CN1127529A

CN1127529A - Agonists and antagonists of human interleukin-10

Info

Publication number: CN1127529A
Application number: CN94192880A
Authority: CN
Inventors: C·-C·周; X·-Y·蔡
Original assignee: Schering Corp
Current assignee: Merck Sharp and Dohme Corp
Priority date: 1993-07-26
Filing date: 1994-07-22
Publication date: 1996-07-24
Also published as: PL312718A1; ZA945434B; SK8396A3; IL110413A0; CZ23396A3; FI960353A; HU9503983D0; HUT73463A; AU7399694A; JPH08507930A; SK150596A3; FI960353A0; WO1995003411A1; NO960309D0; NO960309L; NZ269663A; CA2168110A1; AU681178B2; SG43798A1; KR960704041A

Abstract

Agonists and antagonists of human IL-10 are provided by this invention which are based upon modification of the termini of mature human IL-10. Also provided are compositions and methods for supplying or inhibiting the biological activity of human IL-10. Such compositions may be useful in the treatment of diseases characterized by inappropriate Th responses. Nucleic acids encoding the agonists and antagonists, recombinant vectors and transformed host cells comprising such nucleic acids, and methods for making the agonists and antagonists using the transformed host cells are also provided.

Description

The agonist of human interleukin-10 and antagonist

Background of invention

The present invention relates to agonist and antagonist, its composition and the preparation and the using method of human interleukin-10.These agonists and antagonist are by at the C-terminal of ripe human interleukin-10 and/or N-terminal is introduced amino-acid substitution or disappearance obtains.

Interleukin 10 (IL-10) is the cytokine that can mediate many effects or effect.From mouse and people's cell, all isolated IL-10.IL-10 participates in controlling the CD4 of different classes of or subgroup ⁺The immune response of auxiliary (Th) cell of T.These Th cells can be divided into the different subgroups of distinguishing with its cytokine production form.Two subgroups wherein are called Th1 and Th2 cell.

The Th1 cell clone produces interleukin II (IL-2) and gamma-interferon (IFN-γ), and the Th2 cell clone then is being subjected to antigen or mitogenetic Sugar receptors activation back secretion IL-10, interleukin 4 (IL-4) and interleukin 5 (IL-5) usually.The Th cell clone of this two kind also all produces such as tumor necrosis factor-alpha (TNF-α), interleukin 3 (IL-3) and granulocyte-macrophage colony stimutaing factor cytokines such as (GM-CSF).The 3rd class Th cell (Th0) produces IL-2, IFN-γ, IL-4, IL-5, TNF-α, IL-3 and GM-CSF simultaneously.

The different cytokine production forms of Th1 and Th2 cell have reflected that partly they are to the effect in the reaction of various cause of diseases.For example, the Th1 cell participates in various groups of intra-cellular pathogens are made the cell-mediated reaction of success.These cells also participate in delayed type hypersensitivity.The Th2 cell then interrelates with humoral response, and the feature of humoral response is a production of antibodies.As a rule, the Th reaction that immunity system produces can be removed specific antigen or pathogenic agent most effectively, but not always not like this.

For example, the feature of leishmaniasis is exactly a Th1 reaction defectiveness.This defective can be confirmed with the external test method, as the described assay method of people such as Clerici (J.Clin.Invest.84:1892,1989).Confirmed that by using a kind of like this external test to wash Th1 reaction defective is relevant with endogenous IL-10 level, because in external test, the Th1 function can be restored by adding the anti-IL-10 antibody of neutrality.

Because the feature of leishmaniasis and other diseases is a Th reaction defective, and the Th reaction is improper relevant with endogenous IL-10 effect, so need the agonist of IL-10 and antagonist to treat these diseases.

Summary of the invention

The present invention has satisfied above-mentioned needs by some compositions and method are provided, and these compositions and method provide or suppress the biological activity of people IL-10.

More particularly, the invention provides the antagonist of people IL-10, this antagonist comprises because 157 lysine residue is replaced by acidic amino acid residue, or owing to lacking the one-tenth acquaintance IL-10 that one or more amino-acid residue obtains modifying in the zone that contains about 12 carboxyl terminal residues.

The amino-acid sequence of three above-mentioned embodiments is limited by 1,2 and No. 3 in sequence list order.

The present invention further provides the nucleic acid of a kind of people IL-10 antagonist of coding, this antagonist comprises because 157 lysine residue is replaced by acidic amino acid residue, or owing to lacking the one-tenth acquaintance IL-10 that one or more amino-acid residue obtains modifying in the zone that contains about 12 carboxyl terminal residues.The present invention also provides recombinant vectors that contains described nucleic acid and the host cell that contains described recombinant vectors.

The present invention also provides the method for a kind of people IL-10 antagonist of preparation, this antagonist comprises because 157 lysine residue is replaced by acidic amino acid residue, or owing to lacking the one-tenth acquaintance IL-10 that one or more amino-acid residue obtains modifying in the zone that contains about 12 carboxyl terminal residues, described method comprises: under the condition of the nucleic acid of expressing the described antagonist of coding, cultivate a kind of above-mentioned host cell.

The present invention further provides and suppress the bioactive method of IL-10, this method comprises: the cell that has the IL-10 acceptor is contacted with the people IL-10 antagonist of significant quantity, described antagonist comprises because 157 lysine residue is replaced by acidic amino acid residue, or owing to lacking the one-tenth acquaintance IL-10 that one or more amino-acid residue obtains modifying in the zone that contains about 12 carboxyl terminal residues.

The present invention further provides the agonist of people IL-10, this agonist comprises owing to 1-11 one-tenth acquaintance IL-10 that the amino terminal amino acid residue obtains modifying of disappearance.

The present invention also provides nucleic acid, the recombinant vectors that contains described nucleic acid and the transformed host cell of the described agonist of coding, the method for the described antagonist of preparation and the pharmaceutical composition that contains one or more kind IL-10 agonists or antagonist and a kind of pharmaceutically acceptable carrier.

Detailed Description Of The Invention

All reference that this paper quoted all are incorporated herein by reference in full.

Antagonist of the present invention can be used for treating disease, is the leishmaniasis of feature with the Th1 reaction defective relevant with endogenous IL-10 for example.Immunosuppression or IL-10 that these antagonists also can be used for treating with the IL-10 mediation excessively produce diseases associated such as B cell lymphoma.In addition, these antagonists can be used for being intended to illustrate the research of the IL-10 mechanism of action and the appropriate design of medicine, because they demonstrate the strong receptors bind effect that is separated with effector function.In the time of on being fixed on solid phase carrier, these antagonists can be used for the IL-10 acceptor that affinity purification has lacked the easy molten form of striding the film district.

The viral IL-10 albumen of Epstein-Barr virus (EBV) (BCRFI or vIL-10) also has the biological activity of IL-10, by inference it can with the IL-10 receptors bind.By inference, with regard to regard to the ability that infects in the host, duplicates and/or keep, the expressed vIL-10 activity of EBV makes this virus obtain advantage on certain survival ability.The ability that vIL-10 negative regulation T cell and NK cell IFN-γ generate, with and B cell viability reinforcing effect, prompting vIL-10 can suppress antiviral immunity, strengthens the potential that EBV transforms human B cell simultaneously.

So IL-10 antagonist of the present invention also can be used for effectively strengthening the antiviral immunity at EBV (might also have other viruses).(J.Clin.Immuno1.12:239,1992) such as Howard are seen in the more detailed narration of relevant IL-10 antagonist potential use.

The embodiment of back discloses the suddenly change Three Represents embodiment of IL-10 antagonist of the present invention.In embodiment, the lysine residue that becomes 157 of acquaintance IL-10 orders is by glutaminic acid residue displacement (No. 1 order) therein.In another embodiment, the carboxyl-terminal deletion of people IL-10 three (No. 2 orders) or four (No. 3 orders) amino-acid residues.These are called K157E, C Δ 3 and C Δ 4 antagonists below antagonist.

Term used herein " becomes acquaintance IL-10 " and is defined as a kind of protein that lacks leader sequence, and this albumen (a) has and No. 4 essentially identical amino-acid sequences of the defined order of order; (b) have the biological activity identical with natural IL-10.This term comprises having one or more conservative amino acid replacement (Grantham, Science185:862,1974) but do not influence bioactive natural allelic variant and other varients substantially.These conservative substitutions relate to some groups of synonym amino acid, and are for example of people's such as Lee United States Patent (USP) 5,017,691.

Should be appreciated that,, also can carry out other to the C-terminal of people IL-10 and modify to produce other antagonists though above-mentioned embodiment is preferred at present.For example, can replace glutaminic acid residue to replace 157 lysine residue with asparagicacid residue, thereby produce effective antagonist.So term used herein " acidic amino acid residue " is defined as and both comprises asparagicacid residue, also comprises glutaminic acid residue.

The degree of disappearance also can be greater or lesser.Can lack one or more amino-acid residue that comprises about 12 carboxyl terminal residues.About 8 terminal residues of preferred disappearance more preferably lack 3 or 4 terminal residues.

Now be surprised to find that, also can lack 11 amino-acid residues at the most from the aminoterminal that becomes acquaintance IL-10.According to measuring in the following MC/9 mastocyte stimulation assay method, the brachymemma varient that these may have the pharmacokinetic property different with IL-10 itself has into the biological activity of acquaintance IL-10.So these varients can be used for treating to the responsive any indication of IL-10 treatment itself, also can be used for the front to the more described purposes of antagonist of the present invention, as affinity purification.

Its amino-acid sequence shortens because these varients have the biological activity of people IL-10, and this paper also is referred to as " people IL-10 agonist ".

But, it is believed that 12 cysteine residues is essential to biological activity.In fact, the active varient of lifeless matter of disappearance generation that comprises 12 residues of this cysteine residues.So N-terminal disappearance is limited to the disappearance of one or more residue in 11 residues.

This aminoterminal disappearance can be modified with above-mentioned carboxyl terminal and be combined, and produces the antagonist that has following characteristics but may have the different pharmaceutical kinetic property.These antagonists also are parts of the present invention.

The nucleic acid of coding IL-10 agonist and antagonist also is a part of the present invention.Certainly, those skilled in the art can clearly recognize, because genetic code has degeneracy, many different nucleic acid can both encode each agonist and antagonist are arranged.Used specific cryptosystem can be selected so that make up and realize optimum expression in protokaryon or eukaryotic system.

Preferably, modify the cDNA of coding people IL-10 as (Nucleic Acids Res.19:2471,1991) as described in the people such as Daugherty, with polymerase chain reaction method (PCR) (people such as Saiki, Science 239:487,1988) nucleic acid of preparation coding agonist and antagonist.This cDNA is well known in the art, can prepare with standard method, for example as described in the open WO91/00349 of international patent application.The clone who comprises coding people IL-10 order also has been preserved in American type culture collection, and (Maryland), preserving number is 68191 and 68192 for ATCC, Rockville.

In addition, also can be with known site-directed mutagenesis technology modifying DNA.Referring to people such as for example Gillman, Gene 8:81,1979; People such as Roberts, Nature 328:731,1987; Or Innis (Ed.), 1990, PCR Protocols:A Guide to Methods andApplicationS, Academic Press, New YorK, NY.

Nucleic acid of the present invention also can chemosynthesis, for example with people's such as Matteucci phosphoramidite (phosphoramidite) solid phase vector (J.Am.Chem.Soc.103:3185,1981), method (J.Boil.Chem.764:17078,1989) or other known method of people such as Yoo.

Contain the recombinant vectors of above-mentioned nucleic acid, with this carrier transformed host cells and prepare agonist and the method for antagonist, also be a part of the present invention.

If the end of DNA and carrier all contains the restriction site of consistency, then be easy to the DNA of one of coding agonist and antagonist is inserted one of many known expression vectors.If can not do like this, then must modify, that is, digest the single stranded DNA overhang that produces by the restriction endonuclease cutting again the end of DNA and/or carrier, produce flat end, perhaps reach same effect by fill and lead up the strand end with suitable archaeal dna polymerase.

In addition, can be by nucleotide sequence (joint) being connected to the terminal site that produces any needs of going up.These joints can comprise the special oligonucleotide order that limits required restriction site.If desired, can also modify carrier and dna fragmentation after cutting with method that adds the homopolymer tail or PCR method.

The feature of antagonist of the present invention is to have the people IL-10 receptor binding affinity similar to people IL-10 itself, but biologically active not basically.Preferably, the biological activity that these antagonists are measured with the standard test method is about 10% less than people IL-10's, more preferably less than about 1%.

In the cell that has the IL-10 acceptor, these antagonists generally reach about 25% IL-10 biological activity inhibition at least.The inhibition degree is preferably at least about 50%, more preferably at least about 75%.Actual inhibition degree can change along with the particular organisms activity of being surveyed.

Agonist and antagonist also can be with suitable method chemosynthesis, for example with getting rid of solid-phase synthesis, part solid phase method, fragment condensation or classical solution synthetic method.The polypeptide of chemosynthesis is preferably with solid phase method of peptide synthesis preparation, for example as Merrifield (J.Am.Chem.Soc.85:2149,1963; Science 232:341,1986) and people such as Atherton (Solid Phase PeptideSynthesis:A Practical Approach, 1989, IRL Press, Oxford) described.

How no matter agonist and antagonist prepare, and can carry out purifying, for example use HPLC method, gel filtration method, ion-exchange and partition chromatography, counter-current distribution and/or other known methods.

Make one or more plant IL-10 agonist or antagonist or its pharmacologically acceptable salt and mix, can make pharmaceutical composition with physiologically acceptable carrier.

The available pharmaceutical carrier can be any consistency non-toxic substance that is suitable for using the present composition to the patient.Can contain sterilized water, alcohol, fat, wax and inert solid in the carrier.Also can mix pharmaceutically useful auxiliary agent (buffer reagent, dispersion agent) in the pharmaceutical composition.In general, the composition that is used for this class medicine of parenteral administration is known (for example Remington ' sPharmaceutical Science, 18th Ed., Mack Publishing Company, Easton, PA, 1990).Often preferred unit dose package, for example unit dose package of sterile form.

The preferred administered parenterally of agonist and antagonist, i.e. intraperitoneal, intravenously, subcutaneous or intramuscularly or infusion, or any acceptable other general methods.In addition, also can use antagonist (referring to people such as for example Urquhart, Ann.Rev.Pharmacol.Toxicol.24:199,1984 with implantable or injectable drug delivery system; Lewis, Ed., ControlledRelease of Pesticides and Pharmaceuticals, l981, Plenum Press, New York, New YorK; United States Patent (USP) 3,773,919 and 3,270,960).Also can be with protecting antagonist not to be subjected to the known preparation of gastrointestinal protein enzyme effect to carry out oral.Other sees also Langer, Science249:1527,1990.

Agonist and anti-antagonistic agent can also be used with the gene therapy technology of standard, be included in direct injection nucleic acid in the tissue, use recombinant viral vector or liposome and implant transfectional cell (referring to for example Rosenberg, J.Clin.Oncol.10:180,1992).

Agonist and antagonist can be used separately, and also can be usually used in treating with Th reaction defective with one or more kinds be that other medicament compatibilities of the illness of feature are used.For example, can use jointly with antagonist such as interleukin 12 (IL-12) or gamma-interferon medicines such as (IFN-γ).If replace IL-10 treatment or prevention insulin-dependent diabetes, then can use (seeing common unsettled U. S. application submission on October 1st, 07/955,523,1992) jointly with Regular Insulin, S-Neoral, prednisone or azathioprine with agonist.

One or more plant other medicaments this use jointly can with the using of agonist or antagonist common () or successively (before or after it) carry out.All medicaments of being used all should be present in patient's body with the level that is enough to produce result of treatment.In general, if second kind of medicament is to use in the half life of first kind of medicament greatly, then these two kinds of medicaments are considered to use jointly.

For specific condition, the ordinary skill of determining to belong to this area of the suitable dosage of agonist or antagonist.In general, treatment begins with the smaller dose that is lower than optimal dose.Improve dosage then gradually, up to the best effect that reaches under the particular case.Can in one day, use total per daily dose break into portions when needing for simplicity.

Significant quantity will be obviously to improve one or more clinical parameter, and/or make the statistics that is reflected at of one or more known Th function go up significantly improved dosage, and wherein the production of some function such as IL-2 is narrated in front.This reaction can be carried out external test with the hemocyte of gathering in patient's body, for example as described in the people such as Clerici (the same).This external test can carry out before the treatment beginning, thereby a reference baseline is provided.Improved reaction can compare with it.

For particular patient, the actual amount of application and the frequency of administration of agonist and antagonist and pharmacologically acceptable salt thereof, to adjust according to doctor in charge's judgement, and should consider such as patient's age, physical appearance and body weight, and the factors such as severity of the symptom of being treated.

Embodiment

The present invention can illustrate with the following example.Except as otherwise noted, the per-cent in the solid solid mixture that below provides, liquid liquid mixture and the solidliquid mixture is represented with w/w, volume/volume and weight/volume respectively.

Reagent and general method

Restriction endonuclease derive from Boerhringer Mannheim (Indianapolis, IN), dna ligation kit is then available from Takara Biochem., Inc. (Berkeley, CA).Taq polysaccharase and Pfu polysaccharase derive from Stratagene (La Jolla, CA).Basically as (J.Biochem.106:23,1989) as described in the people such as Tsujimoto, with standard method production recombinant human IL-10 (hIL-10) in Chinese hamster ovary (CHO) cell.Tissue culture medium (TCM), foetal calf serum and glutamine available from Gibco-BRL (Gaithersburg, MD).(Foster City is CA) with standard method synthetic oligonucleotide primer thing to utilize Applied Biosystems380A, 380B or 394 type dna synthesizers.

The standard recombinant dna method is carried out (MolecularCloning:A Laboratory Manual, 2nd Edition, 1989, Cold Spring HarborLaboratory Press, Plainview, New York) substantially by people such as Sambrook are described.

Transfection

The following transient expression that carries out.COS cell (ATCC CRL 1651) is remained in the improved Yi Geershi substratum of Dulbecco (DMEM), be added with 10% foetal calf serum, 6mM glutamine and penicillin/streptomycin in this substratum.Utilize BioRad GENEPULSER ^(Richmond CA), carries out transfection with electroporation.

Handle that by trypsinase-EDTA cell is come off from culture dish, and with cell suspension in fresh culture.Make about 5 * 10 in the 250 μ l volumes ⁶Individual cell mixes with 5 μ g plasmid DNA, carries out electroporation then.Voltage and electric capacity are set in 0.2 volt and 960mFD respectively.

Behind the electroporation, cell transfer in the 10cm culture dish, is contained among the DMEM of serum, at 5%CO at 10ml ₂In cultivated 6 hours down in 37 ℃.Attached to after on the culture dish, the sucking-off substratum is changed serum free medium to cell.After 72 hours, the collection condition substratum is analyzed.

The preparation of antagonist

The reconstruction of wild-type people IL-10cDNA and expression vector

For ease of expressing and operation, be basic hIL-10 carrier (people such as Vieira, Proc.Natl.Acad.Sci.USA88:1172,1991 in order to pCDSR α; Be deposited in GenBank, preserving number is the order of M57627) make template, with the coding region of PCR method generation hIL-10cDNA.But other known cDNA sources may have also been used.

In the 5 ' primer of naming to B1789CC (No. 5 orders), introduce the common rotaring intertranslating start of a Kozak vertebrates district (Kozak, Nucleic Acids Res.20:8125,1987).At 5 ' primer B1789CC with name in the 3 ' primer (No. 6 orders) into A1715CC, introduce a PstI site and an EcoRI site respectively.

Utilize above-mentioned primer, in 0.5ml Eppendorf pipe hIL-10cDNA is carried out PCR, the reaction mixture volume is 50 μ l, and the paraffin oil upper strata of one 50 μ l is arranged.Reaction mixture generally contains 26.5 μ l H ₂O, (final concentration in the reaction solution is 5 μ l Taq (thermus aquaticus) dna polymerase buffer liquid: 10mM Tris-HCl, pH8.8,50mM KCl, 1.5mM MgCl ₂, 0.001% (W/V) gelatin), 200 μ M dNTP, 60ng template DNA, 5 ' primer B1789CC and each 10 picomole of 3 ' primer A1715CC, 0.5 μ l Taq polysaccharase (2 unit).

(Techne, Princeton carry out 30 following circulations in NJ): 95 ℃ of sex change 2 minutes to be reflected at the PHC-1 thermal cycler; Annealed 2 minutes for 42 ℃; 70 ℃ were synthesized 1 minute.After the 30th loop ends, reaction mixture is incubated 9 minutes down in 72 ℃ again and carries out lengthening reaction.

In containing 1.2% agarose Tris-acetate gel of 0.5 μ g/ml ethidium bromide, the PCR mixture is carried out electrophoresis.Downcut dna fragmentation from gel, and use GENECLEAN with expection size ^(La Jolla CA) carries out purifying to test kit.After reclaiming from gel, product D NA is with PstI and EcoRI digestion, by gel electrophoresis and GENECLEAN ^Processing separates, and is cloned among the expression vector pDSRG (ATCC68233) as the PstI/EcoRI restricted fragment, transfer to then expression vector pSV.Sport (Gibco-BRL, Gaithersburg, MD) in.

The carrier that contains hIL-10cDNA is bred in bacillus coli DH 5 alpha strain (Gibco-BRL), and DNA sequence is confirmed with the DNA sequence assay method.Carry out the structure of COS transfection and sudden change hIL-10 carrier for the hIL-10 expression vector on basis in order to pSV.Sport.

The hIL-10cDNA of resynthesis has kept unique BglII site and unique BstEII site, and these two sites all are present among the wild-type cDNA.These two internal limitations sites are used for later on producing sudden change hIL-10cDNA by the box displacement, and the relative position in these two sites is illustrated as follows:

PstI----------BgIII-----------BstEII--------------EcoRI

Carboxyl terminal is modified

Be to produce the C-terminal sudden change antagonist of hIL-10,, and replace respective regions among the above-mentioned pSV.Sport hIL-10DNA with these fragments with the sudden change cDNA fragment in the synthetic BstEII/EcoRI zone corresponding to wild-type hIL-10cDNA of PCR method.

Utilize the hIL-10cDNA order complementary Oligonucleolide primers with above-mentioned resynthesis, and in 3 ' end primer, introduce specified sudden change in advance, produce K157E, C Δ 3 and the C Δ 4 sudden change antagonists of people IL-10 with the PCR method.

Produce three sudden change antagonists with the 5 ' primer of naming to B3351CC, this primer has the amino-acid sequence that order limited No. 7.The order of this 5 ' primer is complementary with the inner order of the people IL-10cDNA of the unique BstEII restriction site that comprises wild-type hIL-10cDNA.The order of 3 ' primer that is used to prepare antagonist is complementary with 3 ' the end order of hIL-10 code cDNA.These primers and limit its order sequence number (SN) as follows:

Mutant primer sequence number (SN)

K157E C3481CC 8

CΔ3 C3482CC 9

CΔ4 B3350CC 10

Utilize above-mentioned primer, in 0.5ml Eppendorf pipe hIL-10cDNA is carried out PCR, the reaction mixture volume is 50 μ l, and the paraffin oil upper strata of one 50 μ l is arranged.Reaction mixture generally contains 26.5 μ l H ₂O, (final concentration in the reaction solution is 5 μ l Pfu dna polymerase buffer liquid: 20mM Tris-HCl, pH8.2,10mM KCl, 2mMMgCl ₂, 6mM (NH ₄) ₂SO ₄, 0.1%Triton x-100,10 μ g/ml do not contain the bovine serum albumin(BSA) (BSA) of nuclease), 200 μ M dNTP, 40ng template DNA, 5 ' primer B3351CC and each 10 picomole of a kind of 3 ' primer, 0.5 μ, 1 pfu polysaccharase (2.5 unit).

(Techne, Phnceton carry out 22 following circulations in NJ): 94 ℃ of sex change 2 minutes to be reflected at the PHC-1 thermal cycler; Annealed 2 minutes for 50 ℃; 72 ℃ of Synthetic 2s minute.After the 22nd loop ends, reaction mixture is incubated 7.5 minutes down in 72 ℃ again and carries out lengthening reaction.

By phenol-CHCl ₃Extraction and ethanol sedimentation are handled the PCR mixture, then successively with BstEII and EcoRI digestion.In 1% agarose that contains 0.5 μ g/ml ethidium bromide/Tris-acetate gel, the restriction digestion product is carried out electrophoresis.Downcut the dna fragmentation of expection size from gel, and by phenol-CHCl ₃Extraction and ethanol sedimentation reclaim.

After from gel, reclaiming, with the respective regions of the wild-type hIL-10DNA in the BstEII/EcoRI restricted fragment displacement pSV.Sport carrier of hIL-10 mutant.Make based on the hIL-10 mutants cDNA of pSV.Sport and in the bacillus coli DH 5 alpha strain, breed, and confirmed with the DNA sequence assay method.Use identical expression vector rotaring redyeing COS cell as mentioned above.

Aminoterminal is modified

Be to produce the N-terminal varient of people IL-10, utilize primer to but without dna profiling, with the modification cDNA fragment in the synthetic PstI/BglII district corresponding to wild-type hIL-10cDNA of PCR method.Respective regions with wild-type hIL-10DNA in the gained fragment displacement pSV.Sport carrier.Obtain some varients like this, in these varients, lacked 7 (varient N Δs 7), 10 (varient N Δs 10), 11 (varient N Δs 11) or the individual residue of 12 (varient N Δs 12) from the N-terminal of wild-type hIL-10.The primer that is used to prepare each varient to and to limit sequence number (SN) of its order as follows:

Varient primer (end) sequence number (SN)

NΔ7 C3352CC(5’) 11

C3355CC(3’) 12

NΔ10 C3353CC(5’) 13

C3354CC(3’) 14

NΔ11 C3483CC(5’) 15

C3485CC(3’) 16

NΔ12 C3484CC(5’) 17

C3486CC(3’) 18

As the front to as described in C-terminal sudden change antagonist synthetic, with right each primer of appointment primer respectively 10 picomole carry out PCR.By phenol-CHCl ₃Extraction and ethanol sedimentation are handled the PCR mixture, then successively with BglII and PstI digestion.In sepharose, the restriction digestion product is carried out electrophoresis as mentioned above, downcut the dna fragmentation of expection size from gel, and by phenol-CHCl ₃Extraction and ethanol sedimentation reclaim.

After from gel, reclaiming, downcut the respective regions of wild-type hIL-10DNA in the pSV.Sport carrier by PstI/BglII digestion, and be connected into the PstI/BglII restricted fragment of hIL-10 varient, thereby with the respective regions among these fragments displacement wild-types hIL-10DNA.Breed as mentioned above, confirm and use hIL-10 mutants cDNA based on pSV.Sport.

The N Δ 7 of gained, N Δ 10, N Δ 11 and N Δ 12 varients have the amino-acid sequence that 8-160,11-160,12-160 and the 13-160 residue of No. 4 orders are limited respectively.

Has the people IL-10 antagonist that N-terminal is modified

Aforesaid method combined then be easy to prepare the antagonist that aminoterminal and carboxyl terminal all have modification.For example, N Δ 7/K157E antagonist can prepare with following method: use 5 ' primer B3351CC and 3 ' primer C3481CC to carry out PCR as mentioned above, produce the pSV.Sport of the cDNA that contains encoded K 157E antagonist.After using 5 ' primer C3352CC and 3 ' primer C3355CC preparation as mentioned above and isolating N Δ 7 varient fragments, respective regions with K157E mutant DNA in the PstI/BglII restricted fragment displacement pSV.Sport carrier of this varient, method is to downcut this zone by PstI/BglI digestion, and is connected into the displacement fragment.

Metabolic marker

With carrying coding people IL-10; Antagonist K157E, C Δ 3 or C Δ 4; Or the cDNA of agonist varient N Δ 7, N Δ 10, N Δ 11 or N Δ 12 inserts segmental expression vector pSV.Sport, rotaring redyeing COS cell as mentioned above.Then cell is being contained in the 10cm culture dish in the blood serum medium and cultivating 48-72 hour.After this time cultivating, culture dish washes twice with phosphate buffered saline buffer (PBS), again under 37 ℃ at 5%CO ₂The middle cultivation 30 minutes, the no methionine(Met) DMEM substratum that is added with dialyse FBS and glutamine of the 8ml/ of cultivation use this time culture dish.Substratum in each culture dish of sucking-off is used 500 μ l instead and is contained 250-300 μ Ci ³⁵S-methionine(Met) (DuPont NEN, Boston, MA; Than 43.3mCi/ml alive) no methionine(Met) substratum.

With cell under 37 ℃ at 5%CO ₂The middle cultivation 5 hours adds 10 μ l1.5mg/ml L-methionine(Met)s storage liquid then, and carries out 30 minutes tracking in culture dish.Collect the conditioned medium of mark, and in the 10-20% gel, carrying out SDS-PAGE (SDS PAGE under the non-reduced condition; Laemmli, Nature227:680,1970), carry out radioautograph with standard method and Kodak XAR film behind the gel drying.

Radioautograph demonstrates people IL-10; The different mark bands of antagonist K157E, C Δ 3 and C Δ 4 and agonist N Δ 7 and N Δ 10, all bands all move at the about 16-18 kilodalton of apparent molecular weight place.Under identical transfection and cell culture condition, the expression level of three people IL-10 carboxyl terminal sudden change antagonists is all lower slightly, than the low approximately 2-4 of IL-10 doubly.The expression and the IL-10 of aminoterminal agonist varient N Δ 7 are similar, and the expression level of N Δ 10 varients is lower about 4 times than IL-10.The expression of varient N Δ 11 and N Δ 12 is too low, can not detect with present method.

Elisa assay

Be further quantitative analysis sudden change antagonist and the level of people IL-10 in the COS cell conditioned medium, carry out enzyme-linked immunosorbent assay (ELISA) as (Immunol.Rev.127:5,1992) as described in the people such as Abrams substantially.Make two kinds of monoclonal antibodies that are specific to different epitopes on the people IL-10 with standard method, these two kinds of antibody are named respectively and are 9D7 and 12G8, and are used separately as trapping agent and detection agent.In this is measured, test the conditioned medium of serial dilution, and make standard substance with the recombinant human IL-10 of purifying.The limit of detection of this assay method is about 1ng/ml, cultivates after 72 hours, generally measures the IL-10 level in substratum in the 100-300ng/ml scope.

Find that with aforesaid method IL-10 and the relative level of antagonist and the result of metabolic marker gained has good dependency, show that the epitope of being discerned by used monoclonal antibody is not in saltation zone.In typical a mensuration, the expression level of measured people IL-10, K157E, C Δ 3, C Δ 4, N Δ 7, N Δ 10, N Δ 11 and N Δ 12 is respectively 133,80,63,48,139,28,23 and 6.5ng/ml.

Biological assay

Utilize mouse hypertrophy cell and human peripheral blood mononuclear cell (PBMC), studied people IL-10 and representative IL-10 sudden change antagonist.

Substantially stimulate mensuration as the described mastocyte that carries out of people (J.Exp.Med.173:507,1991) such as people such as O ' Garra (Int.Immunol.2:821,1990) and Thompson-Snipes.Briefly, on 96 hole microtiter plates, every hole adds 5 * 10 ³Individual MC/9 cell (ATCCCRL8306) is measured (RPMI-1640 in the substratum in 100 μ l, contain 10% foetal calf serum (FBS), 50 μ M beta-mercaptoethanols, 2mM glutamine and penicillin/streptomycin), handled 48 hours with people IL-10 or a kind of IL-10 antagonist of different amounts.(Sigma, St.Louis MO), and are incubated 3-5 hour with titer plate to add 25 μ 15mg/ml MTT (bromination 3-(4,5-dimethylthiazole-2-yl)-2,5-phenylbenzene tetrazolium) then in every hole.Measure photoabsorption then with the 10%SDS dissolved cell that contains 10mMHCl, and at the 570nm place.

People IL-10 and varient N Δ 7, N Δ 10 and N Δ 11 have activity in this mensuration, but observe varient N Δ 12 activity are not arranged.Carboxyl terminal sudden change antagonist does not all have activity, even be like this during test down up to the concentration of 375ng/ml (approximately be produce strong active IL-10 amount 100 times) yet.

In order to measure the IL-10 antagonist to by lipopolysaccharides (LPS) inductive cytokine synthetic restraining effect, obtain human peripheral blood mononuclear cell (PBMC) by healthy donors, and separated (Boyum, Scand.J.Clin.Invest.Supp1:77,1966) with FICOLL  gradient centrifugation.The PBMC sample aliquot is transferred in each apertures of 96 hole microtiter plates (10 ⁵Individual cell/aperture, 200 μ l RPMI-1640 substratum wherein contain 5%FBS, penicillin/streptomycin, non-essential amino acid, Sodium.alpha.-ketopropionate and 2mM glutamine).

In some aperture, add people IL-10, add or do not add the IL-10 antagonist (measuring) of 100 times of molar excess (10nM) simultaneously by ELISA with fixed 100pM concentration.In every aperture, add immediately then LPS (Sigma) to final concentration be 80ng/ml.Utilize the substratum cultivated respectively with the COS cell of carrier of expressing IL-10 or plasmid pSV.Sport transfection, positive and negative IL-10 is contrasted parallel the insulation.With the thinner of back one conditioned medium that contrasts as all samples.All measure all parallel secondary that carries out, and are confirmed with the mensuration that continues with the cell of different batches.

Titer plate is placed 5%CO ₂Humid atmosphere in 37 ℃ of down insulations 24 hours, collect supernatant liquor then, store down in-20 ℃ and analyze after being provided with.Utilize ELISA test kit (R﹠amp; D Systems, Minneapolis MN) measures the level of IL-6, IL-1 α and TNF α in the collected sample by the specification sheets of manufacturers.

In measuring, this finds that all antagonists all make the plain synthetic of IL-10 pair cell suppress active and reverse, and are as shown in table 1.

Table 1

The active per-cent of remaining IL-10 ^*

Sample IL-6 IL-1 α TNF-α

Damping fluid 100 100 100

Antibody 000

K157E 21 27 51

CΔ3 12 13 39

CΔ4 19 27 61

* in the presence of three kinds of IL-10 antagonists of the anti-IL-10 monoclonal antibody of neutrality that contrasts damping fluid, saturation capacity and 100 times of molar excess, measure people IL-10 to the plain synthetic restraining effect of designated cell.

Do not exist the similar mensuration of carrying out with the different IL-10 sudden change antagonists of measuring under the IL-10 condition to show that it is active that all antagonists do not have the synthetic inhibition of cytokine.Concentration all can not detect the inhibition activity up to any antagonist of 100pM (comprising 100pM).

For of the influence of research IL-10 antagonist, carried out mixed lymphocyte reacion (MLR) and measured the T cytoactive.Separation of human PBMC as mentioned above.(Sigma, St.Louis MO) handled 20 minutes, and making stimulates PBMC with the 50mg/ml ametycin down in 37 ℃ with cell.

In each aperture of 96 hole microtiter plates, mix reply PBMC and irritation cell each about 1 * 10 ⁵Individual, add a kind of in the people IL-10 of different amounts or K157E, C Δ 3 or C Δ 4 antagonists simultaneously, cumulative volume is 200 μ l (three parts of parallel tests).Cell is used 5%CO under 37 ℃ ₂Cultivated 6 days, then in the culture of every aperture pulsed add 1 μ Ci tritiate thymidine ([ ³H]-TdR, 15.6Ci/mmol, NEN, Boston MA), lasts 16 hours.(Sterline VA) collects lysate on the filter membrane for Skatron, Inc., and (Pharmacia LKB Nuclear Inc., Gaithersburg MD) count with beta-counter with 96 porocyte collectors.

Find that these antagonists can not suppress MLR under the concentration of 1ng/ml.On the contrary, people IL-10 makes MLR reach 82% inhibition under this concentration.

Receptors bind is measured

Use ENZYMOBEAD ^(BioRad, Richmond CA), make people IL-10 (purity about 99%) the radiation iodate of purifying to method according to the specification sheets of manufacturers.By making about 4 * 10 in centrifugal 10 minutes with 200 * g ⁵The rotaring redyeing COS cell sedimentation of individual expressing human IL-10 receptor cdna is with binding buffer liquid (PBS, 10% foetal calf serum, 0.1%NaN ₃) washing, and with the concentration of 150pM be suspended in again 200 μ l contain [ ¹²⁵I] in the binding buffer liquid of people IL-10 (is 225 μ Ci/ μ g than radiation), add the COS cell conditioned medium of serial dilution simultaneously, the cDNA of one of this COS cell expressing coding people IL-10 or sudden change antagonist of the present invention.

In 4 ℃ down cultivate 2 hours after, under uniform temp with cell centrifugal 10 minutes with 200 * g.Shift out supernatant liquor then, each cell precipitation is suspended in the binding buffer liquid that does not contain mark IL-10 again, be layered on and be contained in the 200 μ ls of lengthening in the little centrifuge tube and contain in the binding buffer liquid of 10% glycerine, under 4 ℃ with the centrifugal 1O of 200 * g minute, quick freezing in liquid nitrogen.Then cell precipitation is cut in the counter tube, at CLINIGAMMA ^Counting in 1272 counters (PharmaciaLKB).Under the unmarked people IL-10 of 500 to 1000 times of molar excess leaves, carry out combination, thereby measure non-specific binding.

The results are shown in table 2.As can be seen from Table 2, in the receptors bind competition, all IL-10 antagonists are nearly all the same with IL-10 itself effective.

Table 2

Radio-labeled IL-10 bonded suppresses ^*

Sample IC ₅₀(pM)

People IL-10 100

K157E 136±65

CΔ3 172±28

CΔ4 120±9

* data shown in are 2 independent mean values of measuring, reach the 50% unmarked people IL-10 that suppresses or the concentration of assigned I L-10 antagonist with combining of cell receptor for the radiolabeled people IL-10 that sends as an envoy to.

It will be apparent to those skilled in the art that and under the prerequisite of main idea of the present invention and scope, can make many modifications and change the present invention.Specific embodiments described here only provides with way of example, and the present invention is only limited by appending claims.

Sequence list

(1) general information

(i) applicant: Schering Corp

(ii) denomination of invention: the agonist of human interleukin-10 and antagonist

(iii) Ser.No.: 18

(iv) mailing address:

(A) addressee: Schering-Plough Corporation

(B) street: One Giralda Farms

(C) city: Madison

(D) state: New Jersey

(E) country: USA

(F) postcode: 07940

(v) computer-reader form:

(A) media type: floppy disk

(B) computer: Apple Macintosh

(C) operating system: Macintosh 7.1

(D) software: Microsoft Word5.1a

(vi) the application's data

(A) application number: (B) applying date (C) classification: (vii) request for data formerly: U.S. Patent application 08/098,943 (viii) proxy's information: (A) name: Lunn, Paul, (2) No. 1 order informations of G.: (i) ordinal characteristics: (A) length: 160 amino acid (B) type: amino acid (D) topological framework: linearity is molecule type (ii): peptide (xi) order is described: No. 1 order: Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro1 5 10 15Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30Val?Lys?Thr?Phe?Phe?Gln?Met?Lys?Asp?Gln?Leu?Asp?Asn?Leu?Leu?Leu

35 40 45Lys?Glu?Ser?Leu?Leu?Glu?Asp?Phe?Lys?Gly?Tyr?Leu?Gly?Cys?Gln?Ala

50 55 60Leu?Ser?Glu?Met?Ile?Gln?Phe?Tyr?Leu?Glu?Glu?Val?Met?Pro?Gln?Ala65 70 75 80Glu?Asn?Gln?Asp?Pro?Asp?Ile?Lys?Ala?His?Val?Asn?Ser?Leu?Gly?Glu

85 90 95Asn?Leu?Lys?Thr?Leu?Arg?Leu?Arg?Leu?Arg?Arg?Cys?His?Arg?Phe?Leu

100 105 110Pro?Cys?Glu?Asn?Lys?Ser?Lys?Ala?Val?Glu?Gln?Val?Lys?Asn?Ala?Phe

115 120 125Asn?Lys?Leu?Gln?Glu?Lys?Gly?Ile?Tyr?Lys?Ala?Met?Ser?Glu?Phe?Asp

150 155 160 (2) No. 2 order informations of 130 135 140Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Glu Ile Arg Asn145: (i) ordinal characteristics: (A) length: 157 amino acid (B) type: amino acid (D) topological structure: linear (ii) molecule type: peptide (xi) order is described: No. 2 orders: Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro1 5 10 15Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30Val?Lys?Thr?Phe?Phe?Gln?Met?Lys?Asp?Gln?Leu?Asp?Asn?Leu?Leu?Leu

35 40 45Lys?Glu?Ser?Leu?Leu?Glu?Asp?Phe?Lys?Gly?Tyr?Leu?Gly?Cys?Gln?Ala

85 90 95Asn?Leu?Lys?Thr?Leu?Arg?Leu?Arg?Leu?Arg?Arg?Cys?His?Arg?Phe?Leu

100 105 110Pro?Cys?Glu?Asn?Lys?Ser?Lys?Ala?Val?Glu?Gln?Val?Lys?Asn?Ala?Phe

115 120 125Asn?Lys?Leu?Gln?Glu?Lys?Gly?Ile?Tyr?Lys?Ala?Met?Ser?Glu?Phe?Asp

150 155 (2) No. 3 order informations of 130 135 140Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys145: (i) ordinal characteristics: (A) length: 156 amino acid (B) type: amino acid (D) topological structure: linear (ii) molecule type: peptide (xi) order is described: No. 3 orders: Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro1 5 10 15Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30Val?Lys?Thr?Phe?Phe?Gln?Met?Lys?Asp?Gln?Leu?Asp?Asn?Leu?Leu?Leu

35 40 45Lys?Glu?Ser?Leu?Leu?Glu?Asp?Phe?Lys?Gly?Tyr?Leu?Gly?Cys?Gln?Ala

85 90 95Asn?Leu?Lys?Thr?Leu?Arq?Leu?Arg?Leu?Arg?Arg?Cys?His?Arg?Phe?Leu

100 105 110Pro?Cys?Glu?Asn?Lys?Set?Lys?Ala?VaL?Glu?Gln?Val?Lys?Asn?Ala?Phe

115 120 125Asn?Lys?Leu?Gln?Glu?Lys?Gly?Ile?Tyr?Lys?Ala?Met?Ser?Glu?Phe?Asp

150 155 (2) No. 4 order informations of 130 135 140Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met145: (i) ordinal characteristics: (A) length: 160 amino acid (B) type: amino acid (D) topological structure: linear (ii) molecule type: peptide (xi) order is described: No. 4 orders: Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro1 5 10 15Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30Val?Lys?Thr?Phe?Phe?Gln?Met?Lys?Asp?Gln?Leu?Asp?Asn?Leu?Leu?Leu

35 40 45Lys?Glu?Ser?Leu?Leu?Glu?Asp?Phe?Lys?Gly?Tyr?Leu?Gly?Cys?Gln?Ala

50 55 60Leu?Ser?Glu?Met?Ile?Gln?Phe?Tyr?Leu?Glu?Glu?Val?Met?Pro?Gln?Ala65 70 75 80Glu?Asn?Gln?Asp?Pro?Asp?Ile?Lys?Ala?His?Val?ASh?Ser?Leu?Gly?Glu

85 90 95Asn?Leu?Lys?Thr?Leu?Arg?Leu?Arg?Leu?Arg?Arg?Cys?His?Arg?Phe?Leu

100 105 110Pro?Cys?Glu?Asn?Lys?Ser?Lys?Ala?Val?Glu?Gln?Val?Lys?Asn?Ala?Phe

115 120 125Asn?Lys?Leu?Gln?Glu?Lys?Gly?Ile?Tyr?Lys?Ala?Met?Ser?Glu?Phe?Asp

130 135 140Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn145 150 155 160 ( 2 ) 5： ( i ) ： ( A ) ：60 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：5：GTCGACTGCA GCCGCCACCA TGCACAGCTC AGCACTGCTC TGTTGCCCTGG TGTTGCCTGG TCCTCCTGAC 60 ( 2 ) 6： ( i ) ： ( A ) ：41 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：6：ACGTCGAATT CTCAGTTTCG TATCTTCATT GTCATGTAGG C 41 ( 2 ) 7： ( i ) ： ( A ) ：85 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：7：GGACTTTAAG GGTTACCTGG GTTGCCAAGC CTTGTCTGAG ATGATCCAGT TTTATCTAGA 60GGAGGTGATG CCCCAAGCTG AGAAC 85 ( 2 ) 8： ( i ) ： ( A ) ：49 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：8：AGCTGAATTC AGTTTCGTAT CTCCATTGTC ATGTAGGCTT CTATGTAGT 49 ( 2 ) 9： ( i ) ： ( A ) ：40 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：9：AGCTGAATTC ACTTCATTGT CATGTAGGCT TCTATGTAGT 40 ( 2 ) 10： ( i ) ： ( A ) ：37 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：10：AGCTGAATTC ACATTGTCAT GTAGGCTTCT ATGTAGT 37 ( 2 ) 11： ( i ) ： ( A ) ：91 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：11：GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTGCTCTGTT GCCTGGTCCT 60CCTGACTGGG GTGAGGGCCT CTGAGAACAG C 91 ( 2 ) 12： ( i ) ： ( A ) ：90 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：12：GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGCAGCTGTT 60CTCAGAGGCC CTCACCCCAG TCAGGAGGAC 90 ( 2 ) 13： ( i ) ： ( A ) ：82 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：13：GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTGCTCTGTT GCCTGGTCCT 60CCTGACTGGG GTGAGGGCCA GC 82 ( 2 ) 14： ( i ) ： ( A ) ：81 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：14： GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGCAGCTGGC 60CCTCACCCCA GTCAGGAGGA C 81 ( 2 ) 15： ( i ) ： ( A ) ：82 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：15：GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTGCTCTGTT GCCTGGTCCT 60CCTGACTGGG GTGAGGGCCT GC 82 ( 2 ) 16： ( i ) ： ( A ) ：78 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：16GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGA AGTGGG TGCAGGCCCT 60CACCCCAGTC AGGAGGAC 78 ( 2 ) 17： ( i ) ： ( A ) ：81 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：17：GACGACGGTG GCTGCAGCCG CCACCATGCA CAGCTCAGCA CTGCTCTGTT GCCTGGTCCT 60CCTGACTGGG GTGAGGGCCA C 81 ( 2 ) 18： ( i ) ： ( A ) ：75 ( B ) ： ( C ) ： ( D ) ： ( xi ) ：18：GGCATCTCGG AGATCTCGAA GCATGTTAGG CAGGTTGCCT GGGAAGTGGG TGGCCCTCAC 60CCCAGTCAGG AGGAC 75

Claims

1. the antagonist of a people IL-10, this antagonist comprises because 157 lysine residue is replaced by acidic amino acid residue, or owing to lacking the one-tenth acquaintance IL-10 that one or more amino-acid residue obtains modifying in the zone that contains about 12 carboxyl terminal residues.

2. the antagonist of claim 1, wherein N-terminal has lacked 1-11 amino-acid residue.

3. the antagonist of claim 1, this antagonist have the amino-acid sequence that is limited by 1,2 or No. 3 order.

4. the nucleic acid of the antagonist of the people IL-10 that encodes, described antagonist comprises because 157 lysine residue is replaced by acidic amino acid residue, or owing to lacking the one-tenth acquaintance IL-10 that one or more amino-acid residue obtains modifying in the zone that contains about 12 carboxyl terminal residues.

5. the nucleic acid of claim 4, a kind of aminoterminal of this nucleic acid encoding has lacked the antagonist of 1-11 amino-acid residue.

6. the nucleic acid of claim 4, the people IL-10 antagonist of this nucleic acid encoding have the amino-acid sequence that is limited by 1,2 or No. 3 order.

7. recombinant vectors that comprises the nucleic acid of claim 4, this carrier can instruct described expression of nucleic acids.

8. host cell, this host cell contains the recombinant vectors of claim 7.

9. method of producing the antagonist of people IL-10, described antagonist comprises because 157 lysine residue is replaced by acidic amino acid residue, or owing to lacking the one-tenth acquaintance IL-10 that one or more amino-acid residue obtains modifying in the zone that contains about 12 carboxyl terminal residues, described method comprises: the host cell of cultivating claim 8 under the condition of express nucleic acid.

10. the method for claim 9, a kind of aminoterminal of wherein said nucleic acid encoding has lacked the antagonist of 1-11 amino-acid residue.

11. the method for claim 9, the coded antagonist of wherein said nucleic acid have the amino-acid sequence that limits in proper order by 1,2 or No. 3.

12. one kind is suppressed the bioactive method of people IL-10, this method comprises: the cell that has people IL-10 acceptor is contacted with a kind of people IL-10 antagonist of significant quantity, antagonist comprises because 157 lysine residue is replaced by acidic amino acid residue, or owing to lacking the one-tenth acquaintance IL-10 that one or more amino-acid residue obtains modifying in the zone that contains about 12 carboxyl terminal residues.

13. the method for claim 12, wherein the aminoterminal of antagonist has lacked 1-11 amino-acid residue.

14. the method for claim 12, wherein antagonist has the amino-acid sequence that limits in proper order by 1,2 or No. 3.

15. the antagonist of a people IL-10, this antagonist comprise owing to having lacked 1-11 the one-tenth acquaintance IL-10 that the aminoterminal amino-acid residue obtains modifying.

16. the antagonist of claim 15 has wherein lacked 7,10 or 11 oxygen base acid residues.

17. the nucleic acid of the people IL-10 antagonist of encoding, this antagonist comprise owing to 1-11 one-tenth acquaintance IL-10 that the aminoterminal amino-acid residue obtains modifying of disappearance.

18. the nucleic acid of claim 17, this nucleic acid encoding have lacked the antagonist of 7,10 or 11 amino-acid residues.

19. a recombinant vectors that comprises the nucleic acid of claim 17, this carrier can instruct expression of nucleic acids.

20. a host cell, this host cell comprises the recombinant vectors of claim 19.

21. a method of producing people IL-10 agonist, this agonist comprise owing to 1-11 one-tenth acquaintance IL-10 that the aminoterminal amino-acid residue obtains modifying of disappearance, described method comprises: the host cell of cultivating claim 20 under the condition of express nucleic acid.

22. the method for claim 21, wherein said nucleic acid encoding has lacked the agonist of 7,10 or 11 amino-acid residues.

23. pharmaceutical composition, said composition comprises the agonist of a kind of people IL-10 of a kind of pharmaceutically acceptable carrier and significant quantity (a), this agonist comprises owing to 1-11 one-tenth acquaintance IL-10 that the aminoterminal amino-acid residue obtains modifying of disappearance, or (b) antagonist of a kind of people IL-10, this antagonist comprises because 157 lysine residue is replaced by acidic amino acid residue, or owing to lacking the one-tenth acquaintance IL-10 that one or more amino-acid residue obtains modifying in the zone that contains about 12 carboxyl terminal residues.

24. the pharmaceutical composition of claim 23, wherein the aminoterminal of antagonist has lacked 1-11 amino-acid residue.

25. the pharmaceutical composition of claim 23, wherein antagonist has the amino-acid sequence that limits in proper order by 1,2 or No. 3.

26. the pharmaceutical composition of claim 23 has wherein lacked 7,10 or 11 amino-acid residues in the agonist.

27. the application of the antagonist of a people IL-10 in suppressing the IL-10 biological activity, this antagonist comprises because 157 lysine residue is replaced by acidic amino acid residue, or owing to lacking the one-tenth acquaintance IL-10 that one or more amino-acid residue obtains modifying in the zone that contains about 12 carboxyl terminal residues.

28. the antagonist of a people IL-10 is used for suppressing the application of the bioactive medicine of IL-10 in preparation, described antagonist comprises because 157 lysine residue is replaced by acidic amino acid residue, or owing to lacking the one-tenth acquaintance IL-10 that one or more amino-acid residue obtains modifying in the zone that contains about 12 carboxyl terminal residues.