onset still disease, Behcet's disease, special syndrome, psoriatic, relapsing polychondritis, systemic vasculitis, polyarteritis or autoimmune hemolytic anemia and the associated conditions thereof etc. of relying); Organ specificity autoimmune disease and associated conditions thereof (as endocrine system autoimmune disease, blood system autoimmune disease, cardiovascular systems autoimmune disease, respiratory system autoimmune disease, Digestive tract autoimmune disease autoimmune disease, urinary system autoimmune disease, neural system autoimmune disease, autoimmunity illness in eye or autoimmune skin disease and associated conditions thereof etc.) etc.; Transplant rejection and associated conditions etc. thereof; After the percutaneous transluminal coronary angioplasty loose or closed, the PTCA/ stent/bypass surgery postoperative blood vessel of blood vessel again closure/restenosis, get involved that the back blood vessel is loose or closed, the caused vascular disorder of implantable graft and repulsion and associated conditions thereof etc.; Allergic disease: comprise in asthma, rhinallergosis, conjunctivitis, allergic reaction of alimentary canal, pollinosis or anaphylaxis and the associated conditions thereof etc. one or more; Inflammatory diseases: comprise in inflammation after pharyngolaryngitis, urocystitis, pneumonia, myocarditis, myocardosis, meningitis, inflammatory eye disease, pneumonia, silicotuberculosis, sarcoidosis of lung, pulmonary tuberculosis, sacroiliitis and associated conditions thereof (as osteoarthritis, similar rheumatism myelitis and periosteoma forms and associated conditions etc.), the postoperative/wound or atopic dermatitis and the associated conditions thereof etc. one or more; Diabetes and complication thereof: comprise in retinopathy, ephrosis, neuropathy and great vessels disease, diabetic nephropathy or unusual glucose tolerance disease and the associated conditions thereof etc. one or more; Disease in the inflammatory bowel: comprise in Crohn disease or ulcerative colitis and the associated conditions thereof etc. one or more; Circulatory diseases: comprise in chronic heart failure, heart disorder, stenocardia, myocardial infarction, cardiac insufficiency and congestive heart failure, arteriosclerosis, atherosclerosis, hypertension, dvt formation, occlusive peripheral circulatory failure, ischemic circulatory failure, disseminated intravascular coagulation, Raynaud disease, Buerger's disease, portal hypertension, pulmonary hypertension or dialysis property hypertension and the associated conditions thereof etc. one or more; Psoriasis and associated conditions etc. thereof; Hepatopathy: comprise in hepatitis, chronic hepatopathy or liver cirrhosis and the associated conditions thereof etc. one or more; Pancreatic disease: comprise pancreatitis and associated conditions thereof etc.; Neurodegenerative disease: comprise in presenile dementia (Alzheimei), Parkinson's disease, amyotrophic lateral sclerosis or AIDS encephalopathic and the associated conditions thereof etc. one or more; Central nervous system disorder: comprise in cerebrovascular disorder, hematencephalon, cerebral infarction and sequela thereof, cranial injury, spinal injury, cerebral edema, dementia, dysmnesia, the disturbance of consciousness or multiple sclerosis and the associated conditions thereof etc. one or more; Toxicaemia: comprise in septicemia, septic shock, Gram-negative concentration disease or toxin shock syndrome and the associated conditions thereof etc. one or more; Gestosis and associated conditions thereof; Obesity and associated conditions thereof; Hyperlipidaemia: comprise in hyperlipidemia or high cholesterol disease and the associated conditions thereof etc. one or more; Tumour: comprise in malignant lymphoma, stomach and intestinal cancer disease, cancer and the emaciation of following or solid tumor and the associated conditions thereof etc. one or more; Endocrinopathy: comprise in Addison's disease, Cushing's syndrome, melanocytoma or primary aldosteronism and the associated conditions thereof etc. one or more; Creutzfeldt-Jacob disease and associated conditions thereof; Viral infection: comprise in cytomegalovirus infection or influenza virus and the associated conditions thereof etc. one or more; Illness in eye: comprise in glaucoma or high intraocular pressure and the associated conditions thereof etc. one or more; Myasthenia and associated conditions etc. thereof; Confirmed fatigue and associated conditions etc. thereof; Osteopathia: comprise that joint tissue in fracture, refracture, osteoporosis, osteomalacia, bone Behet disease, ankylosing spondylitis or osteoarthritis and the relative disease destroys and associated conditions etc. in one or more.In addition, recombination fusion protein of the present invention can be separately or is united with other active ingredients and to be used for the treatment of.

Known recombination fusion protein of the present invention is in conjunction with the ability of ICOS part, can use it for and join rabbit epidemic disease absorption detection (abbreviation: ELISA) method, radioimmunity detection (abbreviation: RIA) in the routine immunization detection method such as method or histogenic immunity histochemical method, from biological sample, detect the ICOS part such as enzyme such as serum or blood plasma.The invention provides in biological sample the active method of ICOS that detects, this method comprises with recombination fusion protein of the present invention and contacts biological sample and detect or be bonded to ICOS the part activity of ICOS and the content of ICOS part in the detection of biological sample thus.For the ease of detector ligand bonded recombination fusion protein, with this recombination fusion protein of the direct or indirect mark of detectable substance.Suitable detectable substance comprises various enzymes, prothetic group, fluorescent substance, luminophore and radioactive substance etc.The example of suitable enzyme comprises horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes or acetylcholinesterase etc.; Suitable prothetic group complex body example comprises streptavidin/vitamin H and avidin/biotin etc.; The example of suitable fluorescent substance comprises Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine amine fluorescein, dansyl chloride or phycoerythrin etc.; The example of luminophore comprises luminol,3-aminophthalic acid cyclic hydrazide etc.; And the example of suitable radioactive substance comprises ¹²⁵I, ¹³¹I, ³⁵S or ³H etc.

Can also use the ICOS part of recombination fusion protein detection of the present invention from other species except that the mankind, the ICOS part of one or more in mouse, rat, sheep, monkey, ox, pig, horse, rabbit, dog, chimpanzee, baboon, marmoset, macaque or the rhesus monkey etc. for example is because recombination fusion protein can combine with each of these ICOS parts.

Recombination fusion protein of the present invention is external and all can suppress the ICOS activity in vivo; The ICOS activity that can also suppress in addition, other species.Therefore, recombination fusion protein of the present invention can be used, for example, in the cell culture that contains the ICOS part, suppresses the ICOS activity in human subject or in other has the mammalian subject of the ICOS part that recombination fusion protein of the present invention can cross reaction, and described mammalian subject comprises one or more in mouse, rat, sheep, monkey, ox, pig, horse, rabbit, dog, chimpanzee, baboon, marmoset, macaque or the rhesus monkey etc.

2, the drug combination of recombination fusion protein

Recombination fusion protein of the present invention can be united fields such as being used for scientific research, disease treatment or medical diagnosis on disease separately or with other active ingredient.

Can comprise following with the non-limitative example of the Parmaceutical for treating disease of autoimmunization system thing of recombination fusion protein coupling of the present invention: nonsteroid anti-inflammatory drugs (as NSAID), cytokine suppresses antiphlogiston (as CSAID), CDP-571/BAY-10-3356 (peopleization anti-TNF alpha antibodies: Celltech/Bayer company product), anti-Tac (the anti-IL-2R α of peopleization: Protein DesignLabs/Roche company product), IL-4 (being a kind of anti-inflammatory cytokines), Ibuprofen BP/EP (belonging to non-class) because of pure antiphlogiston, adjoin sieve former times health (belonging to non-class) because of pure antiphlogiston, the fragrant acid of two helium, endoxan, S-Neoral, methotrexate, Phenylbutazone, IGIV, Interferon, rabbit-Fu la (trade(brand)name: Avonex7M; Biogen company product), one or more in Interferon, rabbit-Fu, methyl meticortelone, Ultracortene-H, azathioprine, reflunomide or the trypterygine etc.

Can comprise following with the organ-graft refection of recombination fusion protein coupling of the present invention and/or the non-limitative example of graft versus host disease (GVH disease) medicine: azathioprine, endoxan, ciclosporin, suprarenal gland glucocorticosteroid, Prograf be (as Tacrolimus, FK506), mycophenlate mofetil, rapamycin, the smart guanidine element of 15-deoxidation, Tripterysium Glucosides, Brequinar Sodium (be called for short: BQR), Mizoribine (is called for short: MZ), in antilymphocyte antibody, genetically engineered costimulatory molecules inhibitor or all kinds of antimicrobial drugs etc. one or more.

Can comprise following with the non-limitative example of other disease therapeuticing medicine of recombination fusion protein coupling of the present invention: the treatment of inflammatory bowel medicine comprises in Urogastron, reflunomide, ring robe rhzomorph, sulfasalazine, aminosalicylate, Ismipur, metronidazole, aminosallcylic acid, antioxidant, Ultracortene-H, dexamethasone, slowly-releasing aminosalicylic acid, methotrexate, Ciprofloxacin or the lignocaine etc. one or more; The therapeutical agent of cardiovascular disorder comprises cardiotonic drug, diuretic(s), calcium antagonist, beta receptor blocker or the like; Remedies for diabetes comprises among Regular Insulin, Actos, Rosigilidazone, Kinedak, BENFIL, Humulin, Euglucon, Glimicron, Daonil, Nobolin, Monotard, Glucobay, Dimelin, Rastinon, BASILCON, Deamelin S or the Iszilin etc. one or more; The treatment of kidney disease agent comprises in prednisolone (Predonine), Urbason Solubile (Solu-medrol), Betamethasone Valerate (Rinderon), hydrochloric acid Cormelian (Comelian) or the tyropidine Fxa inhibitor etc. one or more; The osteopathia therapeutical agent comprises for example lime carbonate etc. of calcium preparation; Calcitonin preparation; The activity of vitamin d3 preparation; Hormone preparation; Sexual hormoue comprises oestrogenic hormon etc.; PGA1; Bone morphogenic protein (is called for short: BMP); Cell growth factor; Transforming growth factor; Rat parathyroid hormone 1-34 (be called for short: PTH) or the like; And other Remedies for diseases.

3, the using method of recombination fusion protein and composition thereof and requirement

Recombination fusion protein of the present invention can be united use separately or with other active ingredient, comprises being used to prepare the product that is used for the treatment of, diagnoses, detects and study relative disease, comprises medicine, detection reagent, diagnostic reagent etc., especially medicine.

Aspect concrete use, recombination fusion protein of the present invention can use separately, can also use with other many chemical substances.These chemical substances biologically active or have the function of treatment disease whether no matter, comprise subsidiary function as collaborative amplification, antagonism or alleviate the side effect etc. of recombination fusion protein, these chemical substances are to comprise a kind of in pharmaceutically acceptable carrier, food, natural product, chemical synthetic drug, veterinary medicine or the human medication etc.; Preferably include a kind of in pharmaceutically acceptable carrier or the food etc.; Further preferred pharmaceutically acceptable carrier.

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

From concrete classification, said pharmaceutically acceptable carrier is meant the pharmaceutical carrier of medicine and pharmacology field routine, comprises vehicle, as starch, water etc.; Lubricant is as glycerine, Magnesium Stearate etc.; Disintegrating agent is as Microcrystalline Cellulose etc.; Weighting agent is as starch, lactose etc.; Caking agent is as pregelatinized Starch, dextrin, derivatived cellulose, alginate, gelatin and polyvinylpyrrolidone etc.; Osmotic pressure regulator is as glucose, sucrose, sorbyl alcohol and N.F,USP MANNITOL etc.; Thinner is as water etc.; Disintegrating agent is as agar, lime carbonate and sodium bicarbonate etc.; Absorption enhancer is as quaternary ammonium compound etc.; Tensio-active agent is as cetyl alcohol etc.; Absorption carrier is as kaolin and soap clay etc.; Lubricant is as talcum powder, calcium stearate and magnesium and polyoxyethylene glycol etc.; In addition, other assistant agent can also be added, as flavouring agent, sweeting agent etc. in composition.

For example, the active ingredient recombination fusion protein is dissolved, suspendible or (for example be emulsifiable in the suitable aqueous solvent, distilled water, physiological saline and Green's solution etc.) or oil-based solvent in (for example, vegetables oil is sweet oil for example, sesame oil, Oleum Gossypii semen and Semen Maydis oil, propylene glycol etc.) in, can make injection formulations, wherein (for example can contain dispersion agent in the solvent, Polysorbate 80, polyoxyethylene hardened castor oil 60, polyoxyethylene glycol, phenylcarbinol, chlorobutanol, phenol etc.) or osmotic pressure regulator (for example, sodium-chlor, glycerine, the D9-seminose, the D-sorbyl alcohol, glucose etc.) etc. one or more in.In this case, if necessary, can add additive, for example one or more in solubilizing agent (for example, sodium salicylate, sodium-acetate etc.), stablizer (for example, human serum albumin etc.) or pain killer (for example, phenylcarbinol etc.) etc.

Of the present invention and recombination fusion protein can also unite use with the form of composition, particularly with other chemical substance such as medicine animal especially Mammals is comprised that people or other animals treat compositions for use or similar compositions.Described Mammals comprises in people, mouse, rat, sheep, monkey, ox, pig, horse, rabbit, dog, chimpanzee, baboon, marmoset, macaque or the rhesus monkey etc. one or more.For example, recombination fusion protein of the present invention can be added be suitable for to curee's medicinal compositions in.Usually, this medicinal compositions comprises recombination fusion protein of the present invention and pharmaceutically acceptable carrier.

The composition of recombination fusion protein of the present invention particularly pharmaceutical composition can have various forms.These forms comprise for example liquid, semisolid and solid dosage form; Wherein said pharmaceutical composition comprises that the recombination fusion protein for the treatment of significant quantity is an activeconstituents, and one or more pharmaceutically acceptable carriers.The various forms of the composition of described recombination fusion protein of the present invention, for example liquor (for example injection liquid and transfusion), dispersion liquid or suspension, tablet, pill, pulvis, liposome and suppository.The form of recommending depends on administering mode and therepic use.Typical recommendation composition is the form of injection liquid or transfusion, for example with other genetically engineered drug the people is treated the compositions for use similar compositions.The administering mode of recommending is non-enteron aisle (for example intravenously, subcutaneous, intraperitoneal, an intramuscular).Most preferably give and this recombination fusion protein by intramuscular injection or subcutaneous injection.

The pharmaceutical composition of recombination fusion protein generally must be aseptic and stable under the production condition of storage.Said composition can be mixed with solution, microemulsion, dispersion liquid, liposome or other is suitable for the ordered structure of high drug level.By with a kind of of this recombination fusion protein of aequum and required mentioned component or combine to add in the appropriate solvent and then carry out Sterile Filtration and prepare aseptic parenteral solution.Generally speaking, prepare dispersion liquid by this recombination fusion protein being added in the aseptic solvent that contains basic dispersion medium and required above-mentioned other composition.Under the situation of the sterile powder that is used to prepare aseptic parenteral solution, the preparation method of recommendation is vacuum-drying and lyophilized preparation.For example, by passing through to keep required granular size such as the dressing of Yelkin TTS, under the situation of dispersion liquid and, can keeping the adequate liquidity of solution by using tensio-active agent.By comprising that in said composition the medicament (for example Monostearate and gelatin) that postpones to absorb can reach the prolongation absorption of injectable composition.

4, the pharmaceutical dosage form of recombination fusion protein and composition thereof and route of administration

The product of the treatment, diagnosis, detection or the scientific research that are used for immunosuppression and autoimmune disorder or transplant rejection and associated conditions thereof of recombination fusion protein of the present invention and preparation of compositions thereof, wherein the product according to the requirement of beverage, food technology field preparation can carry out immunosuppression and treatment or diagnosis of autoimmune disease or transplant rejection and associated conditions thereof; Can be used in patient's treatment, diagnosis, detection or health care according to the product of the requirement of medical technical field preparation, can either be directly used in the medicine of preparation treatment, diagnosis, detection or health care separately, also can mix with many chemical substances or make up, directly or indirectly be used to prepare the medicine of treatment, diagnosis, detection or health care.Chemical substance described here is above described identical with this section.

In the present invention, required material comprises raw material of the present invention, above-mentioned matching used chemical substance etc., all should adopt the material of food grade or pharmaceutical grade according to practical situation and needs.

The pharmaceutical composition of recombination fusion protein can adopt conventional production method well known in the art to make various formulations, and activeconstituents is mixed with one or more carriers, is made into required formulation then.Described formulation comprises tablet, capsule, granule, suspensoid, emulsion, solution, syrup, injection etc., takes oral or route of administration such as injection (comprising intravenous injection, intravenous drip, intramuscular injection, subcutaneous injection etc.), mucous membrane dialysis are carried out treatment, diagnosis, detection or the scientific research of immunosuppressive action and treatment, diagnosis, detection or the scientific research of treatment autoimmune disorder or transplant rejection and associated conditions thereof.

Recombination fusion protein of the present invention can be with the whole bag of tricks administration known in the art, although route of administration/administering mode of recommending in many therepic use is intravenous injection or transfusion.But the technician will appreciate that route of administration/administering mode changes with required result.In certain embodiments, the carrier that this active compound can avoid snap-out release with this compound of protection is preparation example such as empty release formulation together, comprises that graft, transdermal paste and the micro-capsule transfer system.In addition, can also use biodegradable, biocompatible polymer, for example ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters and poly(lactic acid) etc.Prepare the equal patent applied for of many methods of this preparation or generally known to those skilled in the art (referring to for example Sustained and Controlled Release Drug Delivery Systems, J.R.Robinson edits, Marcel Dekker, Inc., New York, 1978).

In certain embodiments, recombination fusion protein of the present invention can be together oral with for example inert diluent or assimilable edible carrier.This compound (with other compositions, if necessary) can also be wrapped in hard or soft shell gelatin capsules, is pressed into tablet or directly adds in curee's the meals.About oral therapeutic administration, described compound can be added with vehicle and uses with edible tablet, buccal tablet agent, lozenge, capsule, suspension, syrup, wafer or the like form.For to give outside the parenterai administration and compound of the present invention, may need with the material that prevents its inactivation to this compound dressing or with this compound together give with.The active compound that replenishes can also be added in the said composition.In certain embodiments, recombination fusion protein of the present invention and one or more can be used for the treatment of, diagnose, detect ICOS active for other medicine of deleterious disease prepare altogether and/or altogether to.For example, recombination fusion protein of the present invention can combine with one or more treatment, diagnosis, testing product such as the medicine of other target or reagent (for example in conjunction with the recombination fusion protein of other cytokine or cell surface binding molecule or antibody etc.), one or more cytokines, anti-ICOS inhibiting antibody and/or multiple inhibition pharmaceutical chemistry suppress that ICOS produces or active medicine is prepared altogether and/or give altogether with.In addition, can be with one or more recombination fusion proteins of the present invention and two kinds or multiple aforementioned therapies drug combination.This unite use can utilize primely than low dosage should give with treatment, diagnosis, testing product such as medicine or reagent, therefore avoid possible toxicity or the complication relevant with various monotherapies.

Make liquid preparation such as aqua, oil-suspending agent or other liquid preparation such as syrup, elixir etc.; When being used for administered parenterally, can be made into solution, aqua or the oiliness suspension agent etc. of injection.

In the above-described type of service, preferred form is one or more in tablet, capsule, granule, suspensoid, emulsion, solution, syrup or the injection etc., in further preferred capsule, granule, suspensoid, emulsion, solution or the injection etc. one or more, one or more in preferred especially suspensoid, emulsion, solution or the injection etc.

According to object, route of administration, institute's disease for the treatment of and the situation etc. of treatment, dosage every day of variation recombination fusion protein of the present invention.For example, give Mammals, especially grownup (60kg body weight) through vein, the single dose of described recombination fusion protein is about 10～1200mg, preferably about 100mg, preferably administration 1～3 time weekly.Can adjust dose unit so that best required reaction (for example treatment or prevention are replied etc.) to be provided.For example, can single heavy dose of administration can be given in for some time with several divided doses or according to the urgency of treatment situation and be reduced or increase dosage in proportion.It is especially favourable that preparation is easy to the non-enteron aisle composition of the unified dosage unit form of administration and dosage.Dosage unit form used herein refers to be suitable for the physical sepn unit of dosage unit of the mammalian subject of desire treatment; The calculating that each unit contains predetermined amount is used for together producing with required pharmaceutical carrier the active compound of required result of treatment.The specification of dosage unit form of the present invention is determined and is directly depended on the specific characteristic of following (a) this active compound and the particular treatment of desiring to reach or preventive effect and (b) interior in restriction in mixing this technology that is used for the treatment of the individual sensitivity active compound by following.

Medicinal compositions of the present invention can comprise the recombination fusion protein of the present invention of " treatment significant quantity " or " prevention significant quantity "." treatment significant quantity " is meant at the dosage of necessity and effectively reaches the amount of required result of treatment under the time.The treatment significant quantity of recombination fusion protein can cause that at this individuality the factors such as ability of required reaction change according to the patient's condition, age, sex and body weight and this recombination fusion protein such as individuality.The treatment significant quantity also refers to that the useful result of treatment of this recombination fusion protein surpasses the amount of its any toxicity or harmful effect." prevention significant quantity " is meant the amount that effectively reaches required preventive effect under necessary dosage and time.Because preventive dose is used for the ill preceding or early stage curee of disease, the prevention significant quantity is usually less than the treatment significant quantity.The typical non-limiting scope of the treatment of recombination fusion protein of the present invention or prevention significant quantity is 0～20mg/kg, more preferably 1～10mg/kg.Should notice that dose value will change according to disease type of desiring to alleviate and seriousness, that is to say when being used for the patient, the dosage of recombination fusion protein of the present invention or consumption decide according to patient or user's the age and the situation of body weight and physical appearance or patient's symptom usually.In addition; should understand; for any specific curee; should along with the time according to individual need and give with or supervision give with the people's of described composition professional judgement and adjust the given dose system; and the dosage range that this paper sets only be illustrative, the scope or the practice of the composition of can't requirement for restriction protecting.

In sum, recombination fusion protein of the present invention and composition thereof can be used for preparing the product of treatment, diagnosis, detection or the scientific research of immunosuppression and autoimmune disorder or transplant rejection and associated conditions thereof, preferred agents, reagent and food, further preferred agents and reagent, most preferably medicine.

(5) technology speciality

Result of study of the present invention, can deeply be developed as product such as new drug, the reagent etc. of preventing and treating aspects such as autoimmune disorder and transplant rejection with potential applicability in clinical practice, have the advantage that determined curative effect, effective dose are low, have no side effect, compare with similar products at home and abroad and have significant advantage.Autoimmune disorder and transplant rejection are common clinical, frequently-occurring disease, along with the raising of living standards of the people, estimate that the sickness rate of autoimmune disorder and transplant rejection disease will constantly rise.Therefore, the present invention can lay a good foundation for follow-up product research such as new drug, and further produces bigger economic benefit and social benefit.

The invention provides the recombination fusion protein that suppresses mammiferous ICOS molecular activity, but not suppress the active recombination fusion protein of other costimulatory molecules.Described Mammals, same as above.

Recombination fusion protein is ICOS-Ig particularly, has the following advantages: 1. can suppress to remember the function with activating T cell specifically, immune suppression function is strong and special; 2. can mass preparation, by carrying out affinity chromatography with anti-Fc section antibody, staphylococcal protein A,SPA is purifying easily, and detection of fusion proteins is convenient, special, responsive, and preparation cost and use cost are obviously lowered.3. directly produce, not only more absorb, and avoided AIDS, hepatitis virus and other pathogeny microbiological contamination near the suitable body of the situation of natural molecule with the Mammals engineering cell; 4. according to the human source gene design, can not cause internal antibody to produce, safe and reliable; 5. after having merged the Fc section of immunoglobulin (Ig), fusion rotein is not only possessed and ICOS ligand molecular bonded all sites, and can form dimer molecule, strong with ligand binding capacity, transformation period is longer, is more suitable in intravital application, even can passes placenta performance drug effect.

At present, to be the nonspecific inhibitor toxic side effect strong for the immunosuppressor of Shi Yonging clinically, the prepared recombination fusion protein of the inventor can play immunosuppressive action specifically, toxic side effect is little, safe and reliable, and can be in the external mass preparation of carrying out, production cost is lower, help applying, have the value of good clinical application and scientific research and good social benefit and economic benefit.

In sum, through secular pharmacology test, this recombination fusion protein has the activity of immunosuppressive action and treatment autoimmune disorder and transplant rejection and associated conditions thereof, directly use this recombination fusion protein, need not make effect more direct through the metabolism of gi tract bacterium, avoid that gastrointestinal bacterial flora between the individuality is active variantly to be caused the difference of meta-bolites and produce difference on the drug effect, thereby overcome the unmanageable problem of dosage, and improved bioavailability.

In addition, this recombination fusion protein chemical property is stable, the effect of the immunosuppression product of preparation such as medicine and treatment autoimmune disorder and transplant rejection and associated conditions thereof is obvious, so it is more suitable for preparing the especially suitability for industrialized production of medicine and treatment, diagnosis, detection autoimmune disorder and transplant rejection and associated conditions product such as medicine of immunosuppression product.

The activity form of this recombination fusion protein in the gi tract bio-transformation directly uses this recombination fusion protein, and the bioavailability of medicament height can accurately be controlled dosage.The recombination fusion protein stable in properties uses the quality of the pharmaceutical preparations of recombination fusion protein preparation stable.

Recombination fusion protein pharmacological action of the present invention is stronger, and its raw material sources are abundant, and preparation technology is simple, the yield height.

The present invention studies recombination fusion protein targetedly, and this raw material is safe in utilization, takes into account each other, one-object-many-purposes has been brought into play effect to greatest extent, and use range is wide especially, therefore apply easily, can have a tremendous social and economic benefits in the short period of time.

In a word, active adaption of the present invention the need of work in fields such as modern medical service, food, beverage and scientific research and the needs of human nature service, be to be used to prepare the especially safe raw material of medicine of immunosuppression product such as medicine and treatment, diagnosis, detection autoimmune disorder and transplant rejection and associated conditions product thereof.

Description of drawings

Fig. 1 illustrates combining of ligand molecular on hICOS-IgG1 and the ligand expression cell;

Fig. 2 illustrates that hICOS-IgG1 is to the activated lymphocyte inhibition of proliferation;

Fig. 3 illustrates the inhibition of hICOS-IgG1 to the cytokine secretion function of activated lymphocyte.

Embodiment

The present invention studies and discloses a kind of new recombination fusion protein and its production and use, provide a kind of and can be used in preparation and can be used in the especially raw material of medicine and treatment, diagnosis, detection autoimmune disorder and transplant rejection and associated conditions medicine thereof of immunosuppressant product, be convenient to the safe handling in medical industry and fields such as relevant industries such as food, beverage.

That is to say that emphasis of the present invention provides that a kind of genetically engineered is fertile, expression amount is high, be easy to purifying, have the biologic activity that suppresses ICOS and have the recombination fusion protein of height biological safety, this is a kind of new biological immunosuppressant thing.And announce the preparation method that it is main, and the purposes of this product.

(1) following is example with hICOS-IgG1, elaborates the concrete preparation method of recombination fusion protein.

1, the concrete preparation method of hICOS-IgG1 of the present invention:

1. according to N end 21～140 amino acid whose dna sequence dnas of people source ICOS, the design primer clones required ICOS fragment with standard pcr from the DNA of human peripheral blood single nucleus cell; From the plasmid (pIgplus) that contains human IgG Fc, clone the constant fragment of immunoglobulin (Ig) with the same standard PCR method; With the SOC-PCR method required ICOS fragment and the constant fragment of immunoglobulin (Ig) are connected into hICOS-IgG1 recombination fragment, and with two above-mentioned recombination fragments of enzymic digestion of sfiI and NotI and Mammals carrier for expression of eukaryon pSecTag2/Hygro A, utilize the sticky end of the two enzymes of sfiI and NotI that required ICOS fragment is inserted among the pSecTag2/Hygro A, constitute pSecTag2/Hygro A-hICOS-IgG1 recombinant expression vector.

2. changing pSecTag2/Hygro A-hICOS-IgG1 recombinant expression vector over to Chinese hamster ovary cell with liposome method with liposome (or calcium phosphate or electrotransformation) (is called for short: CHO) in the host cell (or other mammalian host cell), screen the CHO host cell of single expression hICOS-IgG1 of the present invention with the medicine antibiotic hygromycin, with a large amount of above-mentioned cells that filter out of cultivating of serum free medium, the collecting cell culture supernatant is purified into hICOS-IgG1 with affinity chromatography from above-mentioned cell culture supernatant.

3. use the high-pressure liquid phase detecting instrument (to be called for short: HPLC) detect hICOS-IgG1 purity, (be called for short: SDS-PAGE) detect the hICOS-IgG1 molecular weight, detect the expression of hICOS-IgG1 with the Western-blot method with the polyacrylamide gel electrophoresis.

4. biologic activity detects and finds that hICOS-IgG1 can suppress the proliferative response of recessive allele people mixed lymphocytes, can also suppress the irritant reaction of anti-cd 3 antibodies to human lymphocyte.

2, hICOS-IgG1 and ICOS ligand-binding activity:

Principle: hICOS-IgG1 can combine with the ICOS ligand specificity of its ligand expression cell surface, under the certain situation of amount of ligand, along with the increase of hICOS-IgG1 content, also increases with its part binding capacity.Fluorescently-labeled anti-IgGFc antibody can combine with part bonded hICOS-IgG1, detects the fluorescence that sends in conjunction with anti-IgGFc antibody by flow cytometer and can react combining of hICOS-IgG1 and its part.

Operation steps: get finite concentration as 1%～50% animal serum such as monkey serum, bovine serum, rabbit anteserum, sheep blood serum etc., the constant segmental acceptor of IgG of sealing ligand expression cell such as Daudi cell (is called for short: FcR), get a certain amount of ligand expression cell as 1 * 10 ⁴～1 * 10 ⁸Individual Daudi cell, the washing back adds the hICOS-IgG1 and the blank of different concns, as (being called for short: PBS) wash hICOS-IgG1 and the blank that adds 1～50ug/ml after 1～5 time respectively with phosphate buffered saline buffer, hatching a few hours under the certain temperature hatched 1～6 hour as 0 ℃～50 ℃, as detecting antibody, analyze ligand-binding activity with the detection reagent such as anti-human IgG of HRP mark with test sets such as flow cytometers.

The result: the fluorescence intensity that the cell of counting some amount sends, increase with hICOS-IgG1 concentration, the fluorescence intensity of detection increases, and prompting hICOS-IgG1 combines with its part.

3, the biologic activity of hICOS-IgG1:

Principle: after lymphocyte was subjected to antigenic stimulation, in the presence of costimulatory molecules, lymphocyte activation also showed biologic activity such as hyperplasia, secrete cytokines.If there is not the participation of ICOS costimulatory molecules, the very fast inactivation of activated lymphocytes, even apoptosis.In the process of recessive allele lymphocytes interactions, add hICOS-IgG1, if lymphocytosis is suppressed or cytokine secretion is suppressed, the common stimulation path that has reflected ICOS is blocked, and illustrates that hICOS-IgG1 has blocking-up ICOS and stimulates the effect of path altogether and can suppress proliferative response between the recessive allele lymphocyte.

Operation steps: appoint and to get two healthy blood donors or other Mammalss peripheral blood mononuclear cell as two different sexes healthy blood donors, add the hICOS-IgG1 that cell culture fluid dilutes as the RPMI-1640 that contains calf serum, mixed culture a few hours, a few hours before stop cultivating, add detection reagent comprise isotope detection reagent as ³H-TdR, count results such as the per minute number of times that glimmers (is called for short: the cpm value).Get the supernatant liquor of above-mentioned mixed culture cell, the ELISA double antibody sandwich method is surveyed its cytokine content.

Result: hICOS-IgG1 can suppress the proliferative response of people or mouse recessive allele mixed lymphocytes, the also secretion of the factor capable of inhibiting cell.

4, the pharmacodynamics activity of hICOS-IgG1:

Principle: sensitization t helper cell subgroup Th2/Th1 is unbalance to be the main pathogenesis of ovum protein allergic asthma mouse model.ICOS stimulates path to participate in regulating t helper cell subgroup Th2/Th1 balance altogether.Treating ovum protein (OVA) allergic asthma mouse with hICOS-IgG1 is the restraining effect that ICOS is stimulated altogether path by in vivo test research hICOS-IgG1.

Operation steps: get body weight and be about 10～40g, the female or male and healthy mouse in about 4～10 ages in week as experimental animal (6～90 every group).Abdominal injection OVA is with sensitized mice.A certain amount of control antibodies of injection in disease group such as the asthma disease group mouse body, the treatment group is injected the hICOS-IgG1 of equivalent with quadrat method in the mouse body after, aerosol excites OVA or nasal cavity drop OVA; Control group gives the control antibodies or the hICOS-IgG1 of equivalent with same method in the mouse body, aerosol excites physiological saline or nasal cavity drop physiological saline.The last aerosol excites physiological saline or nasal cavity drop physiological saline after a few hours, detects the evaluation index respectively organize mouse and comprises that lung-douching fluid (is called for short: BALF), the peripheral blood cells factor, immunoglobulin (Ig), Th subgroup etc.

The result: the serum IgE level of treatment group mouse, the number of inflammatory cells of BALF and IL-4 level, peripheral blood Th2 ratio all obviously lower than disease group, illustrate that hICOS-IgG1 can stimulate path to treat disease such as asthma etc. by ICOS in the blocking-up mouse body altogether.

(2) the several concrete application of hICOS-IgG1 preparation

The present invention prepares powder injection and generally adopts conventional freeze-drying, as solvent, the steps include: to get recombination fusion protein with water, adds vehicle, is dissolved in water, and adds gac, filtration sterilization, and plug is partly rolled in can, and lyophilize, tamponade are rolled lid and are got final product.Used vehicle is selected from one or more in N.F,USP MANNITOL, gelatin hydrolysate, glucose, lactose, the dextran etc.Every bottle contains recombination fusion protein 10～100mg.

The present invention prepares powder injection also can adopt spray-drying process, as solvent, the steps include: to get recombination fusion protein with water, adds or do not add vehicle (vehicle is the same), be dissolved in water, add gac, filtration sterilization, spraying drying, aseptic subpackaged, tamponade is rolled lid and is got final product.Every bottle contains recombination fusion protein 10～100mg.

When the present invention prepared small-volume injection, preparation got final product as solvent with water for injection, also can add appropriate amount of auxiliary materials, and auxiliary material is selected from one or more in ethanol, propylene glycol, glycerine, polyoxyethylene glycol, peruscabin, the N,N-DIMETHYLACETAMIDE.Every contains recombination fusion protein 10～100mg.

The present invention prepares glucose infusion liquid or sodium-chlor transfusion, with water for injection as solvent, add the preparation of an amount of glucose or sodium-chlor and get final product, also can add appropriate amount of auxiliary materials, auxiliary material is selected from one or more in ethanol, propylene glycol, glycerine, polyoxyethylene glycol, peruscabin, the N,N-DIMETHYLACETAMIDE.Every bottle contains recombination fusion protein 10～100mg.

The present invention prepares oral preparations such as tablet, capsule, granule, oral liquid, and auxiliary material can be lactose, starch, dextrin, stearate etc., technology preparation routinely.

In the present invention, the example of above-described embodiment and the following stated all is in order to set forth the present invention better, is not to be used for limiting scope of invention.

The preparation method of embodiment 1, hICOS-IgG1

The source of main agents:

Lipofectamine2000, TRIZOL reagent are available from Invitrogen company; The various tool enzyme is available from Takara company; Totomycin is available from the general biotech company that flies in Shanghai; The DNA purifying reclaims test kit, plasmid DNA is extracted test kit available from the special clean company of dimension; IL-2, IFN-γ ELISA detection kit are available from crystalline substance U.S. biotech company.The pMD-18T cloning vector is preserved by this laboratory available from Invitrogen company, pGL-3-Basic cloning vector, the plasmid pMD-18T-Ig that carries the immunoglobulin Fc fragment gene available from TAKARA company, pSecTag2/Hygro A expression vector; Serum free medium EX302 is available from U.S. JRH company; Protein A affinity purification test kit is available from U.S. sigma company; 293 cells are available from cell institute of the Chinese Academy of Sciences.

(1) clone of ICOS extracellular fragment gene:

According to people source ICOS extracellular region sequence, the primer of design band restriction enzyme site.

With final concentration is the PMA of 20ng/ml and the ionomycin stimulation human peripheral blood mononuclearcell 72h of 200ng/ml, and collecting cell, Trizol extract total RNA, RT-PCR method amplification ICOS extracellular fragment gene 360bp, the pMD-18T cloning vector of packing into.

(2) clone of the Fc fragment gene of Ig:

The primer of design band restriction enzyme site for touching plate, amplifies its Fc section, the pMD-18T cloning vector of packing into plasmid that the Ig gene is housed.

(3) structure of hICOS-IgG1 integrative gene expression vector:

SfiI, XbalI double digestion pMD-18T-ICOS plasmid, it (can be any high efficiency mammalian eukaryotic expression such as pSecTag2/Hygro A, pcDNA4 His MAX etc. that the ICOS extracellular fragment directed cloning that downcuts is gone into pSecTag2/Hygro A expression vector.XbalI, NotI double digestion, plasmid with the directed above-mentioned expression vector of Fc fragment that downcuts, are finished the fusion of ICOS extracellular fragment and Fc section, obtain to carry the secretor type Mammals carrier for expression of eukaryon pSecTag2/Hygro A-ICOSIg of fusion gene.

(4) nucleotide sequencing of hICOS-IgG1:

The recombinant secretor type Mammals carrier for expression of eukaryon pSecTag2/Hygro A-ICOS-Ig that carries fusion gene is checked order, check the exactness of fusion gene open reading frame.

(5) integrative gene expression vector transfection host cell and set up the stably express strain:

With liposome (or calcium phosphate or electrotransformation) will recombinate integrative gene expression vector pSecTag2/HygroA-hICOS-IgG1 (as pSecTag2/Hygro A-hICOS-IgG1 etc.) transfection CHO cell or other host cell (as various derivative type Chinese hamster ovary celIs, 293 cells, various derivative type 293 cells etc.).The selection substratum screening that contains the 800ug/ml antibiotic hygromycin was subjected to transfect cell 14～20 days, and ELISA detects the expression level of hICOS-IgG1, selected the high monoclonal cell of expression level and cultivated the frozen guarantor's kind of part liquid nitrogen; All the other cells enter suspension culture in the serum free medium through after taming.

(6) the ELISA double antibody sandwich method is identified hICOS-IgG1 secreting, expressing amount:

To 3ug/ml, wrap by 96 hole elisa plates 4 ℃ of night incubation with pH9.6 carbonic acid buffer dilution goat anti-human igg antibody; With the sealing of 1% bovine serum albumin, hatched 1 hour for 37 ℃; Adding 72～96 hours supernatant liquor of positive colony cell cultures and 500ng/ml doubling dilution become the pure product of human IgG and the blank of 6 concentration, hatch 1 hour for 37 ℃; Add the anti-human IgG antibody of HRP mark horse, hatched 1 hour for 37 ℃; Add the tmb substrate damping fluid, room temperature is placed colour developing in 5～20 minutes; Read every hole absorbancy with microplate reader with 450nm after the termination reaction, according to the OD value preparation standard curve of human IgG standard substance correspondence, with the expression level of estimation secretory protein.

(7) a large amount of preparations of hICOS-IgG1:

HICOS-IgG1 high expression level Chinese hamster ovary celI with picking out carries out the serum-free suspension culture, and concrete steps are to get 5 * 10 ⁵Individual cell adds in the EX302 substratum that contains 5% calf serum and 500ug/ml Totomycin to be cultivated with the conventional organization cell culture method, when treating cell length to 80%～90% full scale; With passage, with the EX302 culture medium culturing that contains 2% calf serum and 500ug/ml Totomycin during to cell length to 80%～90% full scale; With passage, when continuing with the EX302 culture medium culturing that contains 1% calf serum and 500ug/ml Totomycin to cell length to 80%～90% full scale with passage, cell changed over to shake use the EX302 culture medium culturing in the bottle (or bio-reactor, roll bottle), shake bottle and place on the shaking table and carry out the cell suspension cell cultures with 60～120 rev/mins of rotating speeds, all the other culture condition are with the conventional organization cell culture method.All will find that the cell of death or vigor difference abandons with trypan blue staining monitoring cell viability every day in passage and the suspension culture process each time.Collect cultured cells supernatant in the EX302 substratum, 4 ℃ of short-terms are preserved.

(8) purifying of hICOS-IgG1:

Supernatant liquor with 4 ℃ of short-terms preservations, be 50 in time with molecular weight cut-off, 00～10, the ultra-fine filter of 000Da concentrates, the concentrated solution protein A affinity purification column purification (or ammonium sulphate precipitation method) of flowing through, collect elutriant, 280nm ultraviolet light absorption photometer detects protein content ,-20 ℃ of hICOS-IgG1 that preserve purifying after the packing.

(9) detection of hICOS-IgG1 ligand-binding activity:

1 * 10 ⁶After individual Daudi cell washs secondary with PBS, add the anti-B7RP-1 antibody of PE mark, room temperature effect 30 minutes behind the PBS washing secondary, detects with flow cytometer, and Cellqueat software obtains cell and carries out Daudi cell ICOS ligand molecular phenotype analytical.Behind the FcR with 20% rabbit anteserum sealing Daudi cell, add the positive colony emiocytosis supernatant (2ug/ml, 10ug/ml) and the blank that detect through the ELISA method respectively, hatching 1 hour for 37 ℃, is to detect antibody with the anti-human IgG of HRP mark, uses the flow cytometry analysis ligand-binding activity.

The result shows that hICOS-IgG1 has ligand-binding activity.

(10) hICOS-IgG1 biological activity determination:

10.1 get the mononuclearcell (2 * 10 of two healthy blood donor peripheral bloods ⁶/ ml) each 50ul, the purifying hICOS-IgG1 100ul (final concentration be 209,105,53,27,14,7,0ug/ml) that adds the RPMI-1640 dilution contain 10% calf serum, each concentration triplicate, mixed culture is 96 hours in 96 orifice plates, stop cultivating preceding 16～18 hours, add ³H-TdR, counting cpm value.Get the supernatant liquor of above-mentioned mixed culture cell, the ELISA double antibody sandwich method is surveyed its IL-2, IFN-γ content.

10.2 (be called for short: OVA) with 200ul 1.36%Al (OH) in the 0th, 7,14 day abdominal injection 20ug chicken egg protein ₃Suspension with the healthy BALB/c mouse of sensitization SPF level; The back gives different treatment by different grouping: rose for three days on end in the 21st day, hIgG/OVA organizes tail vein injection 200ug control antibodies human IgG (hIgG), the hICOS-IgG1 of ICOS/OVA group tail vein injection 200ug, and 2mg/ml OVA aerosol excites after 10 minutes; HIgG/Saline group and ICOS/Saline group give hIgG or hICOS-IgG1 tail vein injection and physiological saline aerosol respectively equally and excite.The last aerosol excites and detects lung-douching fluid, the peripheral blood cells factor, immunoglobulin (Ig), the Th subgroup of respectively organizing mouse after 24 hours.With the 1mL physiological saline lavation mouse lung of pre-temperature to 37 ℃, 3 times repeatedly, 4 ℃ temporary; The BALF liquid of collecting with 1200rpm4 ℃ centrifugal 10 minutes, separation of supernatant ,-70 ℃ of preservations are to be measured; The double-antibody sandwich elisa method detects the content of cytokine IL-4 and IFN-γ.Eyeball of mouse is got anticoagulation, gets blood plasma and detects total IgE content; Separate mononuclearcell with lymphocyte separation medium, regulate cell concn to 106/ml, the PMA of 37 ℃ of 5%CO2 and 100ng/ml, the ionomycin of 1ug/ml and the Golgistop of 0.7ul were hatched 6 hours altogether, collecting cell, the CYC-antiCD4 room temperature that adds 5ul was placed 20 minutes, splitting erythrocyte, isolate cell and add Cytofix/Cytoperm liquid, placed 20 minutes for 4 ℃, add Perm/Wash liquid 1ml, centrifugation goes out cell, add FITC-antiIFN-γ, PE-antiIL-4,4 ℃ of lucifuges were placed Perm/Wash liquid washed twice, PBS re-suspended cell 20 minutes.With the homotype control antibodies is control sample.Detect with flow cytometer, Cellqueat software obtains cell, analyzes the positive percentage of CD4+IL-4+ (Th2) and CD4+IFN-γ+(Th1).

Result: hICOS-IgG1 can suppress the secretion of the cytokine of OVA allergic asthma mouse, the content of SERUM IgE and the quantity of Th2 subgroup, illustrates that hICOS-IgG1 can stimulate path by the interior ICOS of inhibition of body altogether.

Embodiment 2, hICOS-IgG1 combine test with the ICOS part

(1) principle

HICOS-IgG1 can combine with the ICOS ligand specificity of its ligand expression cell surface, under the certain situation of amount of ligand, along with the increase of hICOS-IgG1 content, also increases with its part binding capacity.Fluorescently-labeled anti-IgGFc antibody can combine with part bonded hICOS-IgG1, detects the fluorescence that sends in conjunction with anti-IgGFc antibody by flow cytometer and can react combining of hICOS-IgG1 and its part.

(2) operation steps

Behind the FcR with 20% rabbit anteserum sealing Daudi cell, get Daudi cell 1 * 10 ⁶Individual, with the hICOS-IgG1 and the blank that add 2ug/ml, two concentration of 10ug/ml behind the PBS washing secondary respectively, hatched 1 hour for 37 ℃, be detection antibody with the anti-human IgG of HRP mark, use the flow cytometry analysis ligand-binding activity.

(3) result

Counting 1 * 10 ⁴The fluorescence intensity that individual cell sends, (2ug/ml, 10ug/ml), the fluorescence intensity of detection increases (see figure 1), points out hICOS-IgG1 to combine with its part with the increase of hICOS-IgG1 concentration.

Embodiment 3, hICOS-IgG1 Determination of biological activity

(1) principle

After lymphocyte was subjected to antigenic stimulation, in the presence of costimulatory molecules, lymphocyte activation also showed biologic activity such as hyperplasia, secrete cytokines.If there is not the participation of ICOS costimulatory molecules, the very fast inactivation of activated lymphocytes, even apoptosis.In the process of recessive allele lymphocytes interactions, add hICOS-IgG1, if lymphocytosis is suppressed or cytokine secretion is suppressed, the common stimulation path that has reflected ICOS is blocked, and illustrates that hICOS-IgG1 has blocking-up ICOS and stimulates the effect of path altogether and can suppress proliferative response between the recessive allele lymphocyte.

(2) operation steps

Get the peripheral blood mononuclear cell (2 * 10 of two different sexes healthy blood donors ⁶/ ml) each 50ul, the hICOS-IgG1 100ul (final concentration be 209,105,53,27,14,7,0ug/ml) that adds the RPMI-1640 dilution contain 10% calf serum, each concentration triplicate, mixed culture is 96 hours in 96 orifice plates, stop cultivating preceding 16～18 hours, add ³H-TdR, counting cpm value.Get the supernatant liquor of above-mentioned mixed culture cell, the ELISA double antibody sandwich method is surveyed its IL-2, IFN-γ content.

(3) result

HICOS-IgG1 can suppress the proliferative response of people's recessive allele mixed lymphocytes, also can suppress IL-2, IFN-gamma cells factor level (seeing Fig. 2, Fig. 3).

Embodiment 4, the determination of activity of hICOS-IgG1 pharmacodynamics

(1) principle

Sensitization t helper cell subgroup Th2/Th1 is unbalance to be the main pathogenesis of ovum protein allergic asthma mouse model.ICOS stimulates path to participate in regulating t helper cell subgroup Th2/Th1 balance altogether.Treating ovum protein (OVA) allergic asthma mouse with hICOS-IgG1 is the restraining effect that ICOS is stimulated altogether path by in vivo test research hICOS-IgG1.

(2) operation steps

Get body weight and be about 20g, the female Sexual health BALB/c mouse in about 6 ages in week as experimental animal (6 every group).OVA and 200ul 1.36%Al (OH) in the 0th, 7,14 day abdominal injection 20ug ₃Suspension with the sensitization BALB/c mouse.

Rose for three days on end in the 21st day, IgG/OVA organizes (disease group) tail vein injection 200ug control antibodies human IgG (IgG), ICOS/OVA group (treatment group) tail vein injection 200ug hICOS-IgG1, and 2mg/ml OVA aerosol excites after 10 minutes; IgG/Saline group (control group) and ICOS/Saline group (control group) give IgG or hICOS-IgG1 tail vein injection and physiological saline aerosol respectively equally and excite.The last aerosol excites and detects lung-douching fluid (BALF), the peripheral blood cells factor, immunoglobulin (Ig), the Th subgroup of respectively organizing mouse after 24 hours.1mL physiological saline lavation mouse lung with pre-temperature to 37 ℃; The BALF liquid of collecting carries out centrifugal, separation of supernatant, and-70 ℃ of preservations are to be measured; The double-antibody sandwich elisa method detects the content of cytokine IL-4 and IFN-γ.Eyeball of mouse is got anticoagulation, gets blood plasma and detects total IgE content; Separate mononuclearcell with lymphocyte separation medium, regulate cell concn to 106/ml, 37 ℃ of 5%CO ₂Hatched altogether 6 hours with 100ng/ml PMA, 1ug/ml ionomycin and 0.7ul Golgistop, collecting cell, add 5ul CYC-antiCD4 room temperature placement 20 minutes, splitting erythrocyte, isolate cell adding Cytofix/Cytoperm liquid, placed 20 minutes for 4 ℃, add Perm/Wash liquid 1ml, centrifugation goes out cell, add FITC-antiIFN-γ, PE-antiIL-4,4 ℃ of lucifuges were placed Perm/Wash liquid washed twice, PBS re-suspended cell 20 minutes.With the homotype control antibodies is control sample.Detect with flow cytometer, Cellqueat software obtains cell, analyzes the positive percentage of CD4+IL-4+ (Th2) and CD4+IFN-γ+(Th1).

(3) result

The serum IgE level of hICOS-IgG1 treatment group, the number of inflammatory cells of BALF and IL-4 level, peripheral blood Th2 ratio all obviously lower (see figure 3) than disease group.

Embodiment 5 to 11, in order to further specify the formulation preparation method of hICOS-IgG1 of the present invention.

The preparation 1 of embodiment 5, powder injection

Get the hICOS-IgG1 of 100g, add 500ml water for injection, stir and make its dissolving; Add the injection water to 1000ml, add the 1g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Lyophilize gets sterilized powder, is distributed into 1000 bottles.

The preparation 2 of embodiment 6, powder injection

Get the hICOS-IgG1 of 100g, add lactose 50g, add 100ml water for injection, stir and make its dissolving; Add the injection water to 1000ml, add the 1.5g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; Spraying drying gets sterilized powder, is distributed into 1000 bottles.

The preparation 1 of embodiment 7, small-volume injection

Get the hICOS-IgG1 of 10g, add 100ml water for injection, stir and make its dissolving; Add the injection water to 1000ml, with 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 10ml, sterilization gets final product.

The preparation 2 of embodiment 8, small-volume injection

Get the hICOS-IgG1 of 20g, add propylene glycol 30g, add 200ml water for injection, stir and make its dissolving; Add the injection water to 1000ml, add the 1.5g needle-use activated carbon, fully stirred 30 minutes; Decarbonization filtering; With 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 5ml, sterilization gets final product.

The preparation 1 of embodiment 9, glucose infusion liquid

Get the hICOS-IgG1 of 5g, add polyoxyethylene glycol 10g, add glucose 500g, add 2000ml water for injection, stir and make its dissolving; Add the injection water to 5000ml; With 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 100ml, sterilization gets final product.

The preparation 2 of embodiment 10, glucose infusion liquid

Get the hICOS-IgG1 of 2g, add glucose 250g, add 1000ml water for injection, stir and make its dissolving; Add the injection water to 5000ml; With 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 250ml, sterilization gets final product.

The preparation of embodiment 11, sodium-chlor transfusion

Get the hICOS-IgG1 of 4g, add sodium-chlor 90g, add 1000ml water for injection, stir and make its dissolving; Add the injection water to 10000ml; With 0.22 μ m filtering with microporous membrane; The packing embedding, every bottle of 250ml, sterilization gets final product.

The preparation method of embodiment 12, a kind of mICOS-IgG2a

The source of main agents:

Lipofectamine2000, TRIZOL reagent are available from Invitrogen company; The various tool enzyme is available from Takara company; Totomycin is available from the general biotech company that flies in Shanghai; The DNA purifying reclaims test kit, plasmid DNA is extracted test kit available from the special clean company of dimension; IL-2, IFN-γ ELISA detection kit are available from crystalline substance U.S. biotech company.The pMD-18T cloning vector is preserved by this laboratory available from Invitrogen company, pGL-3-Basic cloning vector, the plasmid pMD-18T-Ig that carries the immunoglobulin Fc fragment gene available from TAKARA company, pSecTag2/Hygro A expression vector; Serum free medium EX302 is available from U.S. JRH company; ProteinA affinity purification test kit is available from U.S. sigma company; 293 cells are available from cell institute of the Chinese Academy of Sciences.

(1) clone of ICOS extracellular fragment gene:

According to the ICOS extracellular region sequence in mouse source, the primer of design band restriction enzyme site.

With final concentration is that the PMA of 20ng/ml and the ionomycin of 200ng/ml stimulate mouse peripheral blood mononuclear cell 72h, and collecting cell, Trizol extract total RNA, RT-PCR method amplification ICOS extracellular fragment gene 360bp, the pMD-18T cloning vector of packing into.

(2) clone of the Fc fragment gene of Ig:

(3) structure of mICOS-IgG2a integrative gene expression vector:

(4) nucleotide sequencing of mICOS-IgG2a:

With liposome (or calcium phosphate or electrotransformation) will recombinate integrative gene expression vector pSecTag2/HygroA-mICOS-IgG2a (as pSecTag2/Hygro A-mICOS-IgG2a etc.) transfection CHO cell or other host cells (as various derivative type Chinese hamster ovary celIs, 293 cells, various derivative type 293 cells etc.).The selection substratum screening that contains the 800ug/ml antibiotic hygromycin was subjected to transfect cell 14～20 days, and ELISA detects the expression level of mICOS-IgG2a, selected the high monoclonal cell of expression level and cultivated the frozen guarantor's kind of part liquid nitrogen; All the other cells enter suspension culture in the serum free medium through after taming.

(6) the ELISA double antibody sandwich method is identified fusion rotein secreting, expressing amount:

To 3ug/ml, wrap by 96 hole elisa plates 4 ℃ of night incubation with pH9.6 carbonic acid buffer dilution sheep anti-mouse igg antibody; With the sealing of 1% bovine serum albumin, hatched 1 hour for 37 ℃; Adding 72～96 hours supernatant liquor of positive colony cell cultures and 500ng/ml doubling dilution become the pure product of mouse IgG and the blank of 6 concentration, hatch 1 hour for 37 ℃; Add the anti-mouse IgG antibody of HRP mark horse, hatched 1 hour for 37 ℃; Add the tmb substrate damping fluid, room temperature is placed colour developing in 5～20 minutes; Read every hole absorbancy with microplate reader with 450nm after the termination reaction, according to the OD value preparation standard curve of mouse IgG standard substance correspondence, with the expression level of estimation secretory protein.

(7) a large amount of preparations of mICOS-IgG2a:

MICOS-IgG2a high expression level Chinese hamster ovary celI with picking out carries out the serum-free suspension culture, and concrete steps are to get 5 * 10 ⁵Individual cell adds in the EX302 substratum that contains 5% calf serum and 500ug/ml Totomycin to be cultivated with the conventional organization cell culture method, when treating cell length to 80%～90% full scale; With passage, with the EX302 culture medium culturing that contains 2% calf serum and 500ug/ml Totomycin during to cell length to 80%～90% full scale; With passage, when continuing with the EX302 culture medium culturing that contains 1% calf serum and 500ug/ml Totomycin to cell length to 80%～90% full scale with passage, cell changed over to shake use the EX302 culture medium culturing in the bottle (or bio-reactor, roll bottle), shake bottle and place on the shaking table and carry out the cell suspension cell cultures with 60～120 rev/mins of rotating speeds, all the other culture condition are with the conventional organization cell culture method.All will find that the cell of death or vigor difference abandons with trypan blue staining monitoring cell viability every day in passage and the suspension culture process each time.Collect cultured cells supernatant in the EX302 substratum, 4 ℃ of short-terms are preserved.

(8) purifying of mICOS-IgG2a:

Supernatant liquor with 4 ℃ of short-terms preservations, be 50 in time with molecular weight cut-off, 00～10, the ultra-fine filter of 000Da concentrates, the concentrated solution protein A affinity purification column purification (or ammonium sulphate precipitation method) of flowing through, collect elutriant, 280nm ultraviolet light absorption photometer detects protein content ,-20 ℃ of mICOS-IgG2a that preserve purifying after the packing.

(9) detection of mICOS-IgG2a ligand-binding activity:

1 * 10 ⁶After individual Daudi cell washs secondary with PBS, add the anti-B7RP-1 antibody of PE mark, room temperature effect 30 minutes behind the PBS washing secondary, detects with flow cytometer, and Cellqueat software obtains cell and carries out Daudi cell ICOS ligand molecular phenotype analytical.Behind the FcR with 20% rabbit anteserum sealing Daudi cell, add the positive colony emiocytosis supernatant (2ug/ml, 10ug/ml) and the blank that detect through the ELISA method respectively, hatched 1 hour for 37 ℃, anti-mouse IgG with the HRP mark detects antibody, uses the flow cytometry analysis ligand-binding activity.

The result shows that mICOS-IgG2a has ligand-binding activity.

(10) mICOS-IgG2a biological activity determination:

10.1 get C57/BL mouse and BALB/C spleen mononuclearcell (2 * 10 ⁶/ ml) each 50ul, the purifying mICOS-IgG2a 100ul (final concentration be 209,105,53,27,14,7,0ug/ml) that adds the RPMI-1640 dilution contain 10% calf serum, each concentration triplicate, mixed culture is 96 hours in 96 orifice plates, stop cultivating preceding 16～18 hours, add ³H-TdR, counting cpm value.Get the supernatant liquor of above-mentioned mixed culture cell, the ELISA double antibody sandwich method is surveyed its IL-2, IFN-γ content.

10.2 (be called for short: OVA) with 200ul 1.36%Al (OH) in the 0th, 7,14 day abdominal injection 20ug chicken egg protein ₃Suspension with the healthy BALB/c mouse of sensitization SPF level; The back gives different treatment by different grouping: rose for three days on end in the 21st day, mIgG/OVA organizes tail vein injection 200ug control antibodies mouse IgG (mIgG), the mICOS-IgG2a of ICOS/OVA group tail vein injection 200ug, and 2mg/ml OVA aerosol excites after 10 minutes; MIgG/Saline group and ICOS/Saline group give mIgG or mICOS-IgG2a tail vein injection and physiological saline aerosol respectively equally and excite.The last aerosol excites and detects lung-douching fluid, the peripheral blood cells factor, immunoglobulin (Ig), the Th subgroup of respectively organizing mouse after 24 hours.With the 1mL physiological saline lavation mouse lung of pre-temperature to 37 ℃, 3 times repeatedly, 4 ℃ temporary; The BALF liquid of collecting with 1200rpm4 ℃ centrifugal 10 minutes, separation of supernatant ,-70 ℃ of preservations are to be measured; The double-antibody sandwich elisa method detects the content of cytokine IL-4 and IFN-γ.Eyeball of mouse is got anticoagulation, gets blood plasma and detects total IgE content; Separate mononuclearcell with lymphocyte separation medium, regulate cell concn to 106/ml, the PMA of 37 ℃ of 5%CO2 and 100ng/ml, the ionomycin of 1ug/ml and the Golgistop of 0.7ul were hatched 6 hours altogether, collecting cell, the CYC-antiCD4 room temperature that adds 5ul was placed 20 minutes, splitting erythrocyte, isolate cell and add Cytofix/Cytoperm liquid, placed 20 minutes for 4 ℃, add Perm/Wash liquid 1ml, centrifugation goes out cell, add FITC-antiIFN-γ, PE-antiIL-4,4 ℃ of lucifuges were placed Perm/Wash liquid washed twice, PBS re-suspended cell 20 minutes.With the homotype control antibodies is control sample.Detect with flow cytometer, Cellqueat software obtains cell, analyzes the positive percentage of CD4+IL-4+ (Th2) and CD4+IFN-γ+(Th1).

Result: mICOS-IgG2a can suppress the secretion of the cytokine of OVA allergic asthma mouse, the content of SERUM IgE and the quantity of Th2 subgroup.

Sequence table 1: the nucleotide sequence of human IgG1's of the present invention Fc is as follows:

GAG?CCC?AAA?TCT?TGT?GAC?AAA?ACT?CAC?ACA?TGC?CCA?CCG?TGC?CCA?GCA?CCT

GAA?CTC?CTG?GGG?GGA?CCG?TCA?GTC?TTC?CTC?TTC?CCC?CCA?AAA?CCC?AAG?GAC

ACC?CTC?ATG?ATC?TCC?CGG?ACC?CCT?GAG?GTC?ACA?TGC?GTG?GTG?GTG?GAC?GTG

AGC?CAC?GAA?GAC?CCT?GAG?GTC?AAG?TTC?AAC?TGG?TAC?GTG?GAC?GGC?GTG?GAG

GTG?CAT?AAT?GCC?AAG?ACA?AAG?CCG?CGG?GAG?GAG?CAG?TAC?AAC?AGC?ACG?TAC

CGT?GTG?GTC?AGC?GTC?CTC?ACC?GTC?CTG?CAC?CAG?GAC?TGG?CTG?AAT?GGC?AAG

GAG?TAC?AAG?TGC?AAG?GTC?TCC?AAC?AAA?GCC?CTC?CCA?GCC?CCC?ATC?GAG?AAA

ACC?ATC?TCC?AAA?GCC?AAA?GGG?CAG?CCC?CGA?GAA?CCA?CAG?GTG?TAC?ACC?CTG

CCC?CCA?TCC?CGG?GAT?GAG?CTG?ACC?AAG?AAC?CAG?GTC?AGC?CTG?ACC?TGC?CTG

GTC?AAA?GGC?TTC?TAT?CCC?AGC?GAC?ATC?GCC?GTG?GAG?TGG?GAG?AGC?AAT?GGG

CAG?CCG?GAG?AAC?AAC?TAC?AAG?ACC?ACG?CCT?CCC?GTG?CTG?GAC?TCC?GAC?GGC

TCC?TTC?TTC?CTC?TAC?AGC?AAG?CTC?ACC?GTG?GAC?AAG?AGC?AGG?TGG?CAG?CAG

GGG?AAC?GTC?TTC?TCA?TGC?TCC?GTG?ATG?CAT?GAG?GCT?CTG?CAC?AAC?CAC?TAC

ACG?CAG?AAG?AGC?CTC?TCC?CTG?TCT?CCG?GGT?AAA?TGA

Sequence table 2: the aminoacid sequence of human IgG1's of the present invention Fc is as follows:

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV

VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL

PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL

SLSPGK

Sequence table 3: the 21st～140 nucleotide sequence of ICOS polypeptide of the present invention is as follows:

GAA?ATC?AAT?GGT?TCT?GCC?AAT?TAT?GAG?ATG?TTT?ATA?TTT?CAC?AAC?GGA?GGT

GTA?CAA?ATT?TTA?TGC?AAA?TAT?CCT?GAC?ATT?GTC?CAG?CAA?TTT?AAA?ATG?CAG

TTG?CTG?AAA?GGG?GGG?CAA?ATA?CTC?TGC?GAT?CTC?ACT?AAG?ACA?AAA?GGA?AGT

GGA?AAC?ACA?GTG?TCC?ATT?AAG?AGT?CTG?AAA?TTC?TGC?CAT?TCT?CAG?TTA?TCC

AAC?AAC?AGT?GTC?TCT?TTT?TTT?CTA?TAC?AAC?TTG?GAC?CAT?TCT?CAT?GCC?AAC?TAT

TAC?TTC?TGC?AAC?CTA?TCA?ATT?TTT?GAT?CCT?CCT?CCT?TTT?AAA?GTA?ACT?CTT?ACA

GGA?GGA?TAT?TTG?CAT?ATT?TAT?GAA?TCA?CAA?CTT?TGT?TGC?CAG?CTG?AAG

Sequence table 4: the 21st～140 aminoacid sequence of ICOS polypeptide of the present invention is as follows:

EINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQTLCD

LTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFC

NLSIFDPPPFKVTLTGGYLHIYESQLCCQLK

Sequence table 5: a kind of in the recombination fusion protein of the present invention: the nucleotide sequence of hICOS-IgG1 is as follows:

GAA?ATC?AAT?GGT?TCT?GCC?AAT?TAT?GAG?ATG?TTT?ATA?TTT?CAC?AAC?GGA?GGT

GTA?CAA?ATT?TTA?TGC?AAA?TAT?CCT?GAC?ATT?GTC?CAG?CAA?TTT?AAA?ATG?CAG

TTG?CTG?AAA?GGG?GGG?CAA?ATA?CTC?TGC?GAT?CTC?ACT?AAG?ACA?AAA?GGA?AGT

GGA?AAC?ACA?GTG?TCC?ATT?AAG?AGT?CTG?AAA?TTC?TGC?CAT?TCT?CAG?TTA?TCC

AAC?AAC?AGT?GTC?TCT?TTT?TTT?CTA?TAC?AAC?TTG?GAC?CAT?TCT?CAT?GCC?AAC?TAT

TAC?TTC?TGC?AAC?CTA?TCA?ATT?TTT?GAT?CCT?CCT?CCT?TTT?AAA?GTA?ACT?CTT?ACA

GGA?GGA?TAT?TTG?CAT?ATT?TAT?GAA?TCA?CAA?CTT?TGT?TGC?CAG?CTG?AAG?GAG

CCC?AAA?TCT?TGT?GAC?AAA?ACT?CAC?ACA?TGC?CCA?CCG?TGC?CCA?GCA?CCT?GAA

CTC?CTG?GGG?GGA?CCG?TCA?GTC?TTC?CTC?TTC?CCC?CCA?AAA?CCC?AAG?GAC?ACC

CTC?ATG?ATC?TCC?CGG?ACC?CCT?GAG?GTC?ACA?TGC?GTG?GTG?GTG?GAC?GTG?AGC

CAC?GAA?GAC?CCT?GAG?GTC?AAG?TTC?AAC?TGG?TAC?GTG?GAC?GGC?GTG?GAG?GTG

CAT?AAT?GCC?AAG?ACA?AAG?CCG?CGG?GAG?GAG?CAG?TAC?AAC?AGC?ACG?TAC?CGT

GTG?GTC?AGC?GTC?CTC?ACC?GTC?CTG?CAC?CAG?GAC?TGG?CTG?AAT?GGC?AAG?GAG

TAC?AAG?TGC?AAG?GTC?TCC?AAC?AAA?GCC?CTC?CCA?GCC?CCC?ATC?GAG?AAA?ACC

ATC?TCC?AAA?GCC?AAA?GGG?CAG?CCC?CGA?GAA?CCA?CAG?GTG?TAC?ACC?CTG?CCC

CCA?TCC?CGG?GAT?GAG?CTG?ACC?AAG?AAC?CAG?GTC?AGC?CTG?ACC?TGC?CTG?GTC

AAA?GGC?TTC?TAT?CCC?AGC?GAC?ATC?GCC?GTG?GAG?TGG?GAG?AGC?AAT?GGG?CAG

CCG?GAG?AAC?A?AC?TAC?AAG?ACC?ACG?CCT?CCC?GTG?CTG?GAC?TCC?GAC?GGC?TCC

TTC?TTC?CTC?TAC?AGC?AAG?CTC?ACC?GTG?GAC?AAG?AGC?AGG?TGG?CAG?CAG?GGG

AAC?GTC?TTC?TCA?TGC?TCC?GTG?ATG?CAT?GAG?GCT?CTG?CAC?AAC CAC?TAC?ACG

CAG?AAG?AGC?CTC?TCC?CTG?TCT?CCG?GGT?AAA?TGA

Sequence table 6: a kind of in the recombination fusion protein of the present invention: the aminoacid sequence of hICOS-IgG1 is as follows:

EINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQTLCD

LTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFC

NLSIFDPPPFKVTLTGGYLHIYESQLCCQLKEPKSCDKTHTCPPCP

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV

DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. the application of recombination fusion protein in preparation treatment, diagnosis, detection or research allelgic asthma disease and associated conditions product thereof,

Described this recombination fusion protein is the recombinant protein that functional molecular and inducible co-stimulator merge formation;

Described functional molecular is the part of debond ICOS and does not disturb ICOS and its target molecule bonded one peptide species or albumen that described polypeptide or albumen are the IgG constant regions in the Fc albumen;

The sequence of described inducible co-stimulator is that the N of people ICOS holds the 21st～140 amino acid fragment.