CN1817908A - Recombinant solvent protein derivative, its production and use - Google Patents

Recombinant solvent protein derivative, its production and use Download PDF

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CN1817908A
CN1817908A CN 200510023816 CN200510023816A CN1817908A CN 1817908 A CN1817908 A CN 1817908A CN 200510023816 CN200510023816 CN 200510023816 CN 200510023816 A CN200510023816 A CN 200510023816A CN 1817908 A CN1817908 A CN 1817908A
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derivative
protein derivative
recombinant
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沈茜
王健
张鹏
杨佳荟
张军
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Shanghai Changhai Hospital
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Shanghai Changhai Hospital
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Abstract

A recombinant fused egg albumen derivative, its production and use are disclosed. The procedure is carried out by fusing, expressing, purifying to obtain final product. It can inhibit memory specifically, activate T-type cell and prolong half-life period. It can be used to large-scale prepare immune inhibiting product with high biological activity and safety, treat, diagnose and inspect immune diseases.

Description

A kind of recombinant solvent protein derivative 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 recombinant solvent protein derivative 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 (rheumatoidarthrtis 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 progress 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).The 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.).
With ICOS stimulation approach altogether is the research of the recombination fusion protein of target spot as immunosuppression thing and its production and use aspect, existing preliminary study result.
But by literature search etc., up to the present, do not find with ICOS stimulation approach altogether to be that the recombinant solvent protein derivative of high-affinity/high biologic activity of target spot is as the relevant report of immunosuppression thing and its production and use aspect as yet.
Summary of the invention
The technical problem that will solve required for the present invention is that disclosing a kind of is the recombinant solvent protein derivative and its production and use of the high-affinity/high biologic activity of target spot with ICOS stimulation approach altogether, to overcome the above-mentioned defective that prior art exists.
That is to say, the invention is intended to study a kind of is the recombinant solvent protein derivative and its production and use of the high-affinity/high biologic activity of target spot with ICOS stimulation approach altogether, and then 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 recombinant solvent protein derivative; Two of purpose provides the preparation method of this recombinant solvent protein derivative; Three of purpose provides the purposes of this recombinant solvent protein derivative, so that using and using of aspects such as scientific research, disease treatment, medical diagnosis on disease, disease detection to be provided.Emphasis of the present invention provides that a kind of to stimulate path altogether with ICOS be a kind of novel, efficient, the specific immunosuppression thing of action target spot, i.e. ICOS120 recombinant solvent protein derivative.
(1) about notion and definition
For ease of narration, the present invention is defined as follows with regard to related content: the 1. whole amino acid moleculars of the new people ICOS after the modification that the 120th amino acids of whole amino acid moleculars of people ICOS is carried out obtaining behind the amino-acid substitution, or its variant, fragment or derivative, or segmental the 120th Methionin of partial amino-acid that will contain the people ICOS of the 120th amino acids carries out the partial amino-acid fragment of the new people ICOS that contains the 120th amino acids after the resulting modification behind the amino-acid substitution, or its variant, in fragment or the derivative etc. one or more, unified in this article abbreviating as: ICOS120; Wherein, the preferred amino acids displacement is a conservative amino acid replacement.2. the N of people ICOS holds the 21st～140 segmental amino acid, unified in this article abbreviating as: ICOS 21～1403. with ICOS 21～140The 120th amino acids carry out the new ICOS after the resulting modification behind the amino-acid substitution 21～140, unified in this article abbreviating as: the ICOS120 polypeptide also claims: the ICOS120 ligand binding domain.4. preferred conservative amino acid replacement is with ICOS 21～140The 120th amino acids be replaced into the new ICOS after the resulting modification behind the arginine by Methionin 21～140, i.e. a kind of special ICOS120 polypeptide type in the ICOS120 polypeptide, unified in this article abbreviating as: the ICOS120R polypeptide, its nucleotide sequence is seen sequence table 3, its aminoacid sequence is seen sequence table 4.5. with ICOS120 and functional molecular such as immunoglobulin (Ig) (be called for short: constant fragment Ig) (be called for short: Fc) or its variant, fragment or derivative merge the recombinant protein derivative of formation, unified in this article abbreviating as: recombinant solvent protein derivative, claim again: the ICOS120 recombinant solvent protein derivative also claims: the ICOS120 recombination fusion protein.
" 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 is to comprise basic side chain (Methionin for example, arginine, Histidine etc.), acid side-chain (aspartic acid for example, L-glutamic acid etc.), neutral side chain (glycine for example, aspartic acid, L-glutamic acid, Serine, Threonine, tyrosine, halfcystine etc.), non-polar sidechain (L-Ala for example, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane etc.), branched building block (Threonine for example, Xie Ansuan, Isoleucine etc.) or aromatic side chains (tyrosine for example, phenylalanine, tryptophane, Histidine etc.) etc. one or more in.
(2) 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 Basic Ways; 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, chemical process to wait and prepare recombination fusion protein, even can utilize these means or technology, chemical process to wait to prepare with ICOS stimulation approach altogether be the recombinant solvent protein derivative of the high-affinity/high biologic activity of target spot with ICOS specific inhibitory effect.Wherein, adopt the recombinant solvent protein derivative of this class high-affinity/high biologic activity of engineered means or technology preparation, be meant on gene level, structural analysis of protein by ICOS, change key amino acid sites or the zone relevant with its biologic activity, with its displacement, thereby obtain in whole amino acid moleculars, partial amino-acid fragment or its variant, fragment or the derivative etc. of ICOS of high-affinity/high biologic activity one or more, as ICOS120; Then ICOS120 and functional molecular are merged, as linking to each other with the segmental gene of immunoglobulin part and expression in eukaryotic cell or prokaryotic cell prokaryocyte etc., thereby obtain having the recombinant solvent protein derivative in above-mentioned two-part structure territory.
For reducing production costs, reduce the especially occurrence probability of the untoward reactions such as side effect during drug use of product, and reduce the frequency of its use and dosage etc., thereby save and reduce society and individual's cost expenditure, improve social benefit and economic benefit, the present invention is by preparing binding site corresponding on a kind of part that can cover ICOS or zone or functionally active site or the zone and recombinant solvent protein derivative that have high-affinity/high biologic activity, to reach the effects such as immunosuppression of expection.The exactness of contriver on the product research developing thought also further verified and confirmed to the product and the experimental result of the present invention's research and development.
3, design process
The contriver is an example with the ICOS in people source on the specific design of this recombinant solvent protein derivative research and development of products, the theing contents are as follows of thinking:
By target protein such as ICOS120 congenerous molecule particularly the constant region structural domain of immunoglobulin (Ig) merge mutually, can make recombinant protein obtain new characteristic: 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. have the longer transformation period, 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.
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.
ICOS is the costimulatory molecules on memory and the activating T cell, and the inhibition of ICOS biologic activity can cause remembering or the function of activating T cell suppresses even apoptosis, thereby induces immunological tolerance.The performance of ICOS biologic activity is to constitute ICOS costimulatory molecules path by combining of ICOS and its ligand molecular to realize, if take certain means to stop combining of ICOS and its part then the activity of ICOS just has been suppressed.
Natural ICOS is that a total length is 199 amino acid whose membrane protein molecules, behind the T lymphocyte activation, it is on the cytolemma that ICOS is expressed in the lymphocytic surface of T, utilize genetic engineering technique ICOS to be carried out computer mould is built technology and deletion mutantion is discovered, 21～140 amino acid fragments of N end of ICOS 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 of antagonism ICOS biologic activity.Using gene engineering technique all can be expressed 21～140 amino acid fragments of N end of preparation ICOS, but be had the difficulty that expression amount is not high, subsequent purification is difficult, degrade easily in the inside and outside, the transformation period is short in mammalian cell, intestinal bacteria, yeast.And if utilize recombination fusion protein as this specific immunosuppressive agent, then not only make its function can also make it have the constant segmental feature of immunoglobulin (Ig) with immune antagonism, for example 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.
N end 1～21 amino acid of natural ICOS is the signal peptide fragment, 21～140 amino acid fragments are ligand binding sites of natural ICOS, wherein the 42/83rd, 63/109 amino acid is most important to the structure of keeping natural ICOS ligand binding site, 7 amino acid of 115～121 are its part recognition site, and are most important to the ICOS activity.Using gene engineering technique, it is ICOS that the N of the people ICOS of synthetic holds the 21st～140 amino acid fragment 21～140, can play competition suppresses the ICOS biologic activity in conjunction with the ICOS part effect.7 amino acid of 115～121 are carried out point mutation research, the variation of finding this seven amino acid is very big to the active influence of ICOS, wherein the 120th amino acids is carried out amino-acid substitution and particularly the 120th amino acids is replaced into particularly ICOS120R polypeptide of the ICOS120 polypeptide that obtains behind the arginine by Methionin, with ICOS 21～140Compare, can obviously improve the effect that it suppresses the ICOS biologic activity.In addition, the amino acid encoding gene of ICOS120 and the encoding gene of functional molecular such as constant region for immunoglobulin are merged, promptly obtain ICOS120 recombinant solvent protein derivative such as ICOS120-Ig recombinant solvent protein derivative, can avoid the shortcoming of easy degraded of ICOS120 and difficult purifying.
So, on this basis, the contriver has designed that a kind of novel immunosuppression thing of blocking at ICOS stimulation approach altogether---with ICOS stimulation approach altogether is the recombinant solvent protein derivative of the high-affinity/high biologic activity of target spot, i.e. ICOS120 recombination fusion protein; On this basis, the recombinant protein that the constant fragment of research and development ICOS120 and immunoglobulin (Ig) or its variant or fragment or derivative merge, wherein R 2-R 1The recombinant solvent protein derivative of type, unified in this article abbreviating as: ICOS120-Ig; And further research and develop the constant fragment of ICOS120 polypeptide or ICOS120R polypeptide and immunoglobulin G while 1 or the recombinant protein of its variant or fragment or derivative fusion, wherein have R 2-R 1This recombinant solvent protein derivative of structure type abbreviates as respectively in this article: ICOS120-IgG1 or ICOS120R-IgG1.That is to say that the present invention from many aspects, multi-angle is verified and the exactness that has confirmed the present technique design.
(3) recombinant solvent protein derivative
Recombinant solvent protein derivative provided by the invention is a kind of a kind of novel immunosuppression thing with the preparation of methods such as gene engineering method (comprising engineered means or technology), chemical process.
For people ICOS, wherein the ICOS120 polypeptide is the ligand binding domain of people ICOS, can compete the native ligand in conjunction with ICOS, suppresses the activity of ICOS.That is to say, recombinant solvent protein derivative provided by the invention is a kind of high inhibition activity that obtains in that ICOS120 is modified and/or the recombination fusion protein of part high-affinity, and the height that is included in maintenance ICOS120 suppresses under the situation of activity and/or part high-affinity, other amino-acid substitution at the ICOS120 ligand binding domain also is possible, preferably with the 42nd of ICOS120 ligand binding domain, 83,63,109,52,115～119,121,136,137 amino acid whose displacement is to use amino-acid substitution, further preferred with the 52nd of ICOS120 ligand binding domain, 115～119,121,136,137 amino acid whose displacement is to use amino-acid substitution, preferred again with the 115th～119 of ICOS120 ligand binding domain, 121,136,137 amino acid whose displacement is to use amino-acid substitution, preferred again with the 121st of ICOS120 ligand binding domain, 136,137 amino acid whose displacement is to use amino-acid substitution, and most preferably the 136th amino acid whose displacement with the ICOS120 ligand binding domain is to use amino-acid substitution.
The preferred conservative amino acid replacement of described amino-acid substitution.
ICOS120 recombinant solvent protein derivative of the present invention is that ICOS120 is modified resulting recombinant solvent protein derivative, and promptly functional molecular (is used code R 1Expression) (uses code R with ICOS120 2Expression) merges the recombinant protein of formation.
Described ICOS120 recombinant solvent protein derivative, its structure is as follows:
This recombinant solvent protein derivative has one or more recombinant protein derivative: R that are selected from the following composition form 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 or derivative, streptavidin, polyhistidine or green fluorescent protein (are called for short: one or more GFP) etc.; Continue in preferred Fc albumen or its variant or fragment or the derivative etc. one or more; Continue Fc albumen or in its variant or fragment or the derivative etc. one or more among preferred IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or the IgD etc. again; Fc albumen among further preferred IgG1, IgG2, IgG4, IgA or the IgM etc. or in its variant or fragment or the derivative etc. one or more, continue Fc albumen or in its variant or fragment or the derivative etc. one or more among further preferred IgG1, IgG2 or the IgM etc., further preferably Fc albumen among IgG1 or the IgG2 etc. or in its variant or fragment or the derivative etc. one or more again, most preferably Fc albumen among the IgG1 or in its variant or fragment or the derivative etc. one or more.
Described Fc albumen or its variant or fragment or derivative are selected from following one or more:
(1) Fc aminoacid sequence, the Fc aminoacid sequence shown in the preferred sequence table 1, i.e. human IgG1's Fc aminoacid sequence;
(2) Fc aminoacid sequence, the Fc aminoacid sequence shown in the preferred sequence table 1, i.e. 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 (Clq) binding site; With
(11) 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 2Be ICOS120, derive from the people, be whole amino acid moleculars or its variant, fragment or the derivative that comprises the new people ICOS after the modification that the 120th amino acids of whole amino acid moleculars of people ICOS is carried out obtaining behind the amino-acid substitution, or segmental the 120th amino acids of partial amino-acid that will contain the people ICOS of the 120th amino acids is carried out the partial amino-acid fragment of the new people ICOS that contains the 120th amino acids after the resulting modification behind the amino-acid substitution or in its variant, fragment or the derivative etc. one or more;
The preferred amino acids displacement is a conservative amino acid replacement;
In further preferred ICOS120 polypeptide or its variant, fragment or the derivative etc. one or more are about to ICOS 21～140The 120th amino acids carry out the new ICOS after the resulting modification behind the amino-acid substitution 21～140, or its variant, fragment or derivative etc. in one or more;
Described ICOS120 polypeptide or its variant, fragment or derivative, preferably one or more in following:
(a) any residue of the displacement of the 42nd amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(b) any residue of the displacement of the 83rd amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(c) any residue of the displacement of the 63rd amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(d) any residue of the displacement of the 109th amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(e) any residue of the displacement of the 52nd amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(f) any residue of the displacement of the 115th～119 amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(g) any residue of the displacement of the 121st amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(h) any residue of the displacement of the 136th amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(i) any residue of the displacement of the 137th amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(j) any one described ICOS albumen or its variant, fragment or derivative in above inferior partly (a) to (i) comprise the chemical part that is connected to described protein part;
(k) the proteic a kind of derivative of above inferior part (j) described ICOS, wherein said chemical part is the water-soluble polymers part;
(l) the proteic a kind of derivative of above inferior part (k) described ICOS, wherein said water-soluble polymers partly is a polyoxyethylene glycol;
(m) the proteic a kind of derivative of above inferior part (k) described ICOS, wherein said water-soluble polymers partly is the polyamino acid part; With
(n) the proteic a kind of derivative of above inferior part (k) described ICOS, wherein said water-soluble polymers partly are connected unique N end of described protein part.
In further preferred again ICOS120R polypeptide or its variant, fragment or the derivative etc. one or more are about to ICOS 21～140The 120th amino acids be replaced into the new ICOS after the resulting modification behind the arginine by Methionin 21～140, or its variant, fragment or derivative etc. in one or more;
Most preferably the ICOS120R polypeptide is about to ICOS 21～140The 120th amino acids be replaced into the new ICOS after the resulting modification behind the arginine by Methionin 21～140, its nucleotide sequence is seen sequence table 3, its aminoacid sequence is seen sequence table 4.
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).
From above content, as can be seen: work as R 1Be Fc albumen or its variant or fragment or derivative, R 2During for ICOS120, R 2-R 1The recombinant solvent protein derivative of type, i.e. ICOS120-Ig.
Work as R 1The Fc albumen of the IgG1 in behaviour source or its variant or fragment or derivative, R 2During for the ICOS120 polypeptide, R 2-R 1The recombinant solvent protein derivative of type, i.e. ICOS120-IgG1;
Work as R 1The Fc albumen of the IgG1 in behaviour source or its variant or fragment or derivative, R 2During for the ICOS120R polypeptide, R 2-R 1The recombinant solvent protein derivative of type, i.e. ICOS120R-IgG1, its nucleotide sequence is seen sequence table 5, its aminoacid sequence is seen sequence table 6; That is to say, its structure is specifically: the N end is the 21st～140 segmental amino acid in 199 amino acid of inducible co-stimulator total length, but the 120th amino acids is replaced into arginine by Methionin, and its C end is immunoglobulin G while 1 a constant segmental amino acid.
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: Asparalne, 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, 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, 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, ICOS120 and functional molecular are carried out the bonded method, comprise in engineered means or the methods such as technology, chemical process 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, ICOS120 polypeptide and functional molecular such as Fc structural domain can be merged with various method, so that the ICOS recombinant solvent protein derivative 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 ICOS120 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 ICOS120 polypeptide portion or the two.These different ICOS recombination fusion proteins 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.
The invention provides the ICOS120 recombination fusion protein that suppresses mammiferous ICOS molecular activity, but not suppress the active ICOS120 recombination fusion protein of other costimulatory molecules.
The invention provides the activity that suppresses people ICOS molecule but not the recombinant solvent protein derivative of other costimulatory molecules, preferably the ICOS120 recombinant solvent protein derivative also suppresses not comprise the activity of other Mammals ICOS of people.
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.
Recombinant solvent protein derivative provided by the present invention has the characteristic in active blocking effect of efficient ICOS and constant region for immunoglobulin territory.
ICOS120-Ig provided by the invention comprises a constant region, for example the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD.The constant region of the IgG1 that merges of ICOS120R-IgG1 preferably.
ICOS120 recombination fusion protein of the present invention can be derived for or be connected to another functional molecular (for example another peptide or albumen).Therefore, ICOS120 recombination fusion protein of the present invention comprises the form of ICOS120 recombination fusion protein deutero-described herein and other modification.For example, ICOS120 recombination fusion protein of the present invention can functionally connect (, heredity coupled by chemistry is merged, non-covalent combination or other) to one or more other molecular entities, for example another antibody (for example two specially property antibody or double antibody) 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 ICOS120 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 can detecting the enzyme ICOS120 recombination fusion protein of deriving, described enzyme (is called for short: HRP), notatin (is called for short: GOD) or the like for comprising alkaline phosphatase (being called for short: AKP or ALP), horseradish peroxidase.When with can detect enzyme and derive the ICOS120 recombination fusion protein time, but detect this antibody by adding the other reagent that this enzyme is used to produce the detection reaction product.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 ICOS120 recombination fusion protein of deriving, and by indirect measurement avidin or streptavidin in conjunction with detection.
One type the recombination fusion protein of the present invention of deriving is by crosslinked two or more antibody (same type or dissimilar, for example create bi-specific antibody) preparation.Suitable linking agent comprise those Heterobifunctionals, have two unique reactive groups (for example disuccinimidyl suberate) that have suitable spacer to separate.
(4) preparation method of recombinant solvent protein derivative
Recombinant solvent protein derivative provided by the present invention, be to carry out amino-acid substitution by ligand-binding site point to make it have very high avidity and/or high things activity to the ICOS part to natural ICOS molecule, can be used as the active efficient blocker of ICOS, have that consumption is little, the characteristics of lasting medicine, be that the inhibiting neotype immunosuppressant of specific immunity is arranged, have the good curing prospect.
The preparation method of this recombinant solvent protein derivative may further comprise 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 Mammals carrier for expression of eukaryon (for example pSEC Tag2/Hygro A, pcDNA4/His Max, pcDNA3 etc.), virus vector (for example replication defect type retrovirus, adenovirus and adeno-associated virus), bacterial expression vector, Yeast expression carrier, 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 ICOS120-Ig is example, and its preparation method comprises the steps:
1. merge: the synthetic recombinant dna fragment that can transcribe and can translate into ICOS120-Ig;
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 ICOS120-Ig.
With ICOS120R-IgG1 is example, and its preparation method comprises the steps:
1. merge: the synthetic recombinant dna fragment that can transcribe and can translate into ICOS120R-IgG1;
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 ICOS120R-IgG1.
Can be by recombinant expressed to prepare ICOS120-Ig of the present invention or ICOS120R-IgG1 in host cell.Be preparation ICOS120-Ig or ICOS120R-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 ICOS120-Ig or ICOS120R-IgG1, at first obtain the dna fragmentation of described ICOS120-Ig of coding or ICOS120R-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 ICOS120-Ig or ICOS120R-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 ICOS120-Ig or ICOS120R-IgG1, by standard pcr amplification constant region for immunoglobulin territory.
In case after obtaining the dna fragmentation in the ligands specific calmodulin binding domain CaM of described ICOS gene and rabbit epidemic disease immunoglobulin constant district, can modify these sequences to obtain encode described ICOS120-Ig disclosed herein or ICOS120R-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 the constant region among IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or the IgD etc., one or more in the constant region among IgG1, IgG4, IgA or the IgM 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 ICOS120-Ig or ICOS120R-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 ICOS120-Ig or ICOS120R-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 ICOS120-Ig or ICOS120R-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 ICOS120-Ig or ICOS120R-IgG1 and the carrier or without limits the site be connected with regard to carrying out flush end) gene of described ICOS120-Ig or ICOS120R-IgG1 is inserted this expression vector.In addition, the gene of this ICOS120-Ig or ICOS120R-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 ICOS120-Ig or ICOS120R-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 ICOS120-Ig or ICOS120R-IgG1 gene, recombinant expression vector of the present invention also carries the adjusting sequence that control described ICOS120-Ig or ICOS120R-IgG1 gene are expressed in host cell.Term " adjusting sequence " comprises other expression controlling elements (for example polyadenylation signal) of the described ICOS120-Ig of promotor, enhanser and control or ICOS120R-IgG1 genetic transcription or translation.This adjusting sequence, for example be described in Transcriptional Regulation in Eukaryotes:Concepts such as MichaleCarey, Strategies and Techniques355, 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), adenovirus (adenovirus major late promoter's (be called for short: AdMLP) and the promotor and/or the enhanser of polyoma) 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 ICOS120-Ig or ICOS120R-IgG1, the recombinant expression vector of described ICOS120-Ig of coding or ICOS120R-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 ICOS120-Ig of the present invention or ICOS120R-IgG1 in theory, but most preferably in eukaryotic cell, most preferably in mammalian cell, express ICOS120-Ig or ICOS120R-IgG1, because this eukaryotic cell, particularly mammalian cell more likely assemble and secrete the ICOS120-Ig or the ICOS120R-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 ICOS120-Ig or ICOS120R-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 ICOS120-Ig or ICOS120R-IgG1.Use the standard protein purification method can from this substratum, reclaim ICOS120-Ig or ICOS120R-IgG1.
In the commending system of a recombinant expressed ICOS120-Ig of the present invention or ICOS120R-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 ICOS120-Ig or ICOS120R-IgG1 gene may be operably coupled to the enhancers/promoters regulatory element (for example from SV40, CMV etc. transcribe with the high level that drives described gene, 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 ICOS120-Ig or ICOS120R-IgG1, and from this substratum, reclaim ICOS120-Ig or ICOS120R-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 ICOS120-Ig or ICOS120R-IgG1.
In view of aforementioned, another aspect of the present invention also relates to nucleic acid, carrier and the host cell combination etc. that can be used for recombinant expressed ICOS120-Ig of the present invention or ICOS120R-IgG1.
(5) effect of recombinant solvent protein derivative and purposes
1, general introduction
Further purpose of the present invention provides a kind of raw material that is used to prepare the product with immunosuppressive action and prevention, detection, diagnosis, treatment and research autoimmune disorder or transplant rejection and associated conditions thereof, described product is to comprise in medicine, detection reagent or the diagnostic reagent etc. one or more, especially a kind of medicine.
ICOS120 recombination fusion protein of the present invention can be used for especially Mammals prevention of animal, detects, diagnoses, treats and research " 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 prevent, detects, diagnoses, treats and study 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.ICOS120 recombination fusion protein of the present invention can be subjected to prevention, detection, diagnosis, treatment and investigator with prevention, detection, diagnosis, treatment and research purpose administration of human.Give non-human mammal with ICOS120 recombination fusion protein of the present invention, 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, ICOS120 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 ICOS120 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 one or more in various enzymes, prothetic group, fluorescent substance, luminophore or the radioactive substance etc.The example of suitable enzyme comprises one or more in horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes or the acetylcholinesterase etc.; Suitable prothetic group complex body example comprises one or more in streptavidin/vitamin H or the avidin/biotin etc.; The example of suitable fluorescent substance comprises one or more in Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine amine fluorescein, dansyl chloride or the 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 3Among the H etc. one or more.
Can also use the ICOS part of ICOS120 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.
ICOS120 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, ICOS120 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 recombinant solvent protein derivative
Recombinant solvent protein derivative of the present invention can be separately or is united with other active ingredient and to be used for scientific research, disease prevention, disease detection or fields such as medical diagnosis on disease, disease treatment.
Can comprise following with the non-limitative example of the Parmaceutical for treating disease of autoimmunization system thing of recombinant solvent protein derivative 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 Design Labs/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-beta la (trade(brand)name: Avonex7M; Biogen company product), one or more in interferon-beta, methyl meticortelone, Ultracortene-H, azathioprine, reflunomide or the trypterygine etc.
Can comprise following with the organ-graft refection of ICOS120 recombination fusion protein of the present invention coupling 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, BrequinarSodium (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 ICOS120 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 in cardiotonic drug, diuretic(s), calcium antagonist or the beta receptor blocker etc. one or more; 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 recombinant solvent protein derivative and composition thereof and requirement
Recombinant solvent protein derivative of the present invention can be united use separately or with other active ingredient, comprise and be used to prepare the product that is used to prevent, treat, diagnose, detect and study relative disease, comprise in medicine, detection reagent or the diagnostic reagent etc. one or more, especially medicine.
Aspect concrete use, ICOS120 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 ICOS120 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 ICOS120 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, active ingredient ICOS120 recombination fusion protein is dissolved, suspendible or (for example be emulsifiable in the suitable aqueous solvent, distilled water, physiological saline or 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.), osmotic pressure regulator (for example, sodium-chlor, glycerine, the D9-seminose, in D-sorbyl alcohol or the glucose etc. one or more).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 the ICOS120 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, ICOS120 recombination fusion protein of the present invention can be added be suitable for to curee's medicinal compositions in.Usually, this medicinal compositions comprises ICOS120 recombination fusion protein of the present invention and pharmaceutically acceptable carrier.
The composition of ICOS120 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 ICOS120 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 ICOS120 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 ICOS120 recombination fusion protein by intramuscular injection or subcutaneous injection.
The pharmaceutical composition of ICOS120 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 ICOS120 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 ICOS120 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 recombinant solvent protein derivative 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 recombinant solvent protein derivative 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 ICOS120 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.
ICOS120 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, ICOS120 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, ICOS120 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, ICOS120 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 ICOS120 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 tablet, capsule, granule, suspensoid, emulsion, solution, syrup and injection, further preferred capsule, granule, suspensoid, emulsion, solution and injection, preferred especially suspensoid, emulsion, solution and injection.
According to the object, route of administration of treatment, the disease for the treatment of and situation etc., change dosage every day of ICOS120 recombination fusion protein of the present invention.For example, give Mammals, especially grownup (60kg body weight) through vein, the single dose of described recombinant solvent protein derivative is about 1～1000mg, preferably about 80mg, preferably administration 1～2 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 1. this active compound and the particular treatment of desiring to reach or preventive effect and 2. 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 ICOS120 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 ICOS120 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 ICOS120 recombination fusion protein such as individuality.The treatment significant quantity also refers to that the useful result of treatment of this ICOS120 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 ICOS120 recombination fusion protein of the present invention or prevention significant quantity is 0～20mg/kg, more preferably 1～8mg/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 ICOS120 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, ICOS120 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.
(6) 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 recombinant solvent protein derivative that suppresses mammiferous ICOS molecular activity, but not suppress the active recombinant solvent protein derivative of other costimulatory molecules.Described Mammals, same as above.
Recombinant solvent protein derivative is ICOS120-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 recombinant solvent protein derivative 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 recombinant solvent protein derivative has the activity of immunosuppressive action and treatment autoimmune disorder and transplant rejection and associated conditions thereof, directly use this recombinant solvent protein derivative, need not be through the metabolism of gi tract bacterium, make effect more direct, 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.
Recombinant solvent protein derivative pharmacological action of the present invention is stronger, and its raw material sources are abundant, and preparation technology is simple, the yield height.In addition, this recombinant solvent protein derivative 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 recombinant solvent protein derivative in the gi tract bio-transformation directly uses this recombinant solvent protein derivative, and the bioavailability of medicament height can accurately be controlled dosage.This recombinant solvent protein derivative stable in properties uses the quality of the pharmaceutical preparations of recombinant solvent protein derivative preparation stable.
The present invention studies this recombinant solvent protein derivative 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.
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.
Be example with ICOS120-Ig below, elaborate the concrete Preparation method and use of recombinant solvent protein derivative.
1. the screening of part high-affinity binding site
The homology mould that utilizes Insight II (98.0) the Homology program of MSI/Biosym company to finish the ICOS ligand binding region is built and the foundation of receptor/ligand mixture model and the computer theory screening in mutational site, and carry out biometric authentication with the genetic engineering technique of deletion mutantion and rite-directed mutagenesis, filter out the ICOS120-Ig of ICOS part high-affinity.
The characteristics of this sequence are as follows:
(1) the N terminal sequence is for carrying the 120th upper amino acid metathetical ICOS ligand binding domain sequence, after connect the constant fragments sequence of immunoglobulin (Ig).
(2) to merge the constant fragment of human normal immunoglobulin after the ICOS120 sequence be for stability, transformation period of increasing recombinant protein and make things convenient for subsequent purification and separate.
(3) for the ease of the clone, the head of holding at N adds a sfiI restriction enzyme site, adds a NotI restriction enzyme site after the last terminator codon.
This sequence clone is in carrier pMD18-T.
2.ICOS120-Ig the structure of expression body
(1) selecting pSecTag2 Hygro for use is expression vector, this carrier Mammals eukaryotic expression vector, it is characterized in that: the clonal selection resistance marker is Ampr, expressing and selecting resistance marker is Hygromycin, signal peptide is the secreting signal peptide of immunoglobulin kappa chain, and the insertion site is sfiI-NotI.
(2) from the pMD18-T plasmid, cut out the ICOS120-Ig fragment with sfiI-NotI, be cloned into the sfiI-NotI site of pSecTag2Hygro, construction recombination plasmid;
(3) the recombinant plasmid transformed escherichia coli jm109 competent cell is inoculated on the LB culture plate that contains 100ug/ml Amp, and therefrom selects single clone's bacterium colony, extracting goes out plasmid cuts through the sfiI-NotI enzyme, and the bacterial clone of recombinant gene expression vector is carried in screening; Identify the exactness of nucleotide sequence through sequencing.
(4) from the above-mentioned bacterial clone that carries recombinant gene expression vector, extract plasmid, use the liposome transfection Chinese hamster ovary celI, Hygromycin with 500～800ug/ml filters out the Chinese hamster ovary celI clone who expresses ICOS120-Ig, ELISA detects gained emiocytosis and expresses a kind of albumen that has constant region for immunoglobulin, and expression amount reaches more than the 100ug/ml.
3.ICOS120-Ig acquisition
(1) the target protein express cell is collected supernatant liquor through large scale culturing.Supernatant liquor is through Protein A affinity column, ion exchange column, molecular sieve chromatography, and washing, wash-out are collected elution peak.Elutriant concentrates through ultra-fine filter, and after adding physiological saline redissolved, sampling analysis had obtained ICOS120-Ig, detected through SDS-PAGE and HPLC, and its purity is greater than 95%.
(2) in vitro tests shows, this ICOS120-Ig can obviously suppress recessive allele mixed lymphocytes proliferation test, and it suppresses activity and is better than other similar immunosuppressor.
ICOS120-Ig with present method design and preparation is strong to avidity height, the inhibition biologic activity of part, the preparation efficiency height, and the product purity height has very high ICOS activity inhibition.
4. embodiment combines with the ICOS part in order to explanation ICOS120-Ig
Principle: ICOS120-Ig 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 ICOS120-Ig content, also increases with its part binding capacity.Fluorescently-labeled anti-Ig antibody can combine with part bonded ICOS120-Ig, detects the fluorescence that sends in conjunction with anti-Ig antibody by flow cytometer and can react combining of ICOS120-Ig and its part.
Operation steps: behind the FcR with 20% rabbit anteserum sealing Daudi cell, get Daudi cell 1 * 10 6Individual, with the ICOS120-Ig 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.
Result: counting 1 * 10 4The fluorescence intensity that individual cell sends, (1ug/ml, 5ug/ml), the fluorescence intensity of detection increases, and points out ICOS120-Ig to combine with its part with the increase of ICOS120-Ig concentration.
5. the present embodiment is in order to the biologic activity of explanation ICOS120-Ig
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 ICOS120-Ig, if lymphocytosis is suppressed or cytokine secretion is suppressed, the common stimulation path that has reflected ICOS is blocked, and illustrates that ICOS120-Ig has blocking-up ICOS and stimulates the effect of path altogether and can suppress proliferative response between the recessive allele lymphocyte.
Operation steps: the peripheral blood mononuclear cell (2 * 10 of getting two different sexes healthy blood donors 6/ ml) each 50ul, the ICOS120-Ig 100ul (final concentration be 100,50,25,12.5,6.25,3.125,0ug/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 content.
Result: ICOS120-Ig can suppress the proliferative response of people's recessive allele mixed lymphocytes, also can suppress IL-2, IFN-gamma cells factor level.
6. the present embodiment is in order to the pharmacodynamics activity of explanation ICOS120-Ig
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 ICOS120-Ig is the restraining effect that ICOS is stimulated altogether path by in vivo test research ICOS120-Ig.
Operation steps: get body weight and be about 20mg, the female Sexual health BALB/c mouse in about 6 ages in week as experimental animal (8 every group).In the 0th, 7,14 day abdominal injection 20ug OVA and 200ul 1.36%Al (OH) 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 ICOS120-Ig, and 2mg/ml OVA aerosol excites after 10 minutes; IgG/Saline group (control group) and ICOS/Saline group (control group) give IgG or ICOS120-Ig 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.With 1mL physiological saline lavation mouse lung; The BALF liquid of collecting is centrifugal, separation of supernatant; 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, 37 ℃ of 5%CO 2Hatched altogether 6 hours with 100ng/ml PMA, 1ug/ml ionomycin and 0.7ul Golgistop, collecting cell, add 5ulCYC-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).
The result: the serum IgE level of ICOS120-Ig 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.
7.ICOS120-Ig the several concrete application of preparation
The present invention prepares powder injection and generally adopts conventional freeze-drying, as solvent, the steps include: to get ICOS120-Ig 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 ICOS120-Ig 10～100mg.
The present invention prepares powder injection also can adopt spray-drying process, as solvent, the steps include: to get ICOS120-Ig 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 ICOS120-Ig 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 ICOS120-Ig 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 ICOS120-Ig 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, ICOS120R-IgG1
1. according to people source ICOS ligand binding domain sequence, the design primer, from the DNA of human peripheral blood single nucleus cell, clone required ICOS fragment with standard pcr, with the rite-directed mutagenesis test kit the 120th amino acids in the above-mentioned ICOS fragment is replaced into arginine, from the plasmid (pIgplus) that contains human IgG Fc, clones 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 ICOS120R-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-ICOS120R-IgG1 recombinant expression vector.
2. pSecTag2/Hygro A-ICOS120R-IgG1 recombinant expression vector is changed in Chinese hamster ovary cell (CHO) host cell (or other mammalian host cell) with liposome (or calcium phosphate or electrotransformation) with liposome method, screen the CHO host cell of single expression ICOS120R-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 ICOS120R-IgG1 with affinity chromatography from above-mentioned cell culture supernatant.
3. use high-pressure liquid phase detecting instrument (HPLC) to detect ICOS120R-IgG1 purity, detect the ICOS120R-IgG1 molecular weight, detect the expression of ICOS120R-IgG1 with the Western-blot method with polyacrylamide gel electrophoresis (SDS-PAGE).
4. biologic activity detects and finds that ICOS120R-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.
Embodiment 2, ICOS120R-IgG1 combine test with the ICOS part
(1) principle
ICOS120R-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 ICOS120R-IgG1 content, also increases with its part binding capacity.Fluorescently-labeled anti-IgGFc antibody can combine with part bonded ICOS120R-IgG1, detects the fluorescence that sends in conjunction with anti-IgGFc antibody by flow cytometer and can react combining of ICOS120R-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 ICOS120R-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, (1ug/ml, 5ug/ml), the fluorescence intensity of detection increases, and points out ICOS120R-IgG1 to combine with its part with the increase of ICOS120R-IgG1 concentration.
Embodiment 3, ICOS120R-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 ICOS120R-IgG1, if lymphocytosis is suppressed or cytokine secretion is suppressed, the common stimulation path that has reflected ICOS is blocked, and illustrates that ICOS120R-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 ICOS120R-IgG1 100ul (final concentration be 100,50,25,12.5,6.25,3.125,0ug/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 content.
(3) result
ICOS120R-IgG1 can suppress the proliferative response of people's recessive allele mixed lymphocytes, also can suppress IL-2, IFN-gamma cells factor level.
Embodiment 4, the determination of activity of ICOS120R-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 ICOS120R-IgG1 is the restraining effect that ICOS is stimulated altogether path by in vivo test research ICOS120R-IgG1.
(2) operation steps
Get body weight and be about 20mg, the female Sexual health BALB/c mouse in about 6 ages in week as experimental animal (8 every group).In the 0th, 7,14 day abdominal injection 20ug OVA and 200ul 1.36%Al (OH) 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 ICOS120R-IgG1, and 2mg/ml OVA aerosol excites after 10 minutes; IgG/Saline group (control group) and ICOS/Saline group (control group) give IgG or ICOS120R-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%CO2 and 100ng/mlPMA, 1ug/ml ionomycin and 0.7ul Golgistop were hatched 6 hours altogether, collecting cell, adding 5ul CYC-antiCD4 room temperature placed 20 minutes, splitting erythrocyte, isolate cell and add Cytofix/Cytopern 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 ICOS120R-IgG1 treatment group, the number of inflammatory cells of BALF and IL-4 level, peripheral blood Th2 ratio all obviously lower than disease group.
The preparation of embodiment 5, powder injection
Get the ICOS120R-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 of embodiment 6, powder injection
Get the ICOS120R-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 of embodiment 7, small-volume injection
Get the ICOS120R-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 of embodiment 8, small-volume injection
Get the ICOS120R-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 of embodiment 9, glucose infusion liquid
Get the ICOS120R-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 of embodiment 10, glucose infusion liquid
Get the ICOS120R-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 ICOS120R-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.
Embodiment 12, ICOS120R-IgG1 preparation method 1
Make up the ICOS120R-IgG1 express cell according to method of the present invention, selecting pSecTag2 Hygro for use is expression vector, uses the liposome transfection Chinese hamster ovary celI, cultivates through mass cell, collects culture supernatant.The NaCl of adding 1.5M crosses the protein A post with 20mM phosphoric acid buffer (pH7.4) (balance liquid) pre-equilibration that contains 1.5M NaCl in the supernatant liquor, behind 5 times of column volumes of aforementioned balance liquid washing, with containing 0.1M citrate buffer solution (pH2.5) (elutriant) wash-out, collect elution peak, after elutriant is used 3M Tris damping fluid (pH8.0) balance, cross through damping fluid (20mM PBS, 1.5M the Sepharoes post of NaCl (Ph7.4) pre-equilibration, behind wash-out, sampling analysis has promptly obtained ICOS120R-IgG1.Detect through SDS-PAGE and HPLC, the ICOS120R-IgG1 purity of gained is greater than 95%.
The inhibition activity of table 1, recombinant solvent protein derivative
Derivative Replacement site The inhibiting rate of MLR (%)
Base Amino acid
hICOS-IgG1 - - 100
ICOS83-IgG1 UGC/UCC C 83/S 66
ICOS63-IgG1 UGC/UCC C 63/S 21
ICOS119-IgG1 UGC/UCC C 119/S 6
ICOS42-IgG1 UGC/UCC C 42/S 9
ICOS120R-IgG1 AAA/CGC K 120/R 528
Annotate:
1. described MLR is the proliferative response of recessive allele mixed lymphocytes.
2. described hICOS-IgG1 is meant that the amino acid fragment of the 21st～140 of natural people ICOS and human IgG1's constant fragment merge the recombinant protein that the back forms.
ICOS120R-IgG1 obviously suppresses lymphocytic propagation and cytokine secretion with recessive allele mixed lymphocytes proliferation test, gained ICOS120R-IgG1, compares with hICOS-IgG1 and suppresses activity increase by 500 (seeing Table 1).
Embodiment 13, ICOS120R-IgG1 preparation method 2
According to method of the present invention the restriction enzyme site at sequence two ends is replaced by KpnI and XbalI respectively, with this sequence clone in carrier pMD18-T.Selecting pcDNA4 His Max for use is expression vector, makes up the expression body of ICOS120R-IgG1, extracts plasmid from above-mentioned carrying the recombinant gene expression body, uses the liposome transfection Chinese hamster ovary celI, filters out the Chinese hamster ovary celI clone who expresses ICOS120R-IgG1.
The target protein express cell is through large scale culturing, centrifugal collecting cell, and behind the lysing cell, the centrifuging and taking supernatant liquor.The NaCl of adding 1.5M crosses the protein A post with 20mM phosphoric acid buffer (pH7.4) (balance liquid) pre-equilibration that contains 1.5M NaCl in the supernatant liquor, behind 5 times of column volumes of aforementioned balance liquid washing, with containing 0.1M citrate buffer solution (pH2.5) (elutriant) wash-out, collect elution peak, after elutriant is used 3M Tris damping fluid (pH8.0) balance, add the 10ug/ml enteropeptidase, reacted 2-24 hour.Reaction solution crosses that (behind wash-out, sampling analysis has promptly obtained ICOS120R-IgG1 for 20mM PBS, the Sepharoes post of 1.5M NaCl (Ph7.4) pre-equilibration through damping fluid.Detect through SDS-PAGE and HPLC, the ICOS120R-IgG1 purity of gained is greater than 95%.
ICOS120R-IgG1 obviously suppresses lymphocytic propagation and cytokine secretion with recessive allele mixed lymphocytes proliferation test, gained ICOS120R-IgG1, compares the inhibition activity with hICOS-IgG1 and increases by 500.
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:
E P K S C D K T H T C P P C P A P E L L G G P S V F L F P P K P K D T L M I S R T P E V T C V
V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V S V L
T V L H Q D W L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V Y T L
P P S R D E L T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V
L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L
S L S P G K
Sequence table 3: the 21st～140 nucleotide sequence of ICOS120R 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 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 ICOS120R polypeptide of the present invention is as follows:
E I N G S A N Y E M F I F H N G G V Q I L C K Y P D I V Q Q F K M Q L L K G G Q T L C
D L T K T K G S G N T V S I K S L K F C H S Q L S N N S V S F F L Y N L D H S H A N Y Y
F C N L S I F D P P P F
Figure A20051002381600502
V T L T G G Y L H I Y E S Q L C C Q L K
Sequence table 5: a kind of in the recombination fusion protein of the present invention: the nucleotide sequence of ICOS120R-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 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 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 6: a kind of in the recombination fusion protein of the present invention: the aminoacid sequence of ICOS120R-IgG1 is as follows:
E I N G S A N Y E M F I F H N G G V Q I L C K Y P D I V Q Q F K M Q L L K G G Q T L C D
L T K T K G S G N T V S I K S L K F C H S Q L S N N S V S F F L Y N L D H S H A N Y Y F C
N L S I F D P P P F
Figure A20051002381600512
V T L T G G Y L H I Y E S Q L C C Q L K E P K S C D K T H T C P P C P
A P E L L G G P S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N
W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K
C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V Y T L P P S R D E L T K N Q V S L T
C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V
D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K

Claims (94)

1. recombinant solvent protein derivative, it is characterized in that, this recombinant solvent protein derivative is the recombinant protein that functional molecular and ICOS120 merge formation, described ICOS120 is the whole amino acid moleculars that comprise the new people ICOS after the modification that the 120th amino acids of whole amino acid moleculars of people ICOS is carried out obtaining behind the amino-acid substitution, or its variant, fragment or derivative, or segmental the 120th Methionin of partial amino-acid that will contain the people ICOS of the 120th amino acids carries out the partial amino-acid fragment of the new people ICOS that contains the 120th amino acids after the resulting modification behind the amino-acid substitution, or its variant, in fragment or the derivative one or more.
2. recombinant solvent protein derivative according to claim 1 is characterized in that described amino-acid substitution is a conservative amino acid replacement.
3. recombinant solvent protein derivative according to claim 2 is characterized in that described conservative amino acid replacement is with ICOS 21～140The 120th amino acids be replaced into arginic conservative amino acid replacement by Methionin; Described ICOS 21～140The N that is meant people ICOS holds the 21st～140 segmental amino acid.
4. recombinant solvent protein derivative according to claim 1 is characterized in that, described recombinant solvent protein derivative 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 1, R wherein 1Be functional molecular, R 2Be ICOS120, L is a junction fragment.
5. recombinant solvent protein derivative according to claim 4 is characterized in that described recombinant solvent protein derivative is to have R 2-L-R 1Or R 2-R 1One or more recombinant proteins in the composition form.
6. recombinant solvent protein derivative according to claim 5 is characterized in that described recombinant solvent protein derivative is to have R 2-R 1Recombinant protein in the composition form.
7. according to each described recombinant solvent protein derivative of claim 1～6, it is characterized in that described R 1Be to comprise in polypeptide or the albumen one or more.
8. recombinant solvent protein derivative according to claim 7 is characterized in that, described polypeptide or albumen are to comprise Fc albumen or its variant or fragment or derivative, streptavidin, one or more in polyhistidine or the green fluorescent protein.
9. recombinant solvent protein derivative according to claim 8 is characterized in that, described polypeptide or albumen are one or more in Fc albumen or its variant or fragment or the derivative.
10. recombinant solvent protein derivative according to claim 9, it is characterized in that described Fc albumen or its variant or fragment or derivative are Fc albumen or in its variant or fragment or the derivative one or more that comprise among IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or the IgD.
11. recombinant solvent protein derivative according to claim 10, it is characterized in that described Fc albumen or its variant or fragment or derivative are Fc albumen or in its variant or fragment or the derivative one or more that comprise among IgG1, IgG2, IgG4, IgA or the IgM.
12. recombinant solvent protein derivative according to claim 11, it is characterized in that described Fc albumen or its variant or fragment or derivative are Fc albumen or in its variant or fragment or the derivative one or more that comprise among IgG1, IgG2 or the IgM.
13. recombinant solvent protein derivative according to claim 12 is characterized in that, described Fc albumen or its variant or fragment or derivative are Fc albumen or in its variant or fragment or the derivative one or more that comprise among IgG1 or the IgG2.
14. recombinant solvent protein derivative according to claim 13 is characterized in that, described Fc albumen or its variant or fragment or derivative are Fc albumen or in its variant or fragment or the derivative one or more among the IgG1.
15. recombinant solvent protein derivative according to claim 14 is characterized in that, described Fc albumen or its variant or fragment or derivative are selected from following one or more:
(1) human IgG1's Fc aminoacid sequence;
(2) human IgG1's Fc aminoacid sequence has 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 (Clq) binding site; With
(11) 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.
16. recombinant solvent protein derivative according to claim 9 is characterized in that, described Fc albumen or its variant or fragment or derivative are selected from following one or more:
(1) Fc aminoacid sequence;
(2) Fc aminoacid sequence has 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 (Clq) binding site; With
(11) 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.
17. recombinant solvent protein derivative according to claim 10 is characterized in that, described Fc albumen or its variant or fragment or derivative are selected from following one or more:
(1) Fc aminoacid sequence;
(2) Fc aminoacid sequence has 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 (Clq) binding site; With
(11) 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.
18., it is characterized in that described R according to each described recombinant solvent protein derivative of claim 1～6 2Be in ICOS120 polypeptide or its variant, fragment or the derivative one or more, be about to ICOS 21～140The 120th amino acids carry out the new ICOS after the resulting modification behind the amino-acid substitution 21～140, or its variant, fragment or derivative in one or more in one or more.
19. recombinant solvent protein derivative according to claim 18 is characterized in that, described ICOS120 polypeptide or its variant, fragment or derivative are selected from following one or more:
(a) any residue of the displacement of the 42nd amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(b) any residue of the displacement of the 83rd amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(c) any residue of the displacement of the 63rd amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(d) any residue of the displacement of the 109th amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(e) any residue of the displacement of the 52nd amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(f) any residue of the displacement of the 115th～119 amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(g) any residue of the displacement of the 121st amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(h) any residue of the displacement of the 136th amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(i) any residue of the displacement of the 137th amino acids shown in ICOS120 amino acid sequence of polypeptide gained;
(j) any one described ICOS albumen or its variant, fragment or derivative in above inferior partly (a) to (i) comprise the chemical part that is connected to described protein part;
(k) the proteic a kind of derivative of above inferior part (j) described ICOS, wherein said chemical part is the water-soluble polymers part;
(l) the proteic a kind of derivative of above inferior part (k) described ICOS, wherein said water-soluble polymers partly is a polyoxyethylene glycol;
(m) the proteic a kind of derivative of above inferior part (k) described ICOS, wherein said water-soluble polymers partly is the polyamino acid part; With
(n) the proteic a kind of derivative of above inferior part (k) described ICOS, wherein said water-soluble polymers partly are connected unique N end of described protein part.
20. recombinant solvent protein derivative according to claim 18 is characterized in that, described ICOS120 polypeptide or its variant, fragment or derivative are one or more in ICOS120R polypeptide or its variant, fragment or the derivative; Described ICOS120R polypeptide is meant ICOS 21～140The 120th amino acids be replaced into the new ICOS after the resulting modification behind the arginine by Methionin 21～140
21. recombinant solvent protein derivative according to claim 20 is characterized in that, described ICOS120 polypeptide or its variant, fragment or derivative are the ICOS120R polypeptide.
22., it is characterized in that described junction fragment is the one or more amino acid that comprise in glycine, l-asparagine, Serine, Threonine or the L-Ala according to each described recombinant solvent protein derivative of claim 1～6.
23. recombinant solvent protein derivative according to claim 22 is characterized in that, described junction fragment is to be selected from following 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).
24., it is characterized in that described fusion is that ICOS120 and functional molecular are carried out the bonded method, comprises engineered means or technology according to each described recombinant solvent protein derivative of claim 1～6, or in the chemical process one or more.
25. recombinant solvent protein derivative according to claim 24 is characterized in that, described fusion method is that engineered means or technology directly or by connector connect indirectly.
26. recombinant solvent protein derivative according to claim 25 is characterized in that, described fusion method is that engineered means or technology directly connect.
27. the preparation method according to each described recombinant solvent protein derivative of claim 1～6 is characterized in that, this method comprises the steps:
1. merge: the synthetic recombinant dna fragment that can transcribe and can translate into recombinant solvent protein derivative;
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 recombinant solvent protein derivative.
28. the preparation method of recombinant solvent protein derivative according to claim 27 is characterized in that, described expression vector is to comprise in plasmid or the virus vector one or more.
29. the preparation method of recombinant solvent protein derivative according to claim 28 is characterized in that, described virus vector is to comprise in replication defect type retrovirus, adenovirus or the adeno-associated virus one or more.
30. the preparation method of recombinant solvent protein derivative according to claim 28, it is characterized in that described expression vector is to comprise in Mammals carrier for expression of eukaryon, bacterial expression vector, Yeast expression carrier or the insect expression vector one or more.
31. the preparation method of recombinant solvent protein derivative according to claim 30 is characterized in that, described Mammals carrier for expression of eukaryon is to comprise among pSEC Tag2/Hygro A, pcDNA4/His Max or the pcDNA3 one or more.
32. the preparation method of recombinant solvent protein derivative according to claim 27 is characterized in that, described host cell is the cell that is used to express target protein.
33. the preparation method of recombinant solvent protein derivative according to claim 32, it is characterized in that described host cell is to comprise in Chinese hamster ovary cell, Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell, African green monkey kidney fibroblast-like cells or HEKC and the progeny cell thereof one or more.
34. the preparation method of recombinant solvent protein derivative according to claim 33, it is characterized in that described host cell is one or more in Chinese hamster ovary cell or Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell and the progeny cell thereof.
35. the preparation method of recombinant solvent protein derivative according to claim 34 is characterized in that, described host cell is Chinese hamster ovary cell and progeny cell thereof.
36. the preparation method of recombinant solvent protein derivative according to claim 18 is characterized in that, this method comprises the steps:
1. merge: the synthetic recombinant dna fragment that can transcribe and can translate into the recombinant protein that obtains after ICOS120 and the immunoglobulin (Ig) fusion;
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 the recombinant protein that obtains after described ICOS120 and immunoglobulin (Ig) merge.
37. the preparation method of recombination fusion protein according to claim 36 is characterized in that, described ICOS120 is the ICOS120 polypeptide.
38. the preparation method according to the described recombination fusion protein of claim 37 is characterized in that, described ICOS120 polypeptide is the ICOS120R polypeptide.
39. the preparation method of recombinant solvent protein derivative according to claim 36 is characterized in that, described expression vector is to comprise in plasmid or the virus vector one or more.
40. the preparation method according to the described recombinant solvent protein derivative of claim 39 is characterized in that, described virus vector is to comprise in replication defect type retrovirus, adenovirus or the adeno-associated virus one or more.
41. preparation method according to the described recombinant solvent protein derivative of claim 39, it is characterized in that described expression vector is to comprise in Mammals carrier for expression of eukaryon, bacterial expression vector, Yeast expression carrier or the insect expression vector one or more.
42. the preparation method according to the described recombinant solvent protein derivative of claim 41 is characterized in that, described Mammals carrier for expression of eukaryon is to comprise among pSEC Tag2/Hygro A, pcDNA4/His Max or the pcDNA3 one or more.
43. the preparation method of recombinant solvent protein derivative according to claim 36 is characterized in that, described host cell is the cell that is used to express target protein.
44. preparation method according to the described recombinant solvent protein derivative of claim 43, it is characterized in that described host cell is to comprise in Chinese hamster ovary cell, Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell, African green monkey kidney fibroblast-like cells or HEKC and the progeny cell thereof one or more.
45. preparation method according to the described recombinant solvent protein derivative of claim 44, it is characterized in that described host cell is one or more in Chinese hamster ovary cell or Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell and the progeny cell thereof.
46. the preparation method according to the described recombinant solvent protein derivative of claim 45 is characterized in that, described host cell is Chinese hamster ovary cell and progeny cell thereof.
47. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research autoimmune disorder and associated conditions product thereof.
48. the application of composition in preparation treatment, diagnosis, detection or research autoimmune disorder and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
49. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research transplant rejection and associated conditions product thereof.
50. the application of composition in preparation treatment, diagnosis, detection or research transplant rejection and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
51. according to each described recombinant solvent protein derivative of claim 1～6 preparation treatment, diagnosis, detect or the research percutaneous transluminal coronary angioplasty after 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, application in the caused vascular disorder of implantable graft and repulsion and the associated conditions product thereof.
52. according to the composition of each described recombinant solvent protein derivative of claim 1～6 preparation treatment, diagnosis, detect or the research percutaneous transluminal coronary angioplasty after 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, application in the caused vascular disorder of implantable graft and repulsion and the associated conditions product thereof.
53. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research allergic disease and associated conditions product thereof.
54. the application of composition in preparation treatment, diagnosis, detection or research allergic disease and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
55. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research inflammatory diseases and associated conditions product thereof.
56. the application of composition in preparation treatment, diagnosis, detection or research inflammatory diseases and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
57. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research diabetes and complication and associated conditions product thereof.
58. the application of composition in preparation treatment, diagnosis, detection or research diabetes and complication and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
59. according to each described recombinant solvent protein derivative of claim 1～6 preparation treatment, diagnosis, detect or the research inflammatory bowel in application in disease and the associated conditions product thereof.
60. according to the composition of each described recombinant solvent protein derivative of claim 1～6 preparation treatment, diagnosis, detect or the research inflammatory bowel in application in disease and the associated conditions product thereof.
61. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research circulatory diseases and associated conditions product thereof.
62. the application of composition in preparation treatment, diagnosis, detection or research circulatory diseases and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
63. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research psoriasis and associated conditions product thereof.
64. the application of composition in preparation treatment, diagnosis, detection or research psoriasis and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
65. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research hepatopathy and associated conditions product thereof.
66. the application of composition in preparation treatment, diagnosis, detection or research hepatopathy and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
67. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research pancreatic disease and associated conditions product thereof.
68. the application of composition in preparation treatment, diagnosis, detection or research pancreatic disease and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
69. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research neurodegenerative disease and associated conditions product thereof.
70. the application of composition in preparation treatment, diagnosis, detection or research neurodegenerative disease and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
71. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research central nervous system disorder and associated conditions product thereof.
72. the application of composition in preparation treatment, diagnosis, detection or research central nervous system disorder and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
73. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research toxicaemia and associated conditions product thereof.
74. the application of composition in preparation treatment, diagnosis, detection or research toxicaemia and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
75. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research gestosis and associated conditions product thereof.
76. the application of composition in preparation treatment, diagnosis, detection or research gestosis and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
77. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research obesity and associated conditions product thereof.
78. the application of composition in preparation treatment, diagnosis, detection or research obesity and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
79. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research hyperlipidaemia and associated conditions product thereof.
80. the application of composition in preparation treatment, diagnosis, detection or research hyperlipidaemia and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
81. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research tumour and associated conditions product thereof.
82. the application of composition in preparation treatment, diagnosis, detection or research tumour and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
83. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research endocrinopathy and associated conditions product thereof.
84. the application of composition in preparation treatment, diagnosis, detection or research endocrinopathy and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
85. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research viral infection and associated conditions product thereof.
86. the application of composition in preparation treatment, diagnosis, detection or research viral infection and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
87. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research illness in eye and associated conditions product thereof.
88. the application of composition in preparation treatment, diagnosis, detection or research illness in eye and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
89. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research myasthenia and associated conditions product thereof.
90. the application of composition in preparation treatment, diagnosis, detection or research myasthenia and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
91. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research confirmed fatigue and associated conditions product thereof.
92. the application of composition in preparation treatment, diagnosis, detection or research confirmed fatigue and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
93. according to the application of each described recombinant solvent protein derivative of claim 1～6 in preparation treatment, diagnosis, detection or research osteopathia and associated conditions product thereof.
94. the application of composition in preparation treatment, diagnosis, detection or research osteopathia and associated conditions product thereof according to each described recombinant solvent protein derivative of claim 1～6.
CN 200510023816 2005-02-03 2005-02-03 Recombinant solvent protein derivative, its production and use Pending CN1817908A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115713965A (en) * 2022-10-28 2023-02-24 兰州大学 Computing method for predicting compound-protein affinity based on GECo model

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115713965A (en) * 2022-10-28 2023-02-24 兰州大学 Computing method for predicting compound-protein affinity based on GECo model

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