CN1845937A - Anti-IL-20 antibodies and binding partners and methods of using in inflammation - Google Patents

Abstract

The present invention relates to blocking, inhibiting, reducing, antagonizing or neutralizing the activity of IL-20 polypeptide molecules. IL-20 and IL-22 are cytokines that are involved in inflammatory processes and human disease. The present invention includes anti-IL-20 and anti-IL-22RA antibodies and binding partners, as well as methods for antagonizing IL-20 using such antibodies and binding partners.

Description

Anti-IL-20 antibody and binding partners and the using method in inflammation

Background of invention

Cytokine is the small-sized albumen of solubility, and they mediate multiple different biological action, comprise to the growth of many cell types and the regulation and control of differentiation (referring to, Arai et al. for example, Annu.Rev.Biochem.59:783 (1990); Mosmann, Curr.Opin.Immunol.3:311 (1991); Paul and Seder, Cell 76:241 (1994)).The protein of forming the cytokine group comprises interleukin-, Interferon, rabbit, G CFS, tumour necrosis factor and other regulatory molecules.For example, human interleukin-11 7 is to stimulate the cytokine that adhesion molecule 1, interleukin 8, rHuGM-CSF and prostaglandin E2 are expressed in interleukin 6, the born of the same parents, (Yao et al., J.Immunol.155:5483 (1995) work in the preferential maturation of neutrophilic granulocyte at CD34+ hematopoiesis precursor; Fossiez et al., J.Exp.Med.183:2593 (1996)).

Acceptor in conjunction with cytokine is made up of one or more conformability membranin usually, and they combine with high-affinity with cytokine, and by the tenuigenin part of this receptor subunit this binding events is transduceed to cell.Similarity according to cytokine receptor extracellular aglucon binding domains is divided into broad variety with cytokine receptor.For example, the receptor chain of being responsible for combination and/or transduction Interferon, rabbit effect is the member of II type cytokines receptor family, and the foundation of division is the characteristic extracellular domain of 200 residues.

The activity in vivo that cytokine and their acceptor show has been illustrated for the demand of other cytokines, cytokine receptor, cytokine agonist and cytokine antagonist and they and has been used for clinical potentiality.For example, the activity in vivo that shows of pro-inflammatory cytokine family has been illustrated the demand of short inflammatory molecule antagonist and they has been used for clinical great potential.The present invention has satisfied these demands by the antagonist at pro-inflammatory cytokine IL-20 and IL-22 is provided.This class antagonist of the present invention can be blocked, suppresses, reduction, antagonism or in and the activity of IL-22, IL-20 or IL-20 and IL-22, they comprise the anti-IL-22RA antibody of solubility IL-22RA acceptor and neutrality.The present invention also provides their uses in diseases associated with inflammation and relevant composition and method.

Detailed Description Of The Invention

1. general introduction

The invention provides the soluble receptors that is named as " Zcytor11 " or " IL-22RA " and at the novel use of the neutrality antibody of IL-22RA cytokine receptor, and other invention.The present invention also provides solubility IL-22RA polypeptide fragment and fusion rotein, is used for human struvite and autoimmune disease.Anti-IL-22RA antibody of the present invention and solubility IL-22RA acceptor, comprise the anti-IL-22RA antibody of neutrality of the present invention, can for example psoriasis, arthritic psoriasis, sacroiliitis, endotoxemia, inflammatory bowel (IBD), colitis and other be used for blocking in struvite situation disclosed herein, suppress at the specific human diseases of treatment, reduction, antagonism or and the activity of IL-22 or IL-20 or IL-20 and IL-22.

The illustrative nucleotide sequence of coding people Zcytor11 (IL-22RA) is provided by SEQNO:1; Encoded polypeptide shows in SEQ ID NO:2.IL-22RA is the two a receptor subunit of IL-20 and IL-22.Zcytor11 (IL-22RA) is disclosed in total United States Patent (USP) numbering 5,965,704, total WIPO publication WO 02/12345 and total WIPO publication WO 02/072607.People cDNA clone to coding IL-22RA (SEQ ID NO:1) analyzes the open reading frame of having found 574 amino acid of coding (SEQ ID NO:2), and this open reading frame comprises about 211 amino-acid residues (18-228 residue of SEQ ID NO:2; The cell intracellular domain of the membrane spaning domain of extracellular aglucon binding domains SEQ ID NO:3), about 23 amino-acid residues (the 229-251 residue of SEQ ID NO:2) and about 313 amino-acid residues (the 252-574 residue of SEQ ID NO:2).Like this, molecule of the present invention has comprised the polypeptide of the cytokine binding domains that contains the 18-228 amino-acid residue (SEQ ID NO:3) that comprises SEQ ID NO:2.In an embodiment of soluble receptors of the present invention, the IL-22R of this solubility and the constant region of heavy chain (representing sequence to show in SEQ ID NO:4) merge.Those skilled in the art will appreciate that the border of these structural domains is property roughly.From structural domain terminal deletion residue is possible.

As following description, the invention provides isolated polypeptide, they comprise reference amino acid sequence (also being expressed as SEQ ID NO:3) shown in the 18-228 residue with SEQ ID NO:2 and are 70% at least, are 80% at least or are 90% or greater than 95%, for example 96%, 97%, 98% or greater than 99% or the identical aminoacid sequence of higher degree at least, wherein said isolated polypeptide can with can specific combination comprise shown in the SEQ ID NO:3 antibody generation specific combination of peptide more than the aminoacid sequence.Illustrative polypeptide comprises the polypeptide that comprises amino-acid residue among SEQ ID NO:3 or the SEQ ID NO:3.And the present invention also provides as above disclosed and IL-22 bonded isolated polypeptide (for example human il-22 peptide sequence shown in the SEQ ID NO:6).This human il-22 polynucleotide sequence shows in SEQ ID NO:5.The IL-22 polynucleotide sequence of mouse shows that in SEQ ID NO:10 corresponding polypeptide shows in SEQ ID NO:11.The present invention also provides as above disclosed and IL-20 bonded isolated polypeptide (human IL-2's 0 peptide sequence shown in SEQ ID NO:8 for example; WIPO publication numbering WO99/27103).This human IL-2's 0 polynucleotide sequence shows in SEQ ID NO:7.

The present invention also provides and has comprised shown in the SEQ ID NO:3 isolated polypeptide and the epi-position of at least 15 continuous amino acid residues in the aminoacid sequence.Illustrative polypeptide comprises and comprises SEQ IDNO:3 or its antigenic epitopes or its functional IL-20 or IL-22 binding fragment or be made up of them.And, the present invention also provide as above disclosed combination, blocking-up, inhibition, reduction, antagonism or in and the active isolated polypeptide of IL-22 or IL-20.

The present invention has also comprised variant IL-22RA polypeptide, wherein the aminoacid sequence of this variant polypeptide is selected from following group with the consistence per-cent of amino-acid residue shown in the SEQ ID NO:3: at least 70% consistent, at least 80% unanimity, at least 90% unanimity, at least 95% consistent or greater than 95% unanimity, as 96%, 97%, 98% or greater than 99% or higher consistence, wherein any difference between the corresponding aminoacid sequence of the aminoacid sequence of variant polypeptide and SEQ ID NO:3 causes owing to one or more conserved amino acid is alternative.This conserved amino acid substitutes at this to be described to some extent.And, the present invention also provide as above disclosed can in conjunction with, blocking-up, inhibition, reduction, antagonism or in and IL-22 or the active isolated polypeptide of IL-20.

The present invention also provides antibody and the antibody fragment with this peptide species specific combination.Exemplary antibody comprises neutralizing antibody, polyclonal antibody, mouse monoclonal antibody, from the humanized antibodies and the human monoclonal antibodies of mouse monoclonal antibody.Exemplary antibody fragment comprises F (ab ') ₂, F (ab) ₂, Fab ', Fab, Fv, scFv and atom.Neutralizing antibody preferably can make that the interaction of IL-20 and IL-22 and IL-22RA is blocked in conjunction with IL-22RA, suppresses, reduction, antagonism or neutralization; Or make that IL-20 or IL-22 are blocked with combining of IL-22RA, suppress, reduction, antagonism or the anti-IL-22RA neutralizing antibody of neutral be also included among the present invention.Be the anti-IL-22RA antibody of neutrality of the present invention can in conjunction with, blocking-up, inhibition, reduction, antagonism or in and only a kind of among IL-20 or the IL-22, also combination simultaneously, blocking-up, inhibition, reduction, antagonism or in and IL-20 and IL-22.The present invention also comprises the composition that comprises carrier and peptide described herein, polypeptide or antibody.

In addition, the invention provides pharmaceutical composition, wherein comprise pharmaceutically acceptable carrier and at least one this expression vector or comprise the recombinant virus of this expression vector.The present invention has also comprised the pharmaceutical composition that comprises acceptable carrier on the medicine and polypeptide described herein or antibody.

The invention still further relates to antiidiotypic antibody or antiidiotypic antibody fragment, they can with can and comprise the polypeptide of aminoacid sequence shown in the SEQ ID NO:3 or the antibody or the antibody fragment specificity of its fragment specific combination combines.Exemplary antiidiotypic antibody can combine with the antibody of the polypeptide specific combination that can and be made up of SEQ ID NO:3.

The present invention also provides fusion rotein, wherein comprises IL-22RA polypeptide and immunoglobulin part.In such fusion rotein, immunoglobulin part can be an immunoglobulin heavy chain constant region, as people Fc fragment.The present invention has also comprised the isolated nucleic acid molecule of this fusion rotein of encoding.

The present invention also provides polyclone and monoclonal antibody, they can with the polypeptide that comprises the IL-22RA extracellular domain (for example monomer, all dimer, heterodimer and polymer acceptor comprise soluble receptors) combination.In addition, this antibody can be used for antagonism IL-22RA aglucon, IL-22 (SEQ ID NO:6) and IL-20 (SEQ ID NO:8) separately or together with the IL-22RA receptors bind.

And, overexpression or the rise of IL-20 and IL-22 have been shown in people's psoriasis breakage and the people's atopic dermatitis skin samples, point out identically with IL-20, IL-22 is other diseases associated with inflammation of participant's psoriasis, atopic dermatitis or skin and epithelium also.And as described herein, the overexpression of IL-20 or IL-22 has shown the immunocyte property of participation that epidermal area thickens and shows the psoriasis phenotype in transgenic mice; In addition, inject IL-22 in normal mouse, also shown the immunocyte property of participation that epidermal area thickens and shows the psoriasis phenotype, these phenomenons are by soluble receptors antagonist IL-22RA2 (zcytor16; WIPO publication numbering WO 01/40467) eliminates.Data further point out the IL-22 of short inflammatory to participate in other diseases associated with inflammation of psoriasis, atopic dermatitis or skin and epithelium in these bodies.Therefore, the active antagonist of IL-22 and IL-20 for example IL-22RA soluble receptors and antibody (comprising anti-human il-22 RA monoclonal antibody of the present invention and neutralizing antibody) thereof is useful in the therapeutic treatment of diseases associated with inflammation, particularly can be used as IL-22 and IL-20 separately or the common antagonist in psoriasis treatment.And, the active antagonist of IL-22 for example IL-22RA soluble receptors and antibody (comprising anti-human il-22 RA monoclonal antibody of the present invention and neutralizing antibody) thereof is useful in the therapeutic treatment of other diseases associated with inflammation, for example can be in atopic dermatitis, IBD, colitis, endotoxemia, sacroiliitis, rheumatoid arthritis and arthritic psoriasis adult respiratory disease (ARD), septic shock, multiple organ failure, struvite injury of lung such as asthma or bronchitis, bacterial pneumonia, psoriasis, eczema, the for example combination in the treatment of ulcerative colitis and Crohn disease of atopy or contact dermatitis and inflammatory bowel, blocking-up, suppress, reduce, antagonism or in and IL-22 and IL-20 (individually or the two all can).

With reference to following detailed description, these and other aspects of the present invention will be apparent.In addition, below also listed a plurality of different reference, quoted here as a reference with their integral body.

2. definition

In the following description, some terms have been used in a large number.Provide following definition to help to understand the present invention.

Just as used herein, " nucleic acid " or " nucleic acid molecule " is meant polynucleotide, for example thymus nucleic acid (DNA) or Yeast Nucleic Acid (RNA), oligonucleotide, the fragment of polymerase chain reaction (PCR) generation and the fragment that is produced by connection, shearing, restriction endonuclease effect and the active any way of excision enzyme.Nucleic acid molecule can be by naturally occurring Nucleotide (for example DNA and RNA), and perhaps natural analogue (for example natural α-enantiomorph that has Nucleotide) that has Nucleotide or above the two the such monomer of combination are formed.The Nucleotide of modifying can partly have change at its sugar moieties and/or pyrimidine or purine bases.Sugar-modified comprising, for example use halogen, alkyl, amido and azido-to replace one or poly-hydroxy group more, perhaps sugar can sense etherization or ester.And whole sugar moieties can be replaced by structure three-dimensional or that electronics is similar, for example azasugar and ring-type sugar analogue.The example that base portion is modified comprises the purine or the pyrimidine of alkylating purine and pyrimidine, acylations, perhaps other well-known heterocyclic substituents.Nucleic acid monomer can link to each other by phosphodiester bond or this analogue that connects key.The analogue that phosphodiester bond connects comprises thiophosphatephosphorothioate, phosphorodithioate, seleno phosphoric acid ester, two seleno phosphoric acid ester, phosphoroanilothioate, phosphoranilidate, phosphoramidate or the like.Term " nucleic acid molecule " also comprises so-called " peptide nucleic acid(PNA) ", and it comprises the nucleic acid base that is connected the naturally occurring or modification on the multiamide skeleton.Nucleic acid can be strand or two strands.

Term " complement of nucleic acid molecule " is meant with the reference nucleotide sequence to be compared, and has complementary nucleotide sequence and rightabout nucleic acid molecule.For example sequence 5 ' ATGCACGGG 3 ' and sequence 5 ' CCCGTGCAT 3 ' complementation.

Term " degenerate core nucleotide sequence " is meant compares the nucleotide sequence that comprises or more degenerate codons with the reference nucleic acid molecule of coded polypeptide.Degenerate codon contains different nucleotide triplets, but encodes identical amino-acid residue (be GAU and GAC triplet all encode aspartic acid).

Term " structure gene " is meant the nucleic acid molecule that is transcribed into messenger RNA(mRNA) (mRNA), and this messenger RNA(mRNA) is translated as the peculiar aminoacid sequence of specific polypeptides again.

" isolated nucleic acid molecule " is the nucleic acid molecule that is not incorporated in the organism genomic dna.Dna molecular that for example, separated from the genomic dna of cell, the coding somatomedin is an isolated DNA molecule.Another example of isolated nucleic acid molecule is the nucleic acid molecule that is not incorporated into the chemosynthesis in the organism genome.Isolated nucleic acid molecule is less than the chromosomal global DNA molecule from these species from a certain specific species.

" nucleic acid molecule construct " is meant strand or double-stranded nucleic acid molecule, and it is modified by people's intervention, thereby contains some nucleic acid fragments, and these fragments make up in the non-existent mode of occurring in nature and are in juxtaposition.

" linear DNA " is meant to have free 5 ' and 3 ' terminal non-annularity dna molecular.Can pass through the closed hoop dna molecular, plasmid is for example cut or physical rupture prepares linear DNA molecule by enzyme.

" complementary DNA (cDNA) " is meant the single strand dna that is formed by reversed transcriptive enzyme by the mRNA template.Usually use the initial reverse transcription of part complementary primer with mRNA.Those skilled in the art also use term " cDNA " to represent the double chain DNA molecule of being made up of this single strand dna and complementary dna chain thereof.Term " cDNA " also refers to from the clone of RNA template synthetic cDNA molecule.

" promotor " is meant the nucleotide sequence that instructs structure gene to transcribe.Usually promotor is positioned at 5 ' non-coding region of gene, near the transcription initiation site of structure gene.Sequential element in the promotor that works in transcription initiation, its characteristics that have usually are the nucleotide sequences that have.These promoter elements comprise RNA polymerase binding site, TATA sequence, CAAT sequence, differentiation specificity element (DSEs; McGehee et al., Mol.Endocrinol.7:551 (1993)), ring AMP response element (CREs), serum response element (SREs; Treisman, Seminars in Cancer Biol.1:47 (1990)), glucocorticosteroid reactivity (GREs), and the binding site of other transcription factors, these transcription factors are CRE/ATF (O ' Reilly et al., J.Biol.Chem.267:19938 (1992)) for example, AP2 (Ye etal., J.Biol.Chem.269:25728 (1994)), SP1, cAMP response element binding protein (CREB; Loeken, Gene Expr.3:253 (1993)) and the eight aggressiveness factors (usually referring to Watson et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc.1987) and Lemaigre and Rousseau, Biochem.J.303:1 (1994)).If promotor is an inducible promoter, the rate response of then transcribing is improved in inductor.On the contrary, if promotor is a composition, then transcription rate is not subjected to the regulation and control of inductor.The also known type promotor that checks.

" core promoter " contains the pivotal nucleotide sequence of promotor effect, comprises TATA box and transcriptional start point.According to this definition, may improve activity or make promotor have the active distinguished sequence of organizing specific non-existent the time, core promoter has or does not have detectable activity.

" controlling element " is to regulate the active nucleotide sequence of core promoter.For example, controlling element may contain and cytokine bonded nucleotide sequence, these cytokines make only transcribe or preferentially in specific cell, tissue or organoid, carry out.The controlling element of these types is general relevant with the gene with " cell-specific ", " tissue specificity " or " organoid specificity " phraseology.

" enhanser " is a class controlling element, and they can improve the efficient of transcribing, and regardless of distance or the direction of enhanser with respect to the transcription initiation position.

" allogeneic dna sequence DNA " is meant dna molecular or dna molecular group, and they are not present in the given host cell under natural situation.As long as host cell DNA and non-host cell DNA (being foreign DNA) are combined, then for this particular host cell, belong to and to contain the DNA (source DNA promptly) that comes from this host cell kind in the allogenic dna molecular.For example, the nonhost dna fragmentation that contains coded polypeptide is considered to the allogeneic dna sequence DNA molecule with the dna molecular that the host DNA fragment that comprises transcripting promoter can be operatively connected.On the contrary, the allogeneic dna sequence DNA molecule can comprise the native gene that can be operatively connected with exogenous promoter.As another example, comprise from the dna molecular of the gene of wild-type cell if be introduced in the mutant cells of this wild type gene not, then this dna molecular also is considered to allogenic.

" polypeptide " is meant the polymer of the amino-acid residue that is connected by peptide bond, and though polymer be natural generation or synthetic.Polypeptide less than about 10 amino-acid residues is commonly called " peptide ".

" protein " is to comprise one or the macromole of peptide chain more the more.Protein may also contain non-peptide composition, for example glycosyl.Can add sugar and other non-peptide substituents to this protein by producing proteinic cell, these substituents of adding are different with the difference of cell type.Here protein is described according to proteinic aminoacid shelf structure; Substituting group does not indicate as glycosyl group, but they can exist.

Peptide or polypeptide by the nonhost dna molecule encode are " allogenic " peptide or polypeptide.

" cloning vector " is nucleic acid molecule, for example plasmid, clay or phage, and they have the ability of self-replicating in host cell.Cloning vector contains one or a few limitations restriction endonuclease recognition site usually, and they can make nucleic acid molecule be inserted in the carrier according to confirmable mode, and can not make the important biomolecule of carrier learn afunction; Carrier also contains the nucleotide sequence of encoding marker genes, and this marker gene is applicable to discriminating and selects by the cloning vector cell transformed.Marker gene generally includes the gene that tetracyclin resistance or amicillin resistance are provided.

" expression vector " is the nucleic acid molecule that is coded in the gene of host cell inner expression.Usually, expression vector comprises transcripting promoter, gene and transcription terminator.Genetic expression is under the control of the promotor of being everlasting, and such gene is called as and promotor " operability is connected ".Similarly, if controlling element is regulated the activity of core promoter, then described regulating and controlling sequence is that operability is connected with core promoter.

" recombinant host " is to contain for example cell of cloning vector or expression vector of heterologous nucleic acids molecule.In the context of the present invention, recombinant host example is the cell that produces IL-22RA from expression vector.In contrast, IL-22RA can be produced by the cell as the IL-22RA natural origin, and this cell does not have expression vector.

" integration transformant " is the host cell of reorganization, and wherein allogeneic dna sequence DNA is incorporated in the genomic dna of this cell.

" fusion rotein " is the hybrid protein of being expressed by the nucleic acid molecule of the nucleotide sequence that comprises at least two genes.For example, fusion rotein can comprise the IL-22RA polypeptide to small part with can merge mutually in conjunction with the polypeptide of affinity matrix.This fusion rotein provides a kind of approach that uses affinity chromatography to separate a large amount of IL-22RA.

Term " acceptor " refer to can with the cell-associated protein of bioactive molecules bonded that is called as " aglucon ".This interaction has mediated the effect of aglucon on cell.Acceptor can be film mating type acceptor, cytosol receptor or nuclear receptor; Can be monomer (for example thyrotropin acceptor, B-adrenergic receptor) or polymer (for example pdgf receptor, growth hormone receptor, IL-3 acceptor, GM-CSF acceptor, G-CSF acceptor, erythropoietin receptor and IL-6 acceptor).The feature of film mating type acceptor is the Multidomain structure that comprises extracellular aglucon binding domains and cell internal effect device structural domain (it participates in signal transduction usually).In some film mating type acceptor, this extracellular aglucon binding domains and cell internal effect device structural domain are arranged in to be formed complete and the not homopolypeptide of the acceptor of function is arranged.

Generally speaking, the conformational change that can cause acceptor that combines of aglucon and acceptor causes the interaction between effector structure domain and intracellular other molecules, and it causes the variation of cellular metabolism then.Often comprise gently the moving of increase, cell calcium of genetic transcription, phosphorylation, dephosphorylation, ring AMP output, transfer, cell adhesion, the hydrolysis of inositol lipid and the hydrolysis of phosphatide of film fat with acceptor-metabolism incident that aglucon interaction is relevant.

" soluble receptors " is the receptor polypeptides of debond on cytolemma.Soluble receptors is modal aglucon mating type receptor polypeptides, and they lack strides film and tenuigenin structural domain, and is connected with other of cytolemma, for example by sugared phosphoinositide (gpi).Soluble receptors can comprise extra amino-acid residue, and the purifying of polypeptide for example can be provided or the affinity tag in the site that polypeptide combines with substrate is provided, or the constant region for immunoglobulin sequence.Many cell surface receptors have the relative thing of naturally occurring solubility, and they are produced by proteolysis or are obtained by the alternatively spliced mRNA translation.Soluble receptors can be monomer, equal dimer, heterodimer or polymer, and the subunit that the polymer acceptor generally comprises is no more than 9, preferably is no more than 6, is most preferably not exceeding 3.When receptor polypeptides lacks enough polypeptide fragment parts of striding in film and the cell so that film grappling or signal transduction to be provided respectively, then claim these receptor polypeptides not stride polypeptide section in film and the cell basically.The soluble polypeptide of I class and II type cytokines acceptor generally comprises does not have the extracellular of membrane spaning domain and cell intracellular domain cytokine binding domains.For example, representational soluble receptors comprises the soluble receptors of CRF2-4 (having another name called IL-10RB) (Genbank accession number Z17227), shown in SEQ ID NO:44 and 45; The soluble receptors of IL-10RA (Genbank accession number U00672 and NM_001558) is shown in SEQ ID NO:46; The soluble receptors of pDIRSl (having another name called IL-20RB) (Genbank accession number AY358305) is shown in SEQ ID NO:47; And the soluble receptors of IL-22RA (US patent number 5,965,704), shown in SEQ ID NO:3.Describe in known I class or the II type cytokines sequence which type of sequence and comprise that not have the extracellular of membrane spaning domain and cell intracellular domain cytokine binding domains be that those skilled in the art are known.And those skilled in the art can use genetic code easily to determine the polynucleotide of this soluble receptors polypeptide of coding.

Term " secretory signal sequence " is meant the dna sequence dna of encoded peptide (" secretion peptide "), and this peptide is as the component of bigger polypeptide, can instruct this bigger polypeptide by synthetic its Secretory Pathway of cell.Usually this bigger polypeptide is cut to remove the secretion peptide in the process by the Secretory Pathway transhipment.

" isolated polypeptide " is meant substantially by the cellular component polypeptide that polluted of sugar, fat or other albumen impurity relevant with this polypeptide in physical environment for example.Usually, independently the prepared product of polypeptide contains the described polypeptide of highly purified form, i.e. about at least 80% purity, about at least 90% purity, about at least 95% purity, the purity greater than 95%, for example 96%, 97% or 98% or purer are perhaps greater than 99% purity.A kind of mode that shows that certain protein prepared product contains isolated polypeptide be by this protein prepared product is carried out sodium lauryl sulphate (SDS) polyacrylamide gel electrophoresis, again with Xylene Brilliant Cyanine G manifesting to the single band in gel-colored back.But term " isolating " is not got rid of same polypeptide and is existed with other physical form, for example the form of dimer or variable glycosylation or derivatize.

Use term " aminoterminal " and " carboxyl terminal " to represent the position of polypeptide inside here.When context allowed, these terms were to use with reference to the part of specific sequence or polypeptide, to indicate contiguous or relative position.For example, be positioned at certain sequence of carboxyl terminal with respect to reference sequences in polypeptide, its position still might not be at the carboxyl terminal of whole polypeptide near the carboxyl terminal of this reference sequences.

Term " expression " is meant the biosynthesizing of gene product.For example, for structure gene, express relate to structure gene to mRNA transcribe and mRNA to one or the more the more translation of peptide.

Use the RNA of the variable form that term " splice variant " expression obtains from genetic transcription here.Montage variation is that the RNA that transcribes by use is intramolecular, perhaps is the natural generation in alternative splicing site between the RNA molecule of transcribing respectively comparatively singularly, may cause producing going out several mRNA from same genetic transcription.Can the encode polypeptide of aminoacid sequence of splice variant with change.Also use term " splice variant " to represent the splice variant encoded polypeptide of the mRNA that goes out by genetic transcription here.

Just as used herein, term " immunomodulator " comprises cytokine, stem cell factor, lymphotoxin, costimulatory molecules, Hemopoietic factor or the like, and the synthetic analogues of these molecules.

Term " complement/anti-complement is to (complement/anti-complementpair) " be meant form under proper condition non-covalent connection, stablize paired molecule inequality.For example, vitamin H and avidin (perhaps streptavidin) are the right typical members of complement/anti-complement.Other exemplary complement/anti-complement to comprise acceptor-aglucon to, antibody-antigen (perhaps haptens or epi-position) to, have justice-antisense polynucleotides to or the like.When needing right follow-up the dissociating of complement/anti-complement, complement/anti-complement between binding affinity preferably less than 10 ⁹M ^-1

" antiidiotypic antibody " is the variable region structural domain bonded antibody with immunoglobulin (Ig).In the context of the present invention, antiidiotypic antibody combines with the variable region of anti-IL-22RA antibody, but the epi-position of antiidiotypic antibody Simulation with I L-22RA like this.

" antibody fragment " is the part of antibody, for example F (ab ') ₂, F (ab) ₂, Fab ', the part of Fab or the like.No matter structure how, antibody fragment can combine with the same antigen of being discerned by complete antibody.For example, anti-IL-22RA monoclonal antibody fragment can combine with the epi-position of IL-22RA.

Term " antibody fragment " also comprises can be with the specific antigen bonded synthetic or through the polypeptide of genetic engineering procedure, for example the polypeptide of being made up of variable region of light chain, " Fv " fragment, the recombinant single chain peptide molecule (wherein light chain is linked to each other in (" scFv albumen ") by the peptide connector with variable region of heavy chain) be made up of heavy chain and variable region of light chain and the atom of being made up of the amino-acid residue of simulating the hypervariable region.

" chimeric antibody " is to contain from the variable domains of rodents antibody and the recombinant protein of complementary determining region, and the remainder of this antibody molecule is from people's antibody.

" humanized antibodies " is recombinant protein, and wherein the mouse complementary determining region of monoclonal antibody is transferred to people's variable domains from the heavy chain and the variable region of light chain of rat immune globulin.Be used for being structured in the technical scope that those skilled in that art grasp of the humanized antibodies that human body therapy uses (for example those in conjunction with or and the humanized antibodies of human protein) derived from murine antibody.

Just as used herein, " therapeutic medicament " is molecule or the atom of puting together mutually with antibody moiety, produces the conjugate that can be used for treating.The example of therapeutic medicament comprises medicine, toxin, immunomodulator, sequestrant, boron compound, photolytic activity medicament or dyestuff and radio isotope.

" detectable mark " be can with antibody moiety mutually coupling be created in the molecule or the atom of molecule useful in the diagnosis.The example of detectable mark comprises sequestrant, photolytic activity medicament, radio isotope, fluorescence medicament, paramagnetic ion or other markers.

Term " affinity tag " is used in reference to polypeptide fragment here, and it can be attached on second polypeptide, with purifying or the detection that this second polypeptide is provided, perhaps provides the site for this second polypeptide with combining of substrate.In principle, any had antibody or other specific binding agents can be used as affinity tag for the peptide or the albumen that utilize.Affinity tag comprise poly Histidine, albumin A (Nilsson et al., EMBO is (1985) J.4:1075; Nilsson et al., Methods Enzymol.198:3 (1991)), glutathione s-transferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer etal., Proc.Natl.Acad.Sci.USA 82:7952 (1985)), Substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)), streptavidin binding peptide, perhaps other antigenic epitopes or binding domains.Usually referring to Ford et al., Protein Expression and Purification 2:95 (1991). the dna molecular of coded affinity label can obtain from suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).

" exposed antibody " is complete antibody, and it is opposite with antibody fragment, not with the coupling mutually of therapeutic medicament.Exposed antibody comprises polyclone and monoclonal antibody, and some recombinant antibodies is for example chimeric and the humanized antibodies.

As what here use, term " antibody component " comprises whole antibody and antibody fragment.

" immunoconjugates " is the conjugate of antibody component and therapeutic medicament or detectable label.

As used herein, term " antibody fusion protein " refers to the recombinant molecule that comprises antibody component and IL-22RA polypeptide fraction.The example of antibody fusion protein comprises and comprises IL-22RA extracellular domain and the Fc structural domain or the protein of antigen binding domain.

" target polypeptides " or " target peptide " is one section aminoacid sequence, and it comprises at least one epi-position, at tumour cell or carry on the antigenic cell of infectious factor and express for example on the target cell.The peptide epitopes that the T cell recognition is presented to target polypeptides or target peptide by the major histocompatibility complex molecule, cracking target cell or raise the position that other immunocytes arrive target cells usually, thus kill target cell.

" antigenic polypeptide " is the peptide that can form the MHC-peptide complex in conjunction with the major histocompatibility complex molecule, and this MHC-peptide complex is by the T cell recognition, thereby at mixture inducing cytotoxic lymphocyte responses during to the T presented by cells.Like this, antigenic peptide can combine and induce cytotoxic T cell and reply with suitable major histocompatibility complex molecule, for example lysis or be directed to the specific cell factor that can combine or express this antigenic target cell with this antigen and discharge.Antigenic peptide can be combined under the environment of I class or II class major histocompatibility complex molecule, perhaps on the antigen presenting cell or on target cell.

In eukaryotic cell, rna plymerase ii catalytic structure gene transcription produces mRNA.Can the designing nucleic acid molecule, make it contain the rna plymerase ii template, wherein this rna transcription product has and specific mRNA sequence complementary sequence.This rna transcription product is known as " sense-rna ", and the nucleic acid molecule of encoding antisense RNA is known as " sense-rna gene ".Antisense rna molecule can be suppressed the translation of mRNA in conjunction with the mRNA molecule.

" antisense oligonucleotide that IL-22RA is special " or " IL-22RA antisense oligonucleotide " is the oligonucleotide with certain sequence, this sequence (a) can form stable triplex with the part of IL-22RA gene, and perhaps (b) can form with the mRNA transcription product of IL-22RA gene and stablize two serobilas.

" ribozyme " is the nucleic acid molecule that contains catalytic center.This term comprises RNA enzyme, self-splicing RNA, oneself cutting RNA and the nucleic acid molecule that carries out these catalyzed reactions.The nucleic acid molecule of encoding ribozyme is called as " ribozyme gene ".

" external source homing sequence " is such nucleic acid molecule, and it instructs endogenous ribozyme RNaseP to arrive mRNA in certain cell, and this mRNA is cut by RNaseP.The nucleic acid molecule of encoding exogenous homing sequence is known as " external source homing sequence gene ".

Term " variant IL-22RA gene " is meant that such nucleic acid molecule, its coding have the polypeptide of the aminoacid sequence of SEQID NO:3 modified forms.The conserved amino acid alternate synthetic gene that this variant comprises the polymorphism of naturally occurring IL-22RA gene and contains aminoacid sequence among the SEQ ID NO:3.The variant form of other IL-22RA genes is to contain the insertion of nucleotide sequence described herein or the nucleic acid molecule of disappearance.Can whether under rigorous condition, identify variant IL-22RA gene by for example determining gene with making nucleic acid molecular hybridization with nucleotide sequence shown in the SEQ ID NO:1 or its complementary sequence.

Perhaps, can relatively identify variant IL-22RA gene by sequence.Compare when seeking maximum consistence when two aminoacid sequences, these two amino acid whose amino-acid residues are identical, and then described two aminoacid sequences have " 100% consensus amino acid sequence ".Similarly, compare when seeking maximum consistence when two nucleotide sequences, the nucleotide residue of these two nucleotide sequences is identical, and then described two nucleotide sequences have " 100% nucleotide sequence consistence ".Can use the standard software program, for example those (Madison, the program that is included in the LASERGENE information biology pack processing of Wisconsin) producing is carried out sequence relatively by DNASTAR.Other by determine the best method that compares two Nucleotide or aminoacid sequence for those skilled in the art be well-known (referring to, Peruskiand Peruski for example, The Internet and the New Biology:Tools for Genomicand Molecular Research (ASM Press, Inc.1997), Wu et al. (eds.), " Information Superhighway and Computer Databases of NucleicAcids and Proteins; " in Methods in Gene Biotechnology, pages123-151 (CRC Press, Inc.1997), and Bishop (ed.), Guide toHuman Genome Computing, 2nd Edition (Academic Press, Inc.1998)). the following concrete grammar of describing definite sequence identity.

No matter the special method that is used to identify variant IL-22RA or variant IL-22RA polypeptide how, can carry out functional evaluation to variant gene or by the ability of variant gene encoded polypeptides and anti-IL-22RA antibody specific combination.Can also use biology described herein or biochemistry detection that variant IL-22RA gene or variant IL-22RA polypeptide are carried out functional evaluation in conjunction with the ability of its aglucon IL-22.

Term " allele variant " is used for representing occupying any in two of gene of same chromogene seat or the how variable form here.Allelic variation may cause the phenotypic polymorphism in the colony by the natural generation of sudden change.Transgenation can be the polypeptide of reticent (encoded polypeptides do not have change) or the aminoacid sequence with change of may encoding.Term " allele variant " also is used for representing the allelic variant encoded protein matter by gene here.

Polypeptide or protein that term " orthogenesis homologous protein (ortholog) " expression obtains from species, this polypeptide or protein are from the polypeptide of different plant species or proteinic function counterpart.Sequence difference between the orthologous gene is the result that species form.

" parallelism homologous protein (paralog) " but be the protein of the completely different structurally associated that generates by organism.The parallelism homologous protein is considered to produce by gene redundancy.For example, α-Zhu Danbai, beta-globin and myosin parallelism homologous protein each other.

The present invention includes the functional fragment of IL-22RA gene.Under context of the present invention, " functional fragment " of IL-22RA gene be meant coding IL-22RA polypeptide a part (it be structural domain as herein described or at least with anti-IL-22RA antibody specific combination) nucleic acid molecule.

Because the inaccuracy of standard method of analysis, polymeric molecular weight and length are considered to numerical value roughly.When such numerical value is represented as " approximately " X or " roughly " X, described X refers to be considered to be accurate to ± and 10%.

3.IL-22RA the generation of polynucleotide or gene

The nucleic acid molecule of coding human il-22 RA gene can obtain by using polynucleotide probes screening people cDNA or genomic library according to SEQ ID NO:1.These all are standard and proven technique, can finish by the clone's test kit that uses supplier to provide.Referring to for example Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3rd Edition, John Wiley ﹠amp; Sons 1995; Wu et al., Methods in GeneBiotechnology, CRC Press, Inc.1997; Aviv and Leder, Proc.Nat ' l Acad.Sci.USA 69:1408 (1972); Huynh et al., " Constructingand Screening cDNA Libraries in lgt10 and lgt11, " DNA Cloning:A Practical Approach Vol.I, Glover (ed.), 49 pages (IRL Press, 1985); Wu (1997) 47-52 page or leaf.

Can also pass through polymerase chain reaction (PCR), use Oligonucleolide primers, the nucleic acid molecule of the human il-22 RA gene that obtains encoding based on the nucleotide sequence of IL-22RA gene or cDNA.The general method that uses PCR screening library is by providing below with reference to document: Yu et al. for example, " Use of the Polymerase Chain Reaction to Screen PhageLibraries; " Methods in Molecular Biology, Vol.15:PCRProtocols:Current Methods and Applications, White (ed.), Humana Press, Inc., 1993. and, the technology of using PCR to separate genes involved is for example being described in the following document to some extent: Preston, " Use of DegenerateOligonucleotide Primers and the Polymerase Chain Reaction toClone Gene Family Members; " Methods in Molecular Biology, Vol.15:PCR Protocols:Current Methods and Applications, White (ed.), Humana Press, Inc.1993.Perhaps, can use long common initiation oligonucleotide and nucleotide sequence synthetic nucleic acid molecule described herein, obtain IL-22RA gene (referring to for example Ausubel (1995)).Use the mature technology of polymerase chain reaction can composition length to be at least dna molecular (the Adang et al. of 2kb, Plant Molec.Biol.21:1131 (1993), Bambot et al., PCR Methods and Applications2:266 (1993), Dillon et al., " Use of the Polymerase ChainReaction for the Rapid Construction of Synthetic Genes; " Methods in Molecular Biology, Vol.15:PCR Protocols:CurrentMethods and Applications, White (ed.), the 263-268 page or leaf, (Humana Press, Inc.1993), and Holowachuk et al., PCR Methods Appl.4:299 (1995)).Summarize for the polynucleotide synthetic, referring to for example Glick and Pasternak, Molecular Biotechnology, Principles and Applications ofRecombinant DNA (ASM Press 1994), Itakura et al., Annu.Rev.Biochem.53:323 (1984) and Climie et al., Proc.Nat ' l Acad.Sci.USA 87:633 (1990).

4.IL22-RA the preparation of genetic mutation

The invention provides multiple nucleic acid molecule, comprise DNA and RNA molecule, their IL-22RA polypeptide disclosed herein of encoding.Those skilled in the art can recognize at an easy rate, consider the degeneracy of genetic code, in these polynucleotide molecules considerable sequence variation can be arranged.And the present invention also provides isolating soluble and monomeric, all dimer, heterodimer and polymer receptor polypeptides, and the receptor polypeptides that they comprise at least one and SEQ ID NO:3 is homologous IL-22RA receptor subunit basically.Like this, the present invention relates to comprise the IL-22RA polypeptide encoding nucleic acid molecule and the RNA Equivalent thereof of degenerate core thuja acid shown in the SEQ ID NO:1.

Table 1 is with single-letter coded representation degeneracy nucleotide position." fractionation " is the Nucleotide of alphanumeric codes representative.What " complement " represented is the code of complementary nucleotide.For example code Y represents C or T, and its complement R represents A or G, A and T complementation, and G and C complementation.

Table 1

Nucleotide	Split	Complementary nucleotide	Split
				A	A	T	T
C	C	G	G
				G	G	C	C
T	T	A	A
				R	A|G	Y	C|T
Y	C|T	R	A|G
				M	A|C	K	G|T
K	G|T	M	A|C
				S	C|G	S	C|G
W	A|T	W	A|T
				H	A|C|T	D	A|G|T
B	C|G|T	V	A|C|G
				V	A|C|G	B	C|G|T
D	A|G|T	H	A|C|T
				N	A|C|G|T	N	A|C|G|T

In table 2, provided degenerate codon, comprised for given amino acid whose all possible codon.

Table 2

Amino acid	The single-letter code	Codon	Degenerate codon
				Cys	C	TGC TGT	TGY
Ser	S	AGC AGT TCA TCC TCG TCT	WSN
				Thr	T	ACA ACC ACG ACT	ACN
Pro	P	CCA CCC CCG CCT	CCN
				Ala	A	GCA GCC GCG GCT	GCN
Gly	G	GGA GGC GGG GGT	GGN
				Asn	N	AAC AAT	AAY
Asp	D	GAC GAT	GAY
				Glu	E	GAA GAG	GAR
Gln	Q	CAA CAG	CAR
				His	H	CAC CAT	CAY
Arg	R	AGA AGG CGA CGC CGG CGT	MGN
				Lys	K	AAA AAG	AAR
Met	M	ATG	ATG
				Ile	I	ATA ATC ATT	ATH
Leu	L	CTA CTC CTG CTT TTA TTG	YTN
				Val	V	GTA GTC GTG GTT	GTN
Phe	F	TTC TTT	TTY
				Tyr	Y	TAC TAT	TAY
Trp	W	TGG	TGG
				Ter	·	TAA TAG TGA	TRR
Asn|Asp	B		RAY
				Glu|Gln	Z		SAR
Any	X		NNN

Those skilled in the art should be appreciated that determine coded amino acid might codon the representative degenerated code period of the day from 11 p.m. to 1 a.m introduced some uncertainties.The degenerate codon of Serine (WSN) arginine (AGR) of can encoding in some cases for example, and arginic degenerate codon (MGN) in some cases can encoding serine (AGY).Between coding phenylalanine and leucic codon, also there is similarly relation.The polynucleotide possibility encoding amino acid sequence variant that this degenerate sequence is contained, but those skilled in the art can easily identify this variant sequence with reference to the aminoacid sequence shown in the SEQ ID NO:3.The variant sequence can easily be carried out functional detection according to the description here.

Different species can have " codon use preference ".Usually, referring to Granthamet al., Nucl.Acids Res.8:1893 (1980), Haas et al.Curr.Biol.6:315 (1996), Wain-Hobson et al., Gene 13:355 (1981), Grosjeanand Fiers, Gene 18:199 (1982), Holm, Nuc.Acids Res.14:3075 (1986), Ikemura, J.Mol.Biol.158:573 (1982), Sharp and Matassi, Curr.Opin.Genet.Dev.4:851 (1994), Kane, Curr.Opin.Biotechnol.6:494 (1995), and Makrides, Microbiol.Rev.60:512 (1996).Just as used herein, term " codon use preference " or " preference codon " are terms of the prior art, refer in the cell of certain species the protein translation codon of the most frequent use, will produce preferences to each amino acid whose one or several representative codon in may codon (referring to table 2) of encoding like this.For example Threonine (Thr) can be by ACA, ACC, and ACG or ACT coding, but ACC is a codon the most commonly used in mammalian cell; In other species, for example in insect cell, yeast, virus or the bacterium, may be to different Threonine codons preference to some extent.The preference codon of specific species can be incorporated into by several different methods known in the art in the polynucleotide of the present invention.Introducing preference codon sequence in recombinant DNA can for example make the protein translation in particular cell types or the species more efficiently strengthen proteinic generation.Therefore, degenerated code subsequence disclosed herein can be used as and optimize the template of expressing in that polypeptide is used always and various kinds of cell type disclosed herein and the species in this area.The sequence that contains the preference codon can detect and be used for to express in multiple species to be optimized, and can be according to content disclosed herein to its functional detection.

Can use multiple diverse ways to separate the cDNA of coding IL-22RA,, perhaps use based on one group of disclosed sequence or organize the degeneracy probe more for example by using people cDNA complete or part as probe.Can also utilize primer according to representative human il-22 RA sequences Design disclosed herein to carry out the polymerase chain reaction and obtain cDNA.In addition, can use the cDNA library to transform or transfection host cell, and can use the expression of the antibody test target cDNA of anti-IL-22RA polypeptide.

Those skilled in that art will appreciate that disclosed sequence has been represented the monoallelic of human il-22 RA among the SEQ ID NO:1, and estimate to exist allelic variation and alternative splicing.The allelic variant of this sequence can be cloned by detecting from the cDNA or the genomic library of Different Individual according to the step of standard.The allelic variant of nucleotide sequence disclosed herein comprises the variant that variant that those contain silent mutation and those sudden changes cause aminoacid sequence to change, all in category of the present invention; As the protein of the allelic variant of aminoacid sequence disclosed herein also in category of the present invention.The cDNA molecule that mRNA by variable mode montage produced, kept IL-22RA polypeptide character is also contained in the category of the present invention; Be also included within the category of the present invention by this cDNA and mRNA encoded polypeptides.Can by detecting cDNA or genomic library, clone the allelic variant and the splice variant of these sequences by standard step known in the art from Different Individual or tissue.

Use method discussed above, those skilled in the art can prepare multiple polypeptides, described polypeptide comprises the allelic variant with amino acid whose solubility IL-22RA receptor subunit shown in SEQ ID NO:1 basic homologous solubility IL-22RA receptor subunit or the coding SEQ ID NO:3 or aforementioned polypeptides, and they have kept the aglucon of wild-type IL-22RA acceptor in conjunction with character.This peptide species can also comprise such as here disclosed other polypeptide fragments of generality.

In certain embodiments of the invention, described isolated nucleic acid molecule can be hybridized with the nucleic acid molecule that comprises nucleotide sequence disclosed herein under rigorous condition.For example, this nucleic acid molecule can be hybridized with the nucleic acid molecule that comprises nucleotide sequence shown in the SEQ ID NO:1 under rigorous condition; Perhaps hybridize with the complementary nucleotide sequence that comprises nucleotide sequence shown in the SEQ ID NO:1 or its segmental nucleic acid molecule.

Generally speaking, rigorous condition is chosen to the heat of solution temperature (T than this distinguished sequence under ionic strength of determining and pH _m) approximately low 5 ℃.The temperature (under ionic strength of determining and pH) that Tm is 50% target sequence when hybridization takes place the probe of coupling fully.After the hybridization, can be at rinsing nucleic acid molecule under the rigorous condition or under the highly rigorous condition to remove the not nucleic acid molecule of hybridization.Referring to for example Sambrook et al., Molecular Cloning:A LaboratoryManual, Second Edition (Cold Spring Harbor Press 1989); Ausubelet al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc.1987); Berger and Kimmel (eds.), Guideto Molecular Cloning Techniques, (Academic Press, Inc.1987); And Wetmur, Crit.Rev.Biochem.Mol.Biol.26:227 (1990)).Sequence analysis software is OLIGO 6.0 (LSR for example; Long Lake, MN) and PrimerPremier 4.0 (Premier Biosoft International; Palo Alto, CA), also having the website on the Internet all is the instruments that can obtain, and can be used for analyzing given sequence, and the criterion calculation Tm value of determining according to the user.At the conditionally complete of specific polynucleotide heterozygote adjustment hybridization of using and rinsing in those skilled in that art's limit of power.

The present invention also provides independently IL-22RA polypeptide, and they have similar in fact sequence identity to polypeptide shown in the SEQ ID NO:3, perhaps its orthogenesis homologous protein.Here use term " sequence identity of broadly similar " to represent have at least 70%, at least 80%, at least 90%, at least 95%, for example 96%, 97%, 98% or greater than the polypeptide of 95% sequence identity, perhaps its orthogenesis homologous protein with the sequence shown in the SEQ ID NO:3.For example can use the variant or the orthogenesis homologous protein of IL-22RA acceptor to excite immunne response, produce cross-reacting antibody at human il-22 RA.This antibody can be the peopleization, and modifies according to the description here, and therapeutic ground is used for psoriasis, arthritic psoriasis, IBD, colitis, endotoxemia and other therapeutic application as herein described.

The invention still further relates to the IL-22RA variant nucleic acid molecule that can use following two standards to differentiate out: determine the similarity of aminoacid sequence shown in encoded polypeptide and the SEQ ID NO:3, and hybridization detects.This IL-22RA variant comprises the nucleic acid molecule with following character: (1) keeps the hybridization state with the nucleic acid molecule with nucleotide sequence shown in the SEQ ID NO:1 (perhaps its complement) under rigorous rinsing condition, rinsing preciseness wherein is equivalent to 0.5x-2xSSC and adds 0.1%SDS, at 55-65 ℃, and aminoacid sequence shown in (2) encoded polypeptides and the SEQ ID NO:3 have at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% the sequence identity of (for example 96%, 97%, 98% or 99%).Perhaps, the IL-22RA variant can be characterized as being the nucleic acid molecule with following character: (1) keeps hybridization with the nucleic acid molecule with nucleotide sequence shown in the SEQ ID NO:1 (perhaps its complement) under highly rigorous rinsing condition, wherein said rinsing preciseness is equivalent to 0.1x-0.2x SSC and adds 0.1%SDS, at 50-65 ℃, and aminoacid sequence has at least 70% shown in (2) encoded polypeptides and the SEQ ID NO:3, at least 80%, at least 90%, at least 95% or greater than 95% (for example 96%, 97%, 98% or 99% or higher) sequence identity.

Determine the per-cent sequence identity according to conventional methods.Referring to for example, Altschul et al., Bull.Math.Bio.48:603 (1986), and Henikoff and Henikoff, Proc.Natl.Acad.Sci.USA 89:10915 (1992).In brief, compare two aminoacid sequences, wherein use the breach opening to penalize 10 minutes, breach to extend to penalize 1 minute, utilize " blosum62 " rating matrix (with the single-letter coded representation amino acid of standard) of Henikoff shown in the table 3 and Henikoff (reference is the same) design to optimize the comparison mark.Calculate the per-cent consistence according to following formula then: ([sum of identical match]/[number of the breach of the length of longer sequence+in longer sequence, introduce]) (100) in order to compare two sequences.

Table 3

A R N D C Q E G H I L K M F P S T W Y V

A 4

R -1 5

N -2 0 6

D -2 -2 1 6

C 0 -3 -3 -3 9

Q -1 1 0 0 -3 5

E -1 0 0 2 -4 2 5

G 0 -2 0 -1 -3 -2 -2 6

H -2 0 1 -1 -3 0 0 -2 8

I -1 -3 -3 -3 -1 -3 -3 -4 -3 4

L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4

K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5

M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5

F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6

P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7

S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4

T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5

W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11

Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7

V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

Those skilled in the art are to be understood that many ready-made algorithms can be used for two aminoacid sequences are compared." FASTA " similarity searching algorithm of Pearson and Lipman is the protein comparison method that suits of checking the sequence identity level of being shared between the aminoacid sequence of aminoacid sequence disclosed herein and the IL-22RA variant of inferring.Fasta algorithm is at Pearson and Lipman, and Proc.Nat ' l Acad.Sci.USA 85:2444 (1988), and Pearson describe among the Meth.Enzymol.183:63 (1990) to some extent.In brief, FASTA does not consider that conserved amino acid substitutes, inserts or disappearance, but at first find out inquiry sequence (for example SEQ ID NO:2 or SEQ ID NO:3) and detect zone total between the sequence, thereby distinguishing sequence similarity with the highest consistence density (if ktup variable=1) or the highest consistence pairing density (if ktup=2).By using the relatively similarity of all matching amino acids of amino acid replacement matrix, marked again in 10 zones with the highest consistence density then, the end in zone is carried out " pruning " make it include only those the contributive residue of highest score.If the score that several zones are arranged is greater than " cutoff " value (the predetermined formula by foundation sequence length and ktup numerical value calculates), then check the initiation region of being pruned, determine whether and these zones can be added to come in the roughly comparison that has breach with formation.At last, use Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, the J.Mol.Biol.48:444 (1970) that revises; Sellers, SIAM J.Appl.Math.26:787 (1974), this algorithm allows the existence of aminoacid insertion and disappearance), compare in two aminoacid sequences the zone of the highest scoring.The illustrative parameter that FASTA analyzes is: ktup=1, and the breach opening was penalized 10 fens, and breach extends penalized 1 fen, substituted matrix=blosum62.By revising rating matrix file (" SMATRIX "), these parameters are incorporated in the FASTA program, this explains in the appendix 2 of Meth.Enzymol.183:63 (1990) to some extent at Pearson.

Can also use the sequence identity of FASTA definite kernel acid molecule, disclosed ratio above using.For the comparison of nucleotide sequence, the ktup value can be preferably 3 to 6 between 1 to 6, most preferably be 3, and the setting of other parameters as mentioned above.

The present invention includes coding and compare the nucleic acid molecule of polypeptide with aminoacid sequence disclosed herein with conserved amino acid change.For example, can obtain variant, they contain and have one or more amino acid replacements to sequence shown in the SEQ ID NO:3, wherein alkyl amino acid is replaced with the alkyl amino acid in the IL-22RA aminoacid sequence, aromatic amino acid is replaced with the aromatic amino acid in the IL-22RA aminoacid sequence, sulfur-containing amino acid is replaced with the sulfur-containing amino acid in the IL-22RA aminoacid sequence, hydroxyl amino acid is replaced with the hydroxyl amino acid in the IL-22RA aminoacid sequence, acidic amino acid is replaced with the acidic amino acid in the IL-22RA aminoacid sequence, basic aminoacids is replaced with the basic aminoacids in the IL-22RA aminoacid sequence, and perhaps two alkaline mono carboxylic amino acid are replaced with two alkaline mono carboxylic amino acid in the IL-22RA aminoacid sequence.In common amino acid, for example, the example of " conserved amino acid substitutes " is the amino acid replacement in following each group: (1) glycine, L-Ala, Xie Ansuan, leucine and Isoleucine; (2) phenylalanine, tyrosine and tryptophane; (3) Serine and Threonine; (4) aspartic acid and L-glutamic acid; (5) glutamine and l-asparagine; And (6) Methionin, arginine and Histidine.The BLOSUM62 form is the amino acid replacement matrix, it is from the segmental local multi comparison of about 2000 protein sequences, represented the high conservative zone (Henikoff and Henikoff, Proc.Nat ' l Acad.Sci.USA89:10915 (1992)) of 500 groups of above associated protein.Therefore, the BLOSUM62 alternative frequency conserved amino acid that can be used for determining to be incorporated in the aminoacid sequence of the present invention substitutes.Although might be only according to chemical property design amino acid replacement (as discussed above), statement " conserved amino acid substitutes " preferably refers to BLOSUM62 numerical value greater than-1 represented substituting.For example, if the BLOSUM62 numerical value of an amino acid replacement is 0,1,2 or 3, then should substitutes and guard.According to this system, preferred conserved amino acid alternate feature is that BLOSUM62 numerical value is 1 (for example 1,2 or 3) at least, and more preferred conserved amino acid alternate feature to be BLOSUM62 numerical value be at least 2 (for example 2 or 3).The feature of the particular variant of IL-22RA is, (for example SEQ ID NO:3) compares with amino acid sequence corresponding, have at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% the sequence identity of (for example 96%, 97%, 98% or 99% or higher), wherein the variation of aminoacid sequence causes owing to one or more conserved amino acid substitute.

Can in the IL-22RA gene, introduce conserved amino acid and change by for example with the Nucleotide among the Nucleotide replacement SEQ ID NO:1.Mutagenesis, the joint scanning mutagenesis that can instruct by oligonucleotide, use mutagenesis that the polymerase chain reaction carries out or the like (referring to Ausubel (1995); And McPherson (ed.), Directed Mutagenesis:A Practical Approach (IRL Press 1991)) obtains this " conserved amino acid " variant.Can identify variant IL-22RA polypeptide by the ability of the anti-IL-22RA antibody of its specific combination.

Protein of the present invention also can comprise the amino-acid residue that non-natural exists.The alpha-non-natural amino acid residue comprises, but be not limited to trans-3-methylproline, 2,4-methane proline(Pro), cis-4-Hydroxyproline, trans-4-Hydroxyproline, sarcosine, allothreonine, methylthreonine, the methylol halfcystine, the methylol homocysteine, the nitro glutamine, high glutamine, pipecoline acid, Thiazolidinecarboxylacid acid, dehydroproline, 3-and 4-methylproline, 3,3-dimethyl proline(Pro), Terleu, norvaline, 2-pyridine L-Ala, 3-pyridine L-Ala, 4-pyridine L-Ala and 4-fluorophenylalanine.There is certain methods the amino-acid residue that non-natural exists can be incorporated in the protein known in the art.For example, can use vitro system, wherein nonsense mutation is suppressed by the sub-tRNA of the inhibition of chemical aminoacylation.The method of synthesizing amino acid and aminoacylation tRNA is known in the art.Usually use the cell free system that comprises intestinal bacteria S30 extract and commercial enzyme and other reagent that the plasmid that contains nonsense mutation is transcribed and translated.Use chromatography purification protein.Referring to for example Robertson et al., J.Am.Chem.Soc.113:2722 (1991), Ellman et al., MethodsEnzymol.202:301 (1991), Chung et al., Science 259:806 (1993), and Chung et al., Proc.Nat ' l Acad.Sci.USA 90:10145 (1993).

In the second approach, the mRNA of sudden change and the sub-tRNA microinjection of inhibition (Turcatti et al., J.Biol.Chem.271:19991 (1996)) in the Africa xenopus ovocyte of chemical aminoacylation are translated.In the third method, treat substituted natural amino acid (for example phenylalanine) and exist required non-natural to exist under the condition of amino acid (for example 2-pyridine L-Ala, 3-pyridine L-Ala, 4-pyridine L-Ala or 4-fluorophenylalanine) to cultivate Bacillus coli cells in shortage.The amino acid that this non-natural exists is incorporated in the protein, has substituted its natural counterpart.Referring to Koide et al., Biochem.33:7470 (1994).Can change naturally occurring amino-acid residue into amino-acid residue that non-natural exists by external chemically modified.Chemically modified can be combined with site-directed mutagenesis, to further expand alternate scope (Wynn and Richards, Protein Sci.2:395 (1993)).

A limited number of non-conserved amino acid, can't help amino acid that genetic codon amino acids coding, non-natural exist and alpha-non-natural amino acid and all can replace amino-acid residue among the IL-22RA.

Can be according to step known in the art, site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells for example, Science 244:1081 (1989), Bass et al., Proc.Nat ' l Acad Sci.USA 88:4498 (1991), Coombs and Corey, " Site-Directed Mutagenesis and Protein Engineering; " inProteins:Ahalysis and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc.1998)) identifies the indispensable amino acid in the polypeptide of the present invention.In a kind of technology in back, each the residue place in this molecule all introduces single alanine mutation, detects the biological activity of the mutant molecule that produces, to identify vital amino-acid residue for the activity of this molecule.Can also be referring to Hilton et al., J.Biol.Chem.271:4699 (1996).

Further determine the ligand-binding domain territory of IL-22RA although can use sequential analysis, (for example IL-22RA and aglucon IL-22's combines in conjunction with active at IL-22RA, perhaps with the combining of anti-IL-22RA antibody) in the amino acid that works can also be according to physical analysis such as methods such as nucleus magnetic resonance, crystallography, electron diffraction or photoaffinity labeling to structure, and the contact site amino acid of inferring suddenlyd change to determine.Referring to for example, de Vos et al., Science 255:306 (1992), Smith et al., J.Mol.Biol.224:899 (1992), and Wlodaver et al., FEBS Lett.309:59 (1992).

Can carry out a plurality of amino acid replacements, and use known mutagenesis and screening method to test, for example by Reidhaar-Olson and Sauer disclosed method (Science 241:53 (1988)) or by Bowie and Sauer disclosed method (Proc.Nat ' l Acad.Sci.USA 86:2152 (1989)).In brief, these authors disclose two or more position randomizations that make simultaneously in the polypeptide, select to have the polypeptide of function, and the polypeptide to mutagenesis checks order then, with the method for the scope of the replacement determining to allow on each position to exist.Other operable methods comprise phage display (e.g., Lowman et al., Biochem.30:10832 (1991), Ladner et al., U.S.Patent No.5,223,409, Huse, international publication numbering WO 92/06204) and zone location mutagenesis (Derbyshire etal., Genne 46:145 (1986), and Ner et al., DNA 7:127, (1988)).The cloning by expression that can be used in addition, the IL-22RA aglucon through the IL-22RA of vitamin H or FITC mark.

Disclosed IL-22RA Nucleotide and peptide sequence variant can also pass through Stemmer, Nature 370:389 (1994), Stemmer, Proc.Nat l Acad.Sci.USA91:10747 (1994), and international publication numbering WO 97/20078 disclosed DNA shuffling technology produces.In brief, make parent DNA random fragmentation, use PCR to ressemble then, produce the point mutation of introducing at random, carry out external homologous recombination thus, produce modification D NA molecule.By using one group of parent's dna molecular, for example allelic variant or from the dna molecular of different plant species is introduced extra variability in this process, can make amendment to this technology.By selecting and screen required activity, carry out extra sudden change repeatedly and detection subsequently, can select desirable sudden change and eliminate deleterious change simultaneously, thereby provide rapidly " evolution " for sequence.

Mutafacient system disclosed herein can combine with the screening method of high-throughput, automatization, to detect the activity of the mutagenesis polypeptide of cloning in the host cell.Can from host cell, reclaim the encoding human active polypeptide or with the mutagenized dna molecule of the polypeptide of anti-IL-22RA antibodies, and use modern instrument to check order rapidly.These methods can promptly be determined the importance of single amino acids residue in the target polypeptides, and can be applied on the polypeptide of unknown structure.

The present invention has also comprised " functional fragment " of IL-22RA polypeptide and the nucleic acid molecule of these functional fragments of encoding.Can carry out conventional deletion analysis to nucleic acid molecule, with the functional fragment of the nucleic acid molecule that obtains coding IL-22RA polypeptide.As example, the dna molecular with nucleotide sequence shown in the SEQ ID NO:1 can use the Bal31 nuclease to digest, and obtains a series of nested deletion.According to correct reading frame these fragments are inserted in the expression vector then, separate expressed proteins and detect its ability in conjunction with anti-IL-22RA antibody.With respect to exonuclease digestion, another selection is to use oligonucleotide mediated mutagenesis, lacks or terminator codon to introduce, thereby determines required segmental preparation.Perhaps, can use the special fragment of the synthetic IL-22RA gene in polymerase chain reaction.

The terminal brachymemma research of one or two of relevant Interferon, rabbit has been set forth this general method as example, and this is at Horisberger and Di Marco, and Pgarmac.Ther.66:507 (1995) has general introduction.And, for example describing to some extent in the following document for the standard method that protein function is analyzed: Treuter et al., Molec.Gen.Genet.240:113 (1993), Content et al., " Expression and preliminary deletionanalysis of the 42 kDa 2-5A synthetase induced by humaninterferon; " Biological Interferon Systems, Proceedings ofISIR-TNO Meeting on Interferon Systems, Cantell (ed.), 65-72 page or leaf (Nijhoff 1987), Herschman, " The EGF Receptor; " Control ofAnimal Cell Proliferation, Vol.1, Boynton et al., (eds.) 169-199 page or leaf (Academic Press 1985), Coumailleau et al., J.Biol.Chem.270:29270 (1995); Fukunaga et al., J.Biol.Chem.270:25291 (1995); Yamaguchi et al., Biochem.Pharmacol.50:1295 (1995) and Meisel et al., Plant Molec.Biol.30:1 (1996).

Special sequence disclosed herein is analyzed, one group of exemplary functional fragment is provided, as shown in table 4.Encode the Nucleotide of extra human il-22 RA functional variant structural domain described herein not shown in the table 4, and they can be determined with reference to SEQ ID NO:1.This function fragment comprises the following nucleotide sequences among the SEQ ID NO:1 for example: the corresponding aminoacid sequence of 85-381, the 206-717 of SEQ ID NO:1 and 85-717 Nucleotide and their codings, as sequence 2 and sequence 3 respectively shown in.

Table 4

The IL-22RA feature	Amino-acid residue (SEQ ID NO:2)	Nucleotide (SEQ ID NO:1)
			First Ig structural domain	18-116	85-381
Second Ig structural domain	125-228	206-717
			Two Ig structural domains	18-228	85-717

The invention still further relates to the function fragment of IL-22RA gene, described fragment is compared with aminoacid sequence disclosed herein has amino acid whose change.The IL-22RA genetic mutation can be according to top discussion, by measuring the consistency level of it and disclosed Nucleotide and aminoacid sequence, identifies according to structure.According to structure differentiate another method of variant gene be determine the potential variant IL-22RA gene of coding nucleic acid molecule whether can with for example comprise the nucleic acid molecule of nucleotide sequence shown in the SEQ ID NO:1 and hybridize.

The present invention also comprises the nucleic acid molecule of the part of carrying epi-position of function fragment, antigenic epitopes, the part of carrying epi-position of IL-22RA polypeptide and these function fragments of encoding of using the IL-22RA polypeptide, antigenic epitopes, IL-22RA polypeptide.These fragments can be used for producing polypeptide so that can in conjunction with, blocking-up, inhibition, reduction, antagonism or in and IL-22 activity or IL-20 and IL-22 the two active generation antibody and binding partners." functional " IL-22RA polypeptide or as its fragment determined here, be characterized in blocking, suppress, reduction, antagonism or in and short scorching, propagation of IL-20 or IL-22 or differentiation activity, and can induce or suppress the specialized cell function, perhaps can with anti-IL-22RA antibody, cell, IL-20 or IL-22 specific combination.As described above here, the feature of IL-22RA is to have II type cytokines receptor structure and structural domain described herein.Therefore, the invention still further relates to use comprise below (a) and fusion rotein (b): (a) comprise the peptide molecule of or structural domain how described above; And the function fragment that (b) comprises or more these structural domains.Another polypeptide portion of fusion rotein can be provided by another II type cytokines acceptor, IL-10R for example, IL-13R, IL-20RA, IL-20RB, IL-10RB (CRF2-4), IL-22RA2 is perhaps by helping the non-natural and/or incoherent secreting signal peptide of fusion rotein excretory to provide.

The present invention also provides polypeptide fragment or peptide, and they comprise the part of carrying epi-position of IL-22RA polypeptide described herein.Such fragment or peptide can comprise " immunogenicity epi-position ", and it is a proteic part, and when this whole albumen is used as immunogen, but this part induce antibody is replied.The peptide that carries the immunogenicity epi-position can use the method for standard (referring to for example Geysen et al., Proc.Nat ' l Acad.Sci.USA 81:3998 (1983)) to identify.

On the contrary, polypeptide fragment or peptide can comprise " antigenic epitopes ", and it is an antibody zone of specific combination with it in the protein molecule.Some epi-position is made up of one section linearity or continuous amino acid, and the antigenicity of this epi-position is not destroyed by denaturing agent.In this area, known and to have used the relatively short synthetic peptide of simulated albumin matter epi-position to stimulate generation at this proteinic antibody (referring to for example Sutcliffe et al., Science 219:660 (1983)).Therefore, peptide, antigenic peptide, epi-position and the polypeptide that carries antigenic epitopes of the present invention is useful for producing the antibody that combines with polypeptide described herein, also can be used for identifying and screening anti-IL-22RA monoclonal antibody, described antibody is neutrality, may in conjunction with, blocking-up, inhibition, reduction, antagonism or in and the activity of IL-22 and IL-20 (distinguish or together).This neutralizing monoclonal antibody of the present invention can combine with the IL-22RA antigenic epitopes.Can use the Hopp/Woods hydropathic profile determine in SEQ ID NO:3, to have the zone of the highest antigenicity potentiality (Hopp et al., Proc.Natl.Acad.Sci.78: 3824-3828,1981; Hopp, J.Immun.Meth.88: 1-18,1986 and Triquier et al., Protein Engineering 11: 153-169,1998).This figure based on be the six residue windows that slide.Ignore G, the S and T residue and the H that exposes, Y and the W residue that are covered.Those skilled in the art can determine these zones in IL-22RA.And, according to the Jameson-Wolf curve, use for example DNASTAR Protean program (DNASTAR, Inc.Madison, WI) the IL-22RA antigenic epitopes among Yu Ce the SEQ ID NO:3 can be used as preferred antigenic epitopes, and they can be determined by those skilled in the art.This antigenic epitopes comprises: 1 (Pro) is to 6 (Asp) number amino-acid residue among (1) SEQ ID NO:3; (2) among the SEQ ID NO:3 26 (Ser) to 32 (Pro) number amino-acid residue; (3) among the SEQID NO:3 41 (Lys) to 47 (Asp) number amino-acid residue; (4) among the SEQ ID NO:3 49 (Val) to 62 (Cys) number amino-acid residue; (5) among the SEQ ID NO:3 41 (Lys) to 62 (Cys) number amino-acid residue; (6) among the SEQ ID NO:3 84 (Ala) to 97 (Ser) number amino-acid residue; (7) among the SEQ ID NO:3 103 (Thr) to 108 (Asp) number amino-acid residue; (8) among the SEQ ID NO:3 130 (Arg) to 135 (His) number amino-acid residue; (9) among the SEQ ID NO:3 164 (Gly) to 166 (Lys) number amino-acid residue; (10) among the SEQ ID NO:3 175 (Tyr) to 179 (Glu) number amino-acid residue; (11) among the SEQ ID NO:3 193 (Lys) to 196 (Ala) number amino-acid residue; (12) among the SEQ ID NO:3 203 (Lys) to 209 (Thr) number amino-acid residue.Other epi-position comprises following peptide, and they can be produced through cyanogen bromide cutting back by the total length people IL-RA22 that is not reduced: peptide 6 (SEQ ID NO:56), peptide 7 (SEQ ID NO:57); Peptide 8 (SEQ ID NO:58); Peptide 9 (SEQ ID NO:59); Peptide 10 (SEQ ID NO:60); And peptide 11 (SEQ ID NO:61).Halfcystine forms disulfide linkage, and this might link together peptide 7 (SEQID NO:57) and peptide 10 (SEQ ID NO:60).Especially, SEQ ID NO:56 corresponding to 1 (Pro) of SEQ ID NO:3 to 92 (Met) number amino-acid residue; SEQ ID NO:57 corresponding to 93 (Thr) of SEQ ID NO:3 to 120 (Met) number amino-acid residue; SEQ ID NO:58 corresponding to 121 (Ile) of SEQ ID NO:3 to 160 (Met) number amino-acid residue; SEQ ID NO:59 corresponding to 161 (His) of SEQ ID NO:3 to 185 (Met) number amino-acid residue; SEQ ID NO:60 corresponding to 186 (Ile) of SEQ IDNO:3 to 199 (Met) number amino-acid residue; SEQ ID NO:61 corresponding to 200 (Cys) of SEQ ID NO:3 to 211 (Thr) number amino-acid residue.In addition, comprise the Tyr-60 among the SEQ ID NO:2 for the important residue of aglucon-receptors bind (and the corresponding residue among the SEQ ID NO:3) among the SEQ ID NO:2, Phe-164, Tyr-93, Arg-112, Lys-210, and Glu-211 (and corresponding residue of SEQ ID NO:3).And, directly comprise Tyr-60 and Phe-164 (and corresponding residue of SEQ ID NO:3) among the SEQ ID NO:2 for aglucon-acceptor among the SEQ ID NO:2 in conjunction with important elementary residue (and corresponding residue of SEQ ID NO:3); Secondary residue comprises the Tyr-93 of SEQ ID NO:2, Arg-112, Lys-210, and Glu-211 (and corresponding residue of SEQ ID NO:3).In preferred embodiments, will contain among the SEQ ID NO:2 for the important residue (and corresponding residue of SEQ ID NO:3) of aglucon-receptors bind (for example for the combination of IL-22RA) with neutrality antibody bonded antigenic epitopes of the present invention with IL-20 or IL-22 (separately or).

The peptide and the polypeptide that carry antigenic epitopes can contain at least 4 to 10 amino acid in the aminoacid sequence disclosed herein, at least 10 to 15 amino acid, and perhaps about 15 to about 30 amino acid.As described herein, this peptide and the polypeptide that carries epi-position can be by making the IL-22RA fragmentation or obtaining by chemical peptide is synthetic.And can select epi-position (referring to for example Lane and Stephen, Curr.Opin.Immunol.5:268 (1993), and Cortese et al., Curr.Opin.Biotechnol.7:616 (1996)) by the phage display of random peptide library.Identify epi-position and following document, describe to some extent: Mole for example from the standard method that the little peptide that comprises epi-position prepares antibody, " EpitopeMapping; " in Methods in Molecular Biology, Vol.10, Manson (ed.), pages 105-116 (The Humana Press, Inc.1992), Price, " Production and Characterization of Synthetic Peptide-DerivedAntibodies; " Monoclonal Antibodies:Production, Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages60-84 (Cambridge University Press 1995), and Coligan et al. (eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5and pages 9.4.1-9.4.11 (John Wiley ﹠amp; Sons 1997).

IL-22RA polypeptide for any comprises variant and fusion rotein, and those skilled in the art can be at an easy rate according to the information that provides in top table 1 and the table 2, the polynucleotide sequence of the complete degeneracy of this variant of preparation coding.And those skilled in the art can use the software of standard, according to Nucleotide described herein and aminoacid sequence design IL-22RA variant.

5.IL-22RA the preparation of polypeptide

Polypeptide of the present invention comprises the polypeptide of total length; Soluble and monomeric, equal dimer, heterodimer and polymer acceptor; The total length acceptor; Receptor fragments (for example aglucon binding fragment and antigenic epitopes); Functional fragment and fusion rotein all can prepare in recombinant host cell according to the technology of routine.In order to express the IL-22RA gene, the nucleic acid encoding molecule must be connected with the regulating and controlling sequence operability of control transcriptional expression in expression vector, is incorporated in the host cell then.Except transcription regulating nucleotide sequence such as promotor and enhanser for example, expression vector can comprise the translational control sequence and be suitable for selecting carrying the marker gene of the cell of expression vector.

The expression vector that is suitable for production foreign protein in eukaryotic cell contains the procaryotic DNA element and the antibiotics resistance marker gene of (1) coding bacterium replication orgin usually, so that growth and the selection of expression vector in host bacterium to be provided; (2) the eukaryotic DNA element of control transcription initiation, for example promotor; And the DNA element of (3) control transcription product processing, for example Transcription Termination/polyadenylic acid sequence.As discussed above, expression vector can also comprise the nucleotide sequence of the secretion sequence of encoding, and this secretion sequence directing heterologous polypeptide enters into the Secretory Pathway of host cell.For example, may comprise IL-22RA gene and in the IL-22RA expression vector from the secretion sequence of any secretory gene.

IL-22RA albumen of the present invention can be expressed in mammalian cell.Suitable mammalian host cell example comprises African green monkey kidney cell (Vero; ATCC CRL 1587), human embryonic kidney cell (293-HEK; ATCC CRL 1573), baby hamster kidney cell (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), Madin-Darby canine kidney(cell line) (MDCK) (MDCK; ATCCCCL 34), Chinese hamster ovary cell (CHO-K1; ATCC CCL61; CHO DG44 (Chasinet al., Som.Cell.Molec.Genet.12:555,1986)), rat pituitary cell (GH1; ATCC CCL82), HeLa S3 cell (ATCC CCL2.2), rats'liver oncocyte (H-4-II-E; ATCC CRL 1548), transformed the monkey-kidney cells (COS-1 of SV40; ATCCCRL 1650) and mouse embryonic cell (NIH-3T3; ATCC CRL 1658).

For mammalian hosts, transcribe with the translational control signal and can derive from mammalian virus, for example adenovirus, bovine papilloma virus, monkey disease poison or the like, wherein adjustment signal is relevant with the specific gene with high expression level.Suitable transcribes with the translational control sequence and can also for example obtain Actin muscle, collagen protein, myosin and the metallothionein gene from mammalian genes.

Transcription regulating nucleotide sequence comprises is enough to the synthetic initial promoter region of guide RNA.Suitable eukaryotic promoter comprises promotor (the Hamer et al. of mouse metallothionein(MT) I gene, J.Molec.Appl.Genet.1:273 (1982)), TK promotor (the McKnight of simplexvirus, Cell 31:355 (1982)), SV40 early promoter (Benoist et al., Nature 290:304 (1981)), Rous sarcoma virus promotor (Gorman et al., Proc.Nat ' l Acad.Sci.USA 79:6777 (1982)), cytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and the mouse mammary tumor virus promotor is (totally referring to Etcheverry, " Expression of Engineered Proteinsin Mammalian Cell Culture; " Protein Engineering:Principles andPractice, Cleland et al. (eds.), pages 163-181 (John Wiley ﹠amp; Sons, Inc.1996)).

Perhaps, if prokaryotic promoter is subjected to the regulation and control of eukaryotic promoter, then can use this prokaryotic promoter, phage T3 rna polymerase promoter for example, control expression (the Zhou et al. of IL-22RA gene in mammalian cell, Mol.Cell.Biol.10:4529 (1990), and Kaufman et al., Nucl.Acids Res.19:4485 (1991)).

In certain embodiments, operability links to each other segmental dna sequence dna other genetic elements (generally comprising transcripting promoter and terminator) required with its expression of coding IL22RA soluble receptors polypeptide or IL-RA22 polypeptide in expression vector.This carrier also contains one or more selected marker and one or more replication orgin usually jointly; although one skilled in the art will realize that; in some system; selective marker can provide on different carriers, and duplicating of this foreign DNA can be by providing in the genome that is incorporated into host cell.For promotor, terminator, selective marker, carrier and other selection of components is those of ordinary skills' regular transaction.Many such elements are described in the literature to some extent, and can provide by commercial supplier.A plurality of components of soluble receptors complex body can be by cotransfection on different expression vectors or on single expression vector.The multi-component technology of this marking protein complex body is well-known in this area.

Can use multiple different standard technique, comprise delivery, electroporation of calcium phosphate transfection, liposome-mediated transfection, the mediation of little projectile body or the like, expression vector is introduced in the host cell.Can select and breed transfected cell, so that the recombinant host cell that comprises the expression vector of stable integration in the host cell gene group to be provided.Introduce the technology of carrier and use the description to some extent in the Gene Transfer and Expression Protocols (Humana Press 1991) that for example Ausubel (1995) and Murray edit of technology that dominant selectable marker selects this stable conversion to eukaryotic cell.

For example, a kind of suitable selective marker provides the gene to the microbiotic neomycin resistance.In this case, under the condition of existence such as Xin Meisu types of drug such as G-418, select.Can also use selective system to improve the expression level of target gene, this process is called as " amplification ".Amplification as below carry out: under the situation that low-level selection medicament exists, cultivate transfectant, improve the amount of selecting medicament then, select to produce the high-caliber cell of introducing gene product.Suitable increased selective marker is Tetrahydrofolate dehydrogenase (DHFR), and it makes cell have resistance to methotrexate.Can also use other drug resistant gene (for example hygromycin resistance, multi-drug resistance, tetracycline acetyltransferase).Perhaps can use and introduce the mark that changes phenotype, green fluorescent protein or cell surface protein CD4 for example for example, CD8, I class MHC, P-ALP is opened the cell sorting of cells transfected and untransfected by modes such as FACS sorting or magnetic beads isolation technique.

Can also use viral delivery system to prepare the IL-22RA polypeptide by the mammalian cell of cultivating.The exemplary virus that is used for this purpose comprises adenovirus, retrovirus, simplexvirus, poxvirus and adeno associated virus (AAV).As the adenovirus of double-stranded DNA virus is to study to such an extent that be used for gene transfer vector that heterologous nucleic acids delivers the most fully (summary is referring to Becker etal. now, Meth.Cell Biol.43:161 (1994), and Douglas and Curiel, Science ﹠amp; Medicine 4:44 (1997)).The advantage of adenovirus system comprises can hold relatively large DNA inset, have the paramount titre of growth ability, have the ability and the flexible property that infect extensive mammalian cell types, can use with the ready-made carrier that contains different promoters in a large number.

By the portion gene group of deleted adenovirus, can hold the allogeneic dna sequence DNA of big (can reach 7kb).These insets can be by direct connection or by being incorporated in the viral DNA with cotransfection plasmid generation homologous recombination.Selection is the essential E1 gene of disappearance from virus vector, and this will cause carrier that not reproducible is not provided at host cell under the situation of E1 gene.For example, (ATCC numbers CRL-1573,45504 to people's 293 cells of adenovirus carrier infection, 45505), can be used as the attached cell cultivation or, produce a large amount of albumen (referring to Garnier et al., Cytotechnol.15:145 (1994)) with higher cell density suspension culture.

IL-22RA can also express in other higher eukaryotic cell, for example fowl, fungi, insect, yeast or vegetable cell.Rhabdovirus system provides an efficient manner, clone's IL-22RA gene can be incorporated in the insect cell.Suitable expression vector is based on autographa california (Autographa californica) nuclear polyhedrosis virus (AcMNPV), wherein contain well-known promotor, fruit bat heat shock protein 70 (hsp) promotor for example, autographa california nuclear polyhedrosis virus immediate early gene promotor (ie-1) and delay early stage 39K promotor, baculovirus p10 promotor and fruit bat metallothionein promoter.Second kind of method for preparing recombinant baculovirus is to use the system based on transposon, and (Luckow, et al. describe among the J.Virol.67:4566 (1993) to some extent at Luckow.This system has used transfer vector, the BAC-to-BAC test kit (Life Technologies, Rockville, on sale in MD).Contain the Tn7 transposon among the transfer vector PFASTBAC (LifeTechnologies) that this system uses, can be in the genome of baculovirus with the transgenosis of coding IL-22RA polypeptide, described baculovirus is maintained in the intestinal bacteria as a large-scale plasmid, is called " rod granule ".Referring to Hill-Perkins and Possee, J.Gen.Virol.71:971 (1990), Bonning, et al., J.Gen.Virol.75:1551 (1994), and Chazenbalk, and Rapoport, J.Biol.Chem.270:1543 (1995).In addition, transfer vector can be included in the reading frame endomixis that the DNA of the c of IL-22RA polypeptide of expression or N end and coding epi-position label, for example Glu-Glu epi-position label (Grussenmeyer etal., Proc.Nat ' l Acad.Sci.82:7952 (1985)) forms.Use technology known in the art, the transfer vector that will contain the IL-22RA gene is transformed in the intestinal bacteria, and screening contains the rod granule of the lacZ gene (showing it is recombinant baculovirus) that is interrupted.Using common technology will contain the genomic bacmid dna of recombinant baculovirus then separates.

Can modify sizable degree to exemplary pFastBac.For example, can remove the polyhedrin promotor, and replace with baculovirus basic protein promoter and (also be called as Pcor, p6.9 or MP promotor), this promotor is at the more early expression of baculovirus infection, and shown that it is useful (referring to for example Hill-Perkins and Possee for expression-secretion type albumen, J.Gen.Virol.71:971 (1990), Bonning, et al., J.Gen.Virol.75:1551 (1994), and Chazenbalk andRapoport, J.Biol.Chem.270:1543 (1995)).In this transfer vector construct, can use short or long basic protein promoter.And, can make up such transfer vector, wherein natural IL-22RA secretory signal sequence is replaced with secretory signal sequence from insect protein.For example, from melittin (the Invitrogen Corporation of cast off a skin sterol Transglucosylase (EGT), honeybee; Carlsbad, CA) or baculovirus gp67 (PharMingen:San Diego, secretory signal sequence CA) all can be used in the construct, replace natural IL-22RA secretory signal sequence.

Use recombinant virus or rod granule transfection host cell.Suitable insect host cell comprises the clone that derives from IPLB-Sf-21 (ovary cell line of fall army worm (Spodoptera frugiperda) pupa), Sf9 (ATCC CRL 1711) for example, Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, CA), and fruit bat Schneider-2 cell and derive from the HIGH FIVEO clone (Invitrogen) (United States Patent (USP) numbering 5,300,435) of semilooper (Trichoplusia ni).But the serum-free culture basal growth of commodity in useization and keep these cells.The suitable culture base has Sf900 II ^TM(LifeTechnologies) or ESF 921 ^TM(Expression Systems) (being used for the Sf9 cell); And Ex-cell0405 ^TM(JRH Biosciences, Lenexa, KS) or Express FiveO ^TM(Life Technologies) (being used for the T.ni cell).When using recombinant virus, the inoculum density of cell is 2-5 * 10 roughly usually ⁵Individual cell is 1-2 * 10 with cell cultures to density ⁶Individual cell, add the recombinant virus liquid storage this moment, and infection multiplicity (MOI) is 0.1 to 10, more generally is near 3.

The mature technology of preparation recombinant protein provides in below with reference to document in rhabdovirus system: Bailey et al., " Manipulation of Baculovirus Vectors; " Methods in Molecular Biology, Volume 7:Gene Transfer andExpression Protocols, Murray (ed.), pages 147-168 (The HumanaPress, Inc.1991); Patel et al., " The baculovirus expressionsystem, " DNA Cloning 2:Expression Systems, 2nd Edition, Gloveret al. (eds.), pages 205-244 (Oxford University Press 1995); Ausubel (1995), pages 16-37 to 16-57; Richardson (ed.), and Baculovirus Expression Protocols (The Humana Press, Inc.1995); And Lucknow, " Insect Cell Expression Technology, " in ProteinEngineering:Principles and Practice, Cleland et al. (eds.), pages 183-218 (John Wiley ﹠amp; Sons, Inc.1996).

Also can use the fungal cell, comprise yeast cell, express gene described herein.Interested especially in this respect yeast kind comprises yeast saccharomyces cerevisiae (Saccharomycescerevisiae), Pichia pastoris (Pichia pastoris), and pichia methanolica (Pichia methanolica).Be used for comprising promotor from GAL1 (semi-lactosi), PGK (phosphoglyceric kinase), ADH (ethanol dehydrogenase), AOX (alcohol oxidase), HIS4 (histidinol dehydrogenase) or the like in the suitable promotor that yeast is expressed.Designed many yeast clone carriers, and can obtain at an easy rate.These carriers comprise that the YRp carrier is YRp17 for example based on the carrier of Ylp YIp5 for example, and the YEp carrier is YEp13 and YCp carrier YCp19 for example for example.Be disclosed in for example Kawasaki with the method that prepare recombinant polypeptide the method for foreign DNA transformed saccharomyces cerevisiae (S.cerevisiae) cell and the yeast cell after transforming, United States Patent (USP) numbers 4,599,311, Kawasaki et al., United States Patent (USP) numbering 4,931,373, Brake, United States Patent (USP) numbering 4,870,008, Welch et al., United States Patent (USP) numbering 5,037,743, and Murray et al., in the United States Patent (USP) numbering 4,845,075.Screen cell transformed according to the phenotype of being determined by selective marker, described selective marker is drug resistance or in the ability that does not have certain nutrition (for example leucine) can grow when existing normally.The carrier system that is suitable for using in yeast saccharomyces cerevisiae is the POT1 carrier system, this is by Kawasaki et al. (United States Patent (USP) numbering 4,931,373) open, this carrier system can make cell transformed screen by growing in containing the substratum of glucose.Other be suitable for zymic promotor and terminator comprise those from the glycolytic ferment gene (referring to for example Kawasaki, United States Patent (USP) numbering 4,599,311, Kingsman et al., United States Patent (USP) numbering 4,615,974, and Bitter, United States Patent (USP) numbering 4,977,092) and the promotor and the terminator of alcohol dehydrogenase gene.Also can number 4,990,446,5,063,154,5,139,936 and 4,661,454 referring to United States Patent (USP).

For other yeast, comprise multiple-shaped nuohan inferior yeast (Hansenula polymorpha), schizosaccharomyces pombe (Schizosaccharomyces pombe), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces fragilis (Kluyveromycesfragilis), Ustilago maydis (D C.) Corola. (Ustilago maydis), Pichia pastoris (Pichia pastoris), methyl alcohol pichia (Pichia methanolica), the conversion system of Ji Shi pichia spp (Pichia guillermondii) and maltose candiyeast (Candidamaltosa) is known in this area.Referring to for example Gleeson et al., J.Gen.Microbiol.132:3459 (1986), and Cregg, United States Patent (USP) numbering 4,882,279.Can be according to McKnight et al., method (United States Patent (USP) numbering 4,935,349) use Aspergillus (Aspergillus) cell.The method that transforms product yellow top spore mould (Acremonium chrysogenum) is disclosed in the United States Patent (USP) numbering 5,162,228 at Sumino et al..The method that transforms neurospora (Neurospora) is disclosed in the United States Patent (USP) numbering 4,486,533 at Lambowitz.

For example, use pichia methanolica (Pichia methanolica) to be disclosed in Raymond, United States Patent (USP) numbering 5 as the host who produces recombinant protein, 716,808, Raymond, United States Patent (USP) numbering 5,736,383, Raymond et al., Yeast 14:11-23 (1998), and at international application publication numbering WO 97/17450, WO 97/17451, disclosed among WO 98/02536 and the WO 98/02565.The dna molecular that is used to transform pichia methanolica is prepared as the plasmid of double-stranded circular usually, be preferably in transform before linearizing.For prepare polypeptide in finishing red methanol yeast, promotor in the plasmid and terminator can be to finish red methanol yeast gene, for example finish red methanol yeast alcohol and utilize gene (AUG1 or AUG2).Other useful promotors comprise Protosol synthase (DHAS), hydrogenlyase (FMD), and the promotor of catalase (CAT) gene.In order to help the integration of DNA in host chromosome, the segmental both sides of whole expression that are preferably in plasmid connect the host DNA sequence.The suitable selective marker that is used for finishing red methanol yeast is to finish red methanol yeast ADE2 gene, its Phosphoribosyl-5-aminooimidazole carboxylase (AIRC that encodes; EC 4.1.1.21), it can make the ade2 host cell grow under the situation of VITAMIN B4 not having.For the use minimum large-scale industry metallization processes of expectation methyl alcohol, then can use to have lacked the host cell that two methyl alcohol utilize gene (AUG1 and AUG2).For the production of secretory protein, host cell can lack vacuole protein enzyme gene (PEP4 and PRB1).Use electroporation to promote to contain encode the plasmid of the DNA of target polypeptides to be incorporated in the complete red methanol yeast cell.When electroporation transform to be finished red methanol yeast cell, can use the pulsed electrical field (field intensity is 2.5-4.5kV/cm, is preferably 3.75kV/cm) of exponential attenuation, time constant (t) is 1 to 40 millisecond, most preferably is about 20 milliseconds.

Expression vector can also be incorporated in the protoplastis of plant, in the intact plant tissue or in the isolating vegetable cell.Expression vector is incorporated into that method in the plant tissue comprises direct infection or with the delivery of plant tissue and agrobacterium tumefaciens (Agrobacterium tumefaciens) co-cultivation, little impelling mediation, DNA injection, electroporation or the like.Referring to for example Horschet al., Science 227:1229 (1985), Klein et al., Biotechnology10:268 (1992), and Miki et al., " Procedures for IntroducingForeign DNA into Plants; " Methods in Plant Molecular Biologyand Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press, 1993).

Perhaps, the IL-22RA gene can be expressed in prokaryotic host cell.The suitable promotor that can be used in prokaryotic hosts expressing the IL-22RA polypeptide is well-known for those skilled in the art, and they comprise can discern T4, T3, the promotor of Sp6 and T7 polysaccharase, the P of phage _RAnd P _LPromotor; colibacillary trp, recA, heat shock; lacUV5; tac, lpp-lacSpr, phoA; and lacZ promotor; the promotor of subtilis, the promotor of genus bacillus phage, the int promotor of the promotor of streptomycete, phage, the bla of pBR322 start and give and the CAT promotor of chloramphenicol acetyl-transferase gene.Summary about prokaryotic promoter has Glick, J.Ind.Microbiol.1:277 (1987), Watson et al., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and Ausubel et al. (1995).

Suitable prokaryotic hosts comprises intestinal bacteria and subtilis.Suitable coli strain comprises BL21 (DE3), BL21 (DE3) pLysS, BL21 (DE3) pLysE, DH1, DH4I, DH5, DH5I, DH5IF ', DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (referring to for example Brown (ed.), Molecular BiologyLabfax (Academic Press 1991)).Suitable bacillus subtilis strain comprises BR151, YB886, MI119, MI120, and B170 (referring to for example Hardy, " BacillusCloning Methods; " DNA Cloning:A Practical Approach, Glover (ed.) (IRL Press 1985)).

When on bacterium for example during expression in escherichia coli IL-22RA polypeptide, polypeptide can be retained in the tenuigenin, usually as insoluble particle form, perhaps can be directed to the outer pericentral siphon space of born of the same parents by the secretion sequence of bacterium.For the previous case,, reclaim particle and for example use guanidinium isothiocyanate or urea carries out sex change lysis.Can for example, to the buffer salt solution dialysis, make the polypeptide refolding of sex change and form dimer again by dilution sex change liquid by to containing the solution dialysis of urea and reduced form and Sleep-promoting factor B mixture.For latter event, by ruptured cell (by for example ultrasonic wave or osmotic shock), the content that discharges periplasmic space also reclaims protein, from born of the same parents outside the pericentral siphon space reclamation soluble with the polypeptide that function is arranged, so just saved the needs of sex change and refolding.

The method of marking protein is well-known (referring to for example Williams et al. for those skilled in the art in prokaryotic hosts, " Expression of foreignproteins in E.coli using plasmid vectors and purification ofspecific polyclonal antibodies; " DNA Cloning 2:ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., " Genetic Manipulation andExpression of Antibodies; " Monoclonal Antibodies:Principlesand Applications, page 137 (Wiley-Liss, Inc.1995), and Georgiou, " Expression of Proteins in Bacteria; " in ProteinEngineering:Principles and Practice, Cleland et al. (eds.), page 101 (John Wiley ﹠amp; Sons, Inc.1996)).

The standard method that expression vector is introduced in bacterium, yeast, insect and the vegetable cell provides in for example Ausubel (1995).

By mammal cell line system express and the general method that reclaims the exogenous protein of being produced at for example Etcheverry, " Expression of Engineered Proteins inMammalian Cell Culture; " Protein Engineering:Principles andPractice, Cleland et al. (eds.), (Wiley-Liss provides in Inc.1996) pages 163.The standard technique of the exogenous protein that recovery produces by bacterial system is at for example Grisshammer et al., " Purification of over-produced proteinsfrom E.coli cells; " DNA Cloning 2:Expression Systems, 2ndEdition, Glover et al. (eds.) provides among the pages 59-92 (Oxford UniversityPress 1995).The maturation method of separating recombinant proteins matter is at Richardson (ed.) from rhabdovirus system, and (TheHumana Press describes in Inc.1995) Baculovirus Expression Protocols.

Alternatively, polypeptide of the present invention can synthesize by complete solid phase synthesis, part solid phase method, fragment condensation or classical solution.These synthetic methods are well-known (referring to for example Merrifield for those skilled in the art, J.Am.Chem.Soc.85:2149 (1963), Stewart et al., " Solid Phase PeptideSynthesis " (2nd Edition), (Pierce Chemical Co.1984), Bayerand Rapp, Chem.Pept.Prot.3:3 (1986), Atherton et al., SolidPhase Peptide Synthesis:A Practical Approach (IRL Press 1989), Fields and Colowick, " Solid-Phase Peptide Synthesis; " Methodsin Enzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al., Chemical Approaches to the Synthesis ofPeptides and Proteins (CRC Press, Inc.1997)).The version of complete chemosynthesis strategy, for example " native chemical connection " also is that standard method is (referring to for example Dawson et al. with " expressing protein is connected ", Science 266:776 (1994), Hackenget al., Proc.Nat ' l Acad.Sci.USA 94:7845 (1997), Dawson, Methods Enzymol.287:34 (1997), Muir et al, Proc.Nat ' l Acad.Sci.USA 95:6705 (1998), and Severinov and Muir, J.Biol.Chem.273:16205 (1998)).

At least 6, at least 9 or at least 15 of comprising among the SEQ ID NO:3 of peptide of the present invention and polypeptide adjoin amino-acid residue.As illustration, at least 6, at least 9 or at least 15 of can comprise among the SEQ ID NO:3 of polypeptide adjoin amino-acid residue.In certain embodiments of the invention, polypeptide comprises 20,30,40,50 in these aminoacid sequences, and 100 or more adjoin residue.Encoding the nucleic acid molecule of this peptide and polypeptide can be as the primer and the probe of polymerase chain reaction.

And IL-22RA polypeptide and fragment thereof can be used as monomer, all dimer, heterodimer or polymer are expressed in higher eucaryotic cells.This cell can be used to produce IL-22RA monomer, equal dimer, heterodimer or polymer receptor polypeptides, wherein comprise at least one IL-22RA polypeptide (" acceptor that comprises IL-22RA " or " receptor polypeptides that comprises IL-22RA "), perhaps can in screening system, be used as the detection cell.In one aspect of the invention, the polypeptide of the present invention that comprises the IL-22RA extracellular domain is produced by culturing cell, and this cell is used to screen the aglucon of acceptor, comprises the agonist and the antagonist of natural aglucon IL-22 and natural aglucon.This method summary gets up, and other genetic elements (for example promotor) that the coding cDNA of acceptor or gene and its expression is required are combined, and the expression vector of generation is inserted in the host cell.The cell of selecting to express this DNA and producing functional receptor, and in multiple different screening system, use these cells.Each component of monomer, equal dimer, heterodimer or polymer acceptor complex body can be expressed in same cell.And each component of monomer, equal dimer, heterodimer or polymer acceptor complex body can merge with membrane spaning domain or merge meromixis with other films, thereby can carry out the assembling of complex body and the screening of transfectant as mentioned above.

In order to detect IL-20 of the present invention and IL-22 antagonist polypeptide and antibody, be suitable for express the known acceptor in conjunction with IL-20 or IL-22 of the acceptor comprise IL-22RA or other (for example express IL-22RA/CRF2-4; And IL-20RA, IL-20RB, IL-22RA/IL-20RB, the perhaps cell of IL-20RA/IL-20RB) and the mammalian cell of the receptor-mediated signal of transduceing comprise the cell of expressing other receptor subunits (these receptor subunits may form functional complex with IL22RA or IL-20RA).These subunits may comprise that those belong to Interferon Receptors family or belong to other types II or the subunit of type i cytokine receptor, CRF2-4 (Genbank login numbering Z17227) for example, IL-10R (Genbank login numbering U00672 and NM_001558), IL-22RA (the United States Patent (USP) numbering of owning together 5,965,704), zcytor7 (IL-20RA) (the United States Patent (USP) numbering of owning together 5,945,511), IL-20RA/IL-20RB (WIPO publication numbering WO 01/46232), and IL-9R.The also preferred cell that belongs to same species with the acceptor that will express that uses.In preferred embodiments, the propagation of this cell depends on the hemopoieticgrowth factor that adds.The preferred cell system of this type is people TF-1 clone (ATCC CRL-2003) and AML-193 clone (ATCC CRL-9589), they are the human leukemia cell lines that depend on GM-CSF, and BaF3 (Palacios and Steinmetz, Cell 41:727-734, (1985)), it is the mouse pre B cell system that depends on IL-3.Other clones comprise BHK, COS-1 and Chinese hamster ovary celI.Can carry out the engineering operation to suitable host cells, to produce essential receptor subunit or other required cellular components of cell response for expectation.This method has benefit, because can pair cell system carry out the engineering operation, makes the receptor subunit of its expression from any species, has so just solved the potential restriction that brings owing to species specificity.The species orthologous gene of receptor cdna of can cloning people, and be used in the clone from same species, for example the cDNA with mouse is used for BaF3 clone.Like this, depend on a kind of hemopoieticgrowth factor for example the clone of GM-CSF or IL-3 just can be operated by engineering, make it depend on the cytokine that another works by the IL-22RA acceptor, for example IL-22.

The cell of expressive function acceptor can be used in the shaker test.In multiple different suitable test known in the art.These tests based on be detection to biological response in the target cell.A kind of such test is a cell proliferation test.Cell is cultivated under the condition of testing compound having or do not have, and by for example measuring the tritiated thymus pyrimidine mix or based on 3-(4,5-dimethylthiazole-2-yl)-2,5-phenylbenzene tetrazolium  bromide (MTT) (Mosman, J.Immunol.Meth.65:55-63, (1983)) the colorimetric method of testing of metabolic degradation detect the propagation of cell.Another test form uses the cell of being expressed reporter gene by further engineering operation.This report gene links to each other with promoter element, and described promotor has response for the relevant approach of acceptor, and what this test detected is the activation that reporter gene is transcribed.The preferred promoter element that is used for this purpose is serum response element or SRE, referring to for example Shaw et al., Cell 56:563-572, (1989).Preferred this reporter gene is luciferase gene (deWet et al., Mol.Cell.Biol.7:725, (1987)).Can use the method known in the art (Baumgartner et al. for example, J.Biol.Chem.269:29094-29101, (1994); Schenborn and Goiffin, Promega_Notes41:11,1993), by the expression of luminous detection luciferase gene.Can be from for example Promega Corp., Madison, WI obtains the uciferase activity detection kit.Can use such target cell is screening chemical substance library, cell conditioned medium library, fungi culture medium library, pedotheque library, water sample library or the like.For example, can on target cell, detect by pair cell conditioned medium sample library, to identify the cell that produces aglucon.Use positive cell in the Mammals carrier, to prepare the cDNA library then, the library is divided into set, transfection host cell and expression.Then the culture sample from transfectional cell is detected, next continue to cut apart set, heavily transfection, inferior cultivation and, with the cDNA of the coding aglucon of separating clone the positive cell re-detection.

Several IL-20 there is the clone of replying known in the art, perhaps can makes up such clone, as Baf3/DIRS1/cytoR11 clone (WIPO publication numbering WO02/072607).In addition, also known severally have the clone of replying to IL-22, (Dumontieret al., J.Immunol. 164: 1814-1819,2000; Dumoutier, L.et al., Proc.Nat ' l.Acad.Sci. 97: 10144-10149,2000; Xie MH et al., J.Biol.Chem. 275: 31335-31339,2000; Kotenko SV et al., J. Biol.Chem. 276: 2725-2732,2001), and the clone of expressing IL-22 receptor subunit IL-22RA.For example, following cell is replied IL-22: TK-10 (Xie MHet al., See before.) (people's kidney); SW480 (ATCC numbers CCL-228) (human colon adenocarcinoma); HepG2 (ATCC numbers HB-8065) (people's liver cancer); PC12 (ATCC numbers CRL-1721) (mouse neuronal cell model; The rat pheochromocytoma); And MES13 (ATCC numbers CRL-1927) (mouse mesangial cell system).In addition, some clones of expressing IL-22RA (IL-22 acceptor) also are the candidate cell system that IL-22 is produced response: A549 (ATCC numbers CCL-185) (people's lung cancer); G-361 (ATCC numbers CRL-1424) (Humanmachine tumour); And Caki-1 (ATCC numbers HTB-46) (people's renal cancer).In addition, can also make up the clone that IL-22 is had response, for example, Baf3/cytoR11/CRF2-4 clone described herein (WIPO publication numbering WO 02/12345).These cells can be used for detecting, to measure IL-22RA functional as IL-20 or IL-22 antagonist or anti-inflammatory factors.

Extra screening method provided by the invention comprises use heterozygote receptor polypeptides.These heterozygote polypeptide are divided into two big classes.In the first kind, the cell intracellular domain of IL-22RA links to each other with the aglucon binding domains of second acceptor.The second class heterozygote receptor polypeptides comprises extracellular (aglucon combination) structural domain (SEQ ID NO:3) of IL-22RA and the cell intracellular domain of second acceptor (being preferably hematopoiesis sexual cell factor acceptor), and membrane spaning domain.The heterozygote IL-22RA monomer that belongs to the acceptor of the present invention of second class, all dimer, heterodimer and polymer are expressed known can the generation in the cell of replying the signal of this second acceptor transduction.Generally speaking, this two classes heterozygote acceptor makes and identifies the responsive cells type and become possibility, and this cell can be used to develop the measuring method that detects IL-22 or IL-20.And, in the mensuration of type of competition, can when existing, IL-22 or IL-20 use this raji cell assay Raji soluble receptors antagonist of the present invention.In this mensuration, IL-22 or IL-20 propagation or the active reduction of signal transduction have shown antagonistic activity under the condition that soluble receptors of the present invention exists.And, can also use IL-22RA soluble receptors binding assay (this is a kind of detection method based on cell) measure whether combination of soluble receptors, blocking-up, inhibition, reduction, antagonism or in and the activity of IL-22 or IL-20.

6.IL-22RA the preparation of fusion rotein and conjugate

A general type of IL-22RA analogue is to have the variant that has the aminoacid sequence of sudden change in aminoacid sequence disclosed herein.The IL-22RA analogue of another general type is provided by antiidiotypic antibody described below and fragment thereof.And the recombinant antibodies that comprises the antiidiotype variable domains can be used as analogue (referring to for example Monfardini et al., Proc.Assoc.Am.Physicians 108:420 (1996)).Because the variable domains Simulation with I L-22RA of antiidiotype IL-22RA antibody, these structural domains can provide IL-22RA in conjunction with activity.The method for preparing the antiidiotype catalytic antibody it is known to those skilled in the art that (referring to for example Joron et al., Ann.N Y Acad.Sci.672:216 (1992), Friboulet et al., Appl.Biochem.Biotechnol.47:229 (1994), and Avalle et al., Ann.N Y Acad.Sci.864:118 (1998)).

The method of the another kind of IL-22RA of evaluation analogue provides by using combinatorial library.Make up and the method for screening phage display and other combinatorial library provides Kay et al. for example, Phage Display of Peptides and Proteins (AcademicPress 1996) in following document, Verdine, United States Patent (USP) numbering 5,783,384, Kay, et.al., United States Patent (USP) numbering 5,747,334, and Kauffman et al., United States Patent (USP) numbering 5,723,323.

The IL-22RA polypeptide has in the body and external purposes.As illustration, the soluble form of IL-22RA can be joined in the cell culture medium to suppress effect by the IL-22RA aglucon of cultured cells generation.

The IL-22RA fusion rotein can be used for expressing IL-22RA at recombinant host, and separates the IL-22RA that produces.As described below, specific I L-22RA fusion rotein also has purposes in diagnosis and treatment.One type fusion rotein comprises the peptide that instructs the IL-22RA polypeptide to leave recombinant host cell.In order to instruct the IL-22RA polypeptide to enter into the Secretory Pathway of eukaryotic host cell, in the IL-22RA expression vector, provide secretory signal sequence (also being called as signal peptide, leading peptide, preceding former sequence or presequence).Although secretory signal sequence may be from IL-22RA, suitable signal sequence also can be from other secretory protein or de novo synthesis.Secretory signal sequence and IL-22RA encoding sequence can be operatively connected, and two sequences are connected and the location according to correct reading frame, instruct new synthetic polypeptide to enter into the Secretory Pathway of host cell.Secretory signal sequence is placed in 5 ' end of the nucleotide sequence of coding target polypeptides usually, some secretory signal sequence also may be placed in the target nucleotide sequence other positions (referring to for example Welch et al., United States Patent (USP) numbering 5,037,743; Holland et al., United States Patent (USP) numbering 5,143,830).

Although the secretory signal sequence of IL-22RA or the another kind of albumen that produces by mammalian cell (tissue-type plasminogen activator's signal sequence for example, as numbering 5 at United States Patent (USP), 641, described in 655) be useful in the recombinant mammalian host, expressing IL-22RA, but preferably use the zymic signal sequence for the expression in yeast cell.The example of suitable yeast signal sequence has the yeast signal sequence from yeast mating pheromone alpha factor (by MF α l genes encoding), saccharase (by the SUC2 genes encoding) or acid phosphatase (by the PHO5 genes encoding).Referring to for example Romanos et al., " Expressionof Cloned Genes in Yeast, " DNA Cloning 2:A Practical Approach, 2 ^NdEdition, Glover and Hames (eds.), pages 123-167 (OxfordUniversity Press 1995).

IL-22RA soluble receptors polypeptide can prepare by the brachymemma DNA that expresses Codocyte outer structure territory (for example containing the polypeptide of SEQ ID NO:3 or the respective regions of inhuman acceptor).The extracellular domain polypeptide is not preferably to stride the form preparation of polypeptide fragment in film and the cell basically.In order to instruct receptor domain outwards transportation from host cell, with receptor dna and coding secretion peptide for example another dna fragmentation of t-PA secretion peptide link to each other.The for convenience purifying secreted receptor domain that goes out can extend C-terminal that for example poly is histidine-tagged, P material, Flag ^TMPeptide (Hopp et al., Biotechnology 6:1204-1210, (1988); Can be from Eastman Kodak Co., New Haven, CT obtains) or another polypeptide or protein (having the antibody or other the specific combination factor can supply to utilize for this polypeptide or protein) and receptor polypeptides fusion.And, also can be from the IL-22RA antigenic epitopes of extracellular cytokine binding domains according to top description preparation.

In another method, the receptor extracellular structural domain of IL-22RA or other I classes or II type cytokines acceptor component can with immunoglobulin heavy chain constant region (Fc fragment normally, it comprises two constant region structural domains and hinge area, but lack the variable region) form that merges mutually express (referring to Sledziewski, Az et al., United States Patent (USP) numbering 6,018,026 and 5,750,375).Solubility IL-22RA polypeptide of the present invention comprises this syzygy.A kind of such syzygy shows in SRQ ID NO:4.Such fusion rotein is usually as the multimeric molecule secretion, and Fc part wherein interconnects with disulfide linkage, and two receptor polypeptides are closely close mutually.Such fusion rotein can be used for the homology aglucon is come out from the solution affinity purification; As the instrument of vitro detection, block, suppress or reduce external signal by special titration aglucon; And by with these fusion roteins through the parenteral administration, as intravital antagonist, from circulate, remove away in conjunction with the aglucon in the circulation and with it.Be the purifying aglucon, helping (normally near physiological temp, pH and ionic strength) under acceptor-aglucon bonded condition, in the sample that contains aglucon (for example cell conditioned medium or tissue extract), add the IL-22RA-Ig mosaic.Use the a-protein that is fixed on the solid support (for example insoluble resin pearl) from mixture, to separate mosaic-aglucon complex body then.Use conventional chemical technology for example salt or pH gradient then, the wash-out aglucon.Perhaps, mosaic self can be fixed on the solid support, according to above-mentioned combination and the wash-out of carrying out.Can use mosaic with the regulation and control inflammatory response in vivo, comprise that acute phase replys, for example serum amyloid sample A material (SAA), c reactive protein (CRP) or the like.Mosaic with high binding affinity is by parenteral administration (for example by intramuscular, subcutaneous or intravenous injection).Molecule in the circulation is removed away from circulation in conjunction with aglucon and by normal physiological process.For the use in detection, mosaic is combined with upholder by the Fc zone, use according to the ELISA mode.

For auxiliary anti-IL-22RA of the present invention and the binding partners of separating, can more advantageously use detection system, wherein utilize aglucon bind receptor (perhaps antibody, member that complement/anti-complement is right) or its binding fragment, and commercial biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ).The surface that member that such acceptor, antibody, complement/anti-complement are right or fragment are fixed on the acceptor chip.To the use of doing this instrument at Karlsson, J. Immunol.Methods 145: 229-40,1991 and Cunningham and Wells, J. Mol.Biol. 234: 554-63 is disclosed in 1993.Use amine or sulfydryl chemical method, acceptor, antibody, member or fragment that complement/anti-complement is right is covalently bound on the dextran fiber, and described dextran fiber is attached to above the golden film in the flow cell.Make test sample pass through this pond.If in sample, there is the right opposite member of aglucon, epi-position or complement/anti-complement, then they can be attached to immobilized acceptor, antibody or complement/anti-complement respectively on the member, cause the specific refractory power of medium to change, and this can be detected as the change of golden film surface kytoplasm resonance.The speed that this system can measure out and close in view of the above can the calculations incorporated affinity, and assessment bonded chemistry quantitative relationship.Perhaps, (Palo Alto CA) analyzes aglucon/receptors bind for Ciphergen, Inc. can to use SELDI (TM) technology.And, can use BIACORE technology described above whether in competitive assay, to measure different monoclonal antibodies in conjunction with the identical or different epi-position on the IL-22RA polypeptide; Like this, can be used for subsidiary book invention combination, blocking-up, inhibition, reduction, antagonism or in and the two the epitope mapping of neutralizing antibody of IL-22 or IL-20 and IL-22.

Can also in other detection systems known in the art, use aglucon associativity receptor polypeptides.Such system comprise the Scatchard that measures binding affinity analyze (referring to Scatchard, Ann.NY Acad.Sci. 51: 660-72,1949) and calorimetry (Cunningham et al., Science 253: 545-48,1991; Cunningham et al., Science 245: 821-25,1991).

The present invention also provides multiple other polypeptide syzygys and the relevant polymer protein that comprises or more a plurality of fusion polypeptide.For example, solubility IL-22RA acceptor and dimerizing protein can be prepared into fusion rotein, as disclosed in the United States Patent (USP) numbering 5,155,027 and 5,567,584.The preferred dimerizing protein that is used for this purpose comprises the constant region for immunoglobulin structural domain, for example IgG γ 1 and human kappa light chain.Can in the cell of crossing through genetic engineering procedure, express immunoglobulin (Ig)-solubility IL-22RA fusion rotein, to prepare multiple poly IL-22RA receptor analogs.Supplementary structure territory and solubility IL-22RA acceptor can be merged, it is targeted to (for example cell, IL-22 or the IL-20 of collagen or expression IL-22RA aglucon) on special cell, tissue or the macromole.Can be with IL-22RA polypeptide and two or more a plurality of part (affinity tag and the target structural domain that for example are used for purifying) fusion.The polypeptide syzygy can also comprise one or more a plurality of cleavage site, particularly between structural domain.Referring to Tuan et al., Connective Tissue Research 34: 1-9,1996.

In bacterial cell, more satisfactory usually is with fusion protein form expression, to reduce toxicity, the recovery that improves stability and improve expressing protein with heterologous protein.For example, can be with IL-22RA to comprise the fusion protein form expression of glutathione S-transferase polypeptide.The glutathione S-transferase fusion rotein is normally soluble, and can easily come out by immobilization gsh chromatography column purifying from colibacillary lysate.Use similar methods, use the starch-resin chromatography column IL-22RA fusion rotein that comprises the maltose binding protein polypeptide can be separated, and the fusion rotein that comprises the protein gene AC end of brachymemma can use the IgG-Sepharose purifying.In bacterial cell, heterologous polypeptide is described in following document to some extent with the mature technology of fusion protein form expression, Williams et al. for example, " Expression of Foreign Proteins in E.coli Using PlasmidVectors and Purification of Specific Polyclonal Antibodies; " DNA Cloning 2:A Practical Approach, 2 ^NdEdition, Glover andHames (Eds.), pages 15-58 (Oxford University Press 1995).In addition, also have commercial expression system can supply to utilize.For example, (the Promega Corporation of PINPOINT Xa protein purification system; Madison WI) provides the method for using the resin isolation fusion rotein that comprises avidin, and this fusion rotein is included in during the expression by biotinylated polypeptide.

Useful peptide tag comprises poly histidine-tagged (it has affinity to the nickel resin), c-myc label, caldesmon (using the calmodulin affinity chromatography to separate), Substance P, RYIRS label (it and anti-RYIRS antibodies), Glu-Glu label and FLAG label (it and anti-FLAG antibodies) when the heterologous polypeptide that separates by protokaryon or eukaryotic cell expression.Referring to for example Luo et al., Arch.Biochem.Biophys.329:215 (1996), Morganti et al., Biotechnol.Appl.Biochem.23:67 (1996), and Zheng et al., Gene 186:55 (1997).Encode this peptide tag nucleic acid molecule can (St.Louis MO) obtains from for example Sigma-Aldrich Corporation.

The form of another fusion rotein comprises IL-22RA polypeptide and immunoglobulin heavy chain constant region, Fc fragment normally, and it contains two or three constant region structural domains and hinge area, but does not have the variable region.As illustration, Chang et al., United States Patent (USP) numbering 5,723,125 has been described and has been comprised human interferon and the segmental fusion rotein of human normal immunoglobulin Fc.The C end of Interferon, rabbit is connected with the segmental N end of Fc by the peptide linker part.An example of peptide linker is the peptide that mainly comprises T cell inertia sequence (it is the immunology inert).Exemplary peptide linker has following aminoacid sequence: GGSGG SGGGG SGGGG S (SEQ ID NO:9).In this fusion rotein, illustrative Fc partly is people γ 4 chains, and it is stable in solution, and does not almost have or do not have the complement activation activity.Therefore, the present invention relates to comprise IL-22RA part and the segmental IL-22RA fusion rotein of people Fc, wherein IL-22RA partial C end links to each other with the segmental N end of Fc by peptide linker (peptide that for example comprises aminoacid sequence among the SEQ ID NO:4).This IL-22RA part can be IL-22RA molecule or its fragment.The amino acid and the Fc fragment (for example people Fc fragment) (SEQ ID NO:4) that for example, can comprise SEQ ID NO:3 in the fusion rotein.

In another version, the IL-22RA fusion rotein comprise the IgG sequence, with the covalently bound IL-22RA of IgG sequence aminoterminal part and with the covalently bound signal peptide of aminoterminal of IL-22RA part, wherein the IgG sequence is made up of according to following order following element: hinge area, CH ₂Structural domain and CH ₃Structural domain.Therefore, this IgG sequence lacks CH ₁Structural domain.As described herein, IL-22RA has partly shown the IL-22RA activity, for example with IL-22RA aglucon bonded ability.This preparation comprises the two the general method of fusion rotein of antibody and non-antibody part at LaRochelle et al., describes to some extent among the EP 742830 (WO 95/21258).

The fusion rotein that comprises IL-22RA part and Fc part for example can be used as, and the vitro detection instrument uses.For example, can use the situation that exists of IL-22RA aglucon in the IL-22RA-domain-immunoglobulin fusion proteins detection of biological sample, wherein IL-22RA partly is used for combining with aglucon, and macromole for example a-protein or anti-Fc antibody are used for fusion rotein is attached to solid support.The antagonist that can use this system to identify agonist and disturb IL-22RA aglucon (for example IL-22 or LI-20 and IL-22 the two) and its receptors bind.

The example of other antibody fusion proteins comprises the polypeptide that comprises antigen binding domains and IL-22RA fragment (it contains the IL-22RA extracellular domain).Such molecule can be owing to the active target particular tissues that is used for of the combination of IL-22RA.

The present invention has also comprised multiple other fusion polypeptide.For example, giving that the structural domain of biological function is some or all of can be at IL-22RA of the present invention and exchange between the structural domain of equal value on from another member's of cytokine receptor family function.Can in recombinant host cell, express fusion polypeptide, to prepare multiple IL-22RA syzygy analogue.The IL-22RA polypeptide can with two or more a plurality of part or structural domain (affinity tag and the target structural domain that for example are used for purifying merge).Fusion polypeptide can also comprise one or more a plurality of cleavage site, particularly between structural domain.Referring to for example Tuan et al., Connective Tissue Research34:1 (1996).

The method that can know by those skilled in that art, each component of preparation fusion rotein again with they chemical couplings, prepares fusion rotein so earlier.Perhaps, can use the polynucleotide of known technology, and express this polynucleotide with method described herein according to correct two components of reading frame generation encoding fusion protein.Fusion rotein being carried out the general method of enzymatic and chemical chop describes in the 16-25 page or leaf at for example Ausubel (1995) 16-19.

Can also carry out physical analysis to structure by for example technology such as nucleus magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, and the supposition contact amino acid whose sudden change in site in conjunction with to IL-22RA aglucon agonist further characterizes the IL-22RA binding domains.Referring to for example deVos et al., Science 255:306 (1992), Smith et al., J.Mol.Biol.224:899 (1992), and Wlodaver et al., FEBS Lett.309:59 (1992).

The invention still further relates to the IL-22RA composition of chemically modified, wherein the IL-22RA polypeptide links to each other with polymkeric substance.Illustrative IL-22RA polypeptide is the polypeptide of solubility, and they lack functional membrane spaning domain, for example the polypeptide of being made up of amino-acid residue shown in the SEQ ID NO:3.Common described polymkeric substance is water miscible, and the IL-22RA conjugate for example in the physiological environment, can not precipitate in aqueous environments like this.The example of suitable polymer blend is the polymkeric substance that has an active group through modifying, and for example active ester is used for acidylate, and perhaps aldehyde is used for alkylation.Degree that like this can controlled polymerization.The example of active aldehydes is the polyoxyethylene glycol propionic aldehyde, perhaps single-(C1-C10) alkoxyl group, the perhaps derivative of its aryloxy (referring to for example Harris, et al., United States Patent (USP) numbering 5,252,714).Polymkeric substance can be a branch or not branched.And, can use polymeric blends to prepare the IL-22RA conjugate.

The IL-22RA conjugate that is used for the treatment of can comprise acceptable water-soluble polymers composition on the medicine.Suitable water-soluble polymers comprises polyoxyethylene glycol (PEG), mono methoxy PEG, list-(C1-C10) alkoxyl group PEG, aryloxy PEG, gather-(N-vinyl pyrrolidone) PEG, tresyl mono methoxy PEG, PEG propionic aldehyde, double amber imide base carbonic ether PEG, propylene glycol homopolymer, propylene oxide/ethylene oxide copolymer, polyoxy ethylization polyvalent alcohol (for example glycerine), polyvinyl alcohol, dextran, Mierocrystalline cellulose or other polymkeric substance based on class sugar.The molecular weight of suitable PEG may be from about 600 to about 60000, for example comprises 5,000,12,000,20,000 and 25,000.The IL-22RA conjugate can also comprise the mixture of this water-soluble polymers.

The polyalkylene oxide part that an example of IL-22RA conjugate comprises the IL-22RA part and is connected IL-22RA part N end.PEG is a kind of suitable polyalkylene oxide.As illustration, can modify IL-22RA with PEG, this process is called as " PEGization ".The PEGization of IL-22RA can be undertaken (referring to for example EP 0,154 316 by any PEGization reaction known in the art, Delgado et al., Critical Reviews in Therapeutic DrugCarrier Systems 9:249 (1992), Duncan and Spreafico, Clin.Phalmacokinet.27:290 (1994), and Francis et al., Int J Hematol68:1 (1998)).For example, can use reactive peg molecule undertaken PEGization by acylation reaction or alkylation reaction.In another approach, form the IL-22RA conjugate by condensation activatory PEG, wherein the joint that is activated of the terminal hydroxyl of PEG or amino group replaces (referring to for example Karasiewicz et al., United States Patent (USP) numbering 5,382,657).

The PEGization of being undertaken by acidylate need make the active ester analog derivative of PEG and IL-22RA polypeptide react usually.The example of activatory PEG ester is the ester that PEG and N-hydroxy-succinamide form.Just as used herein, term " acidylate " is included in the connection of the following type between IL-22RA and the water-soluble polymers: acid amides, ammonia (base) manthanoate, urethane or the like.The method for preparing the IL-22RA of PEGization by acidylate comprises following step usually: (a) make IL-22RA polypeptide and PEG (for example active ester of PEG aldehydes derivative) reaction under one or more a plurality of PEG group and the condition that IL-22RA links to each other and (b) obtain reaction product making.Usually, determine the optimum reaction condition of acylation reaction according to known parameter and required result.For example, the ratio of PEG: IL-22RA is big more, and the per-cent of the IL-22RA product of poly PEGization is high more.

PEGization product by acylation reaction is the IL-22RA product of poly-PEGization normally, wherein Methionin ε amino by the acyl group linking group by PEGization.A kind of connection example is an acid amides.Usually IL-22RA at least 95% coverlet that is produced-, two-or three-PEGization, although can form some IL-22RA with higher degree PEGization according to reaction conditions.The purification process of use standard, for example dialysis, ultrafiltration, ion exchange chromatography, affinity chromatography or the like can be separated the IL-22RA of PEGization with link coupled IL-22RA polypeptide not.

The PEGization of being undertaken by alkylation is usually directed to make under the condition that reductive agent exists the terminal aldehyde radical derivative of PEG and IL-22RA to react.The PEG group can pass through-CH ₂-NH group is connected on the polypeptide.

And, also can with in this area and method described herein make anti-IL-22 antibody of the present invention or antibody fragment PEGization.

The derivatization that is undertaken by standard reductive alkylation produce single PEGization product utilization the differential responses of the dissimilar primary amino that can be used for derivatize.Usually be reflected under the pH of the pKa difference between the α amino that can utilize proteinic Methionin ε amino group and N end residue and carry out.By this selective derivatizationization, the water-soluble polymers that control contains active group (for example aldehyde) connects with proteinic.Mainly occur in proteinic N end with the coupling of polymkeric substance, and to for example amino not obviously change of side chain of Methionin of other active groups.The invention provides the single polymer conjugate of IL-22RA of homogeneous basically.

Producing basically, the standard reductive alkylation reaction of the single polymer conjugate of IL-22RA of homogeneous can comprise following step: (a) under the condition of standard reductive alkylation, under the pH condition of the aminoterminal α amino group generation selective modification that is suitable for making IL-22RA, IL-22RA polypeptide and reactive PEG are reacted, and (b) obtain reaction product.The reductive agent that uses in standard reductive alkylation should be stablized in aqueous solution, and can a western Fu Shi alkali that reduction forms in the initiating process of standard reductive alkylation.Illustrative reductive agent comprises sodium borohydride, sodium cyanoborohydride, dimethylin borine, trimethylamine groups borine and pyridine borine.

For single polymkeric substance IL-22RA conjugate group of homogeneous basically, the condition of standard reductive alkylation reaction is the condition that this water miscible polymer moieties is connected with the N end generation selectivity of IL-22RA.Between the α amino group of such reaction conditions general requirement lysine amino group and N end pKa difference is arranged.PH also influences the polymer and the proteinic ratio of use.Usually,, need polymer excessive greatly,, then need many more polymers to reach optimal conditions because the α-group activity of N end is low more to protein if pH is lower.If pH is higher, then polymer: IL-22RA do not need too high because there is the more activated group can be for utilizing.Usually, pH is in the scope of the scope of 3-9 or 3-6.Can use this method preparation to comprise equal dimer, heterodimer or the polymer soluble receptors conjugate of IL-22RA.

The factor that another one will be considered is the molecular weight of water soluble polymerizer.Generally speaking, the molecular weight of polymer is big more, and the number that then may be connected to the polymer molecule on the protein is just few more.For the PEGization reaction, common molecular weight is to arrive between about 100kDa at 2kDa, and approximately 5kDa is between about 50kDa, and perhaps approximately 12kDa arrives between about 25kDa.The molecule ratio of water soluble polymerizer and IL-22RA generally is the scope at 1: 1 to 100: 1.Usually for many PEGization reactions, the molar ratio of water soluble polymerizer and IL-22RA is between 1: 1 to 20: 1, and for single PEGization reaction, ratio was at 1: 1 to 5: 1.

The general method that is used to prepare the conjugate that comprises polypeptide and water soluble polymerizer part is known in the art.Referring to for example Karasiewicz et al., United States Patent (USP) numbering 5,382,657, Greenwald et al., United States Patent (USP) numbering 5,738,846, Nieforthet al., Clin.Pharmacol.Ther.59:636 (1996), Monkarsh et al., Anal.Biochem.247:434 (1997)).This method can be used to prepare the equal dimerization that comprises IL-22RA, assorted dimerization or poly soluble receptors conjugate.

This patent relates to the composition that comprises peptide or polypeptide (soluble receptors for example described herein or antibody).This composition can also comprise carrier.Carrier can be conventional organic or inorganic carrier.The example of carrier comprises water, buffered soln, alcohol, propylene glycol, macrogol, sesame oil, Semen Maydis oil or the like.

7.IL-22RA the separation of polypeptide

With regard to the macromole that pollutes particularly with regard to other protein and the nucleic acid, polypeptide of the present invention can be purified to about at least 80% purity, about at least 90% purity, about at least 95% purity, for example 96%, 97%, 98% or greater than 99% purity, and do not have the factor of infectious and pyrogen.Polypeptide of the present invention also can be purified to the state of pharmaceutical purity, promptly greater than 99.9% purity.In some preparation, the polypeptide of purifying does not almost have other polypeptide, particularly zoogenous other polypeptide.

Can use fractional separation and/or conventional purification process obtain from the IL-22RA of natural origin (for example the people is tissue-derived) purifying prepared product, synthetic IL-22RA polypeptide and from recombinant host cell purifying recombinant il-2 2RA polypeptide and merge the IL-22RA polypeptide.Usually, can use ammonium sulfate precipitation and acid or chaotropic agent to extract, sample is carried out fractional separation.Exemplary purification step can comprise hydroxyapatite, size exclusion chromatography, FPLC and RPHPLC (reversed-phase high-performance liquid chromatography).Suitable chromatography media comprises derivatize dextran, agarose, Mierocrystalline cellulose, polyacrylamide, speciality silica gel or the like.PEI, DEAE, QAE and Q derivative suit.Exemplary chromatography media comprises use phenyl, butyl or octyl group group deutero-medium, Phenyl-Sepharose FF (Pharmacia) for example, Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) or the like; Or acrylic resin Amberchrom CG 71 (Toso Haas) or the like for example.Suitable solid support comprises granulated glass sphere, the resin based on silica gel, celluosic resin, sepharose 4B, Sepharose pearl, polystyrene bead, cross-linked polyacrylamide resin or the like, and they are insoluble under the condition that is used.Can use active group to modify these upholders, these active groups can allow protein to pass through in amino group, carboxylic group, mercapto groups, oh group and/or the sugar moieties connection.

The example of coupling chemical reaction comprises carboxyl and the aminoderivative in cyanogen bromide-activated, N-hydroxy-succinamide activation, epoxide activation, sulfydryl activation, hydrazides activation and the carbodiimide coupling chemical reaction.The solid-phase media of these and other is well-known and widely-used in this area, can obtain from suppliers.Be used for that polypeptide separates and the selection of the ad hoc approach of purifying is conventional design problem, can be partly determine by the character of selected upholder.Referring to for example Affinity Chromatography:Principles ﹠amp; Methods (Pharmacia LKBBiotechnology 1988), and Doonan, Protein PurificationProtocols (The Humana Press 1996).

Other changes in IL-22RA separation and purifying can be designed by those skilled in the art.For example, the IL-22RA antibody that obtains according to following description can be used for separating amounts of protein by immunoaffinity purification.

Polypeptide of the present invention can also be separated by utilizing some special nature.For example, can use immobilized metal absorption (IMAC) chromatography purification to be rich in the albumen of Histidine, comprise that those comprise the histidine-tagged albumen of poly.In brief, at first use divalent-metal ion load gel, form inner complex (Sulkowski, Trends in Biochem.3:1 (1985)).The protein that is rich in Histidine can be adsorbed on this matrix with different affinities according to employed metal ion, then with competitive wash-out, reduce pH or use strong sequestrant wash-out.Other purification process comprises the albumen that uses phytohemagglutinin affinity chromatography and ion exchange chromatography purifying glycosylation (M.Deutscher, (ed.), Meth.Enzymol.182:529 (1990)).In other embodiments of the present invention, can the establishing target polypeptide and affinity tag (for example maltose binding protein, immunoglobulin domains) form fusion polypeptide, help purifying.And, can also utilize the aglucon of IL-22RA extracellular domain to carry out purifying in conjunction with character, for example purifying comprises the soluble receptors of IL-22RA; For example use affinity chromatography, wherein the IL-22 aglucon is attached on the chromatography column, and combination will take place the acceptor that comprises IL-22RA, uses the above-mentioned acceptor of chromatography method wash-out of standard then.

As mentioned above, IL-22RA polypeptide or its fragment can also be prepared by chemosynthesis.The IL-22RA polypeptide can be monomer or polymer, glycosylation or not glycosylation, PEGization or not PEGization, contain or do not have an initial methionine residues.

8. the preparation of anti-IL-22RA protein antibodies

At the antibody of IL-22RA can be by for example using the IL-22RA expression vector product or obtain as antigen by the isolating IL-22RA of natural origin.Useful especially anti-IL-22RA antibody and IL-22RA " specific combination ".If it is at least a that antibody shows in following two kinds of character, think that then antibody is specific combination: (1) antibody with in conjunction with active threshold level in conjunction with IL-22RA, and the related polypeptide of (2) antibody and IL-22RA does not have significant cross reaction.

With regard to first feature, if the binding affinity (K of antibody and IL-22RA polypeptide, peptide or epi-position _a) be 10 ⁶M ^-1Perhaps higher, preferably 10 ⁷M ^-1Perhaps higher, be more preferably 10 ⁸M ^-1Perhaps higher, most preferably be 10 ⁹M ^-1Perhaps higher, then this antibody is specific combination.The binding affinity of antibody can be analyzed (Scatchard, Ann.NY Acad.Sci.51:660 (1949)) by those of ordinary skills by for example Scatchard at an easy rate and measure.With regard to second feature, if use the Western engram analysis method of standard can detect IL-22RA, but can not detect present known polypeptide, then there is not significant cross reaction between this antibody and the relevant peptide molecule.The example of known related polypeptide comprises known cytokine receptor.

Anti-IL-22RA antibody can use the peptide and the polypeptide that carry antigenicity IL-22RA epi-position to be prepared.The peptide of antigenic epitopes and at least 9 or the about 15-30 amino acid that polypeptide contains among the SEQ ID NO:3 or comprises in another aminoacid sequence described herein of carrying of the present invention.But, the peptide or the polypeptide that comprise more a high proportion of aminoacid sequence of the present invention, contain 30-50 amino acid in the complete amino acid sequence of polypeptide of the present invention or contain any length part of polypeptide of the present invention even comprise the peptide and the polypeptide of the whole length amino acid sequence of polypeptide of the present invention, also can be used to induce and IL-22RA bonded antibody.The aminoacid sequence that it is desirable to carry the peptide of epi-position is chosen to make peptide to have significant solvability (promptly this sequence comprises hydrophilic relatively residue, and avoids hydrophobic residue usually) in aqueous solvent.And it also is desirable that the aminoacid sequence that contains proline residue is used for Antibody Preparation.

As illustration, use Jameson-Wolf method (Jameson and Wolf, CABIOS4:181, (1988)), by PROTEAN program (version 3 the .14) (DNASTAR of LASERGENE; Madison WI) implements, and determines the potential antigenic site among the IL-22RA.In analysis, use default parameters.

The Jameson-Wolf method is predicted potential antigenicity determinant by 6 main protein structure prediction sub-routines are combined.In brief, at first use Hopp-Woods method (Hopp et al., Proc.Nat ' l Acad.Sci.USA 78:3824 (1981)) to determine that representative has the aminoacid sequence (parameter: average 7 residues) in maximum local hydrophilic zone.Second step, method (Emini et al., J.Virology 55:836 (the 1985)) gauging surface probability (parameter: surperficial decision threshold (0.6)=1) of use Emini.The 3rd, use Karplus-Schultz method (Karplus and Schultz, Naturwissenschaften 72:212 (1985)) prediction skeleton chain flexibility (parameter: flexible threshold value (0.2)=1).In the 4th and the 5th step of analyzing, data are used secondary structure prediction, wherein use the method (Chou of Chou-Fasman, " Prediction of ProteinStructural Classes from Amino Acid Composition; " Prediction ofProtein Structure and the Principles of Protein Conformation, Fasman (ed.), pages 549-586 (Plenum Press 1990)), and Garnier-Robson, Garnier et al., J.Mol.Biol.120:97 (1978) (Chou-Fasman parameter: conformation form=64 albumen; α region threshold=103; β region threshold=105; Garnier-Robson parameter: α and β decision constant=0).In the 6th sub-routine, flexible parameter and wetting ability/solvent accessibility factor are combined, determine surface profile numerical value, called after " antigenicity index ".At last, the antigenicity index is used peak broadening function, by adding 20%, 40%, 60%, or 80% corresponding peaks, makes main surperficial peak broadening, to consider because the extra free energy that surf zone produces with respect to the migration of interior region.But not with this computing application in any main peaks that is positioned at the spiral intra-zone, as if reason is that the flexibility in spiral zone is lower.

The result of this analysis shows that the following aminoacid sequence among the SEQ ID NO:3 can provide suitable antigenic peptide: can use Hopp/Woods wetting ability synoptic chart determine in SEQ ID NO:3, to have the zone of the highest antigenicity potentiality (Hopp et al., Proc.Natl.Acad. Sci.78: 3824-3828,1981; Hopp, J.Immun.Meth. 88: 1-18,1986 andTriquier et al., Protein Engineering 11: 153-169,1998).This synoptic chart is based on the six residue windows that slide.Ignore G, S and T residue and the H that exposes, Y and W residue that embedding is lived.And, use by for example DNASTAR Protean program (DNASTAR, Inc., Madison according to the Jameson-Wolf curve, WI) the preferred antigenic epitopes of the conduct of the IL-22RA antigenic epitopes among Yu Ce the SEQ ID NO:3, and can determine by those skilled in the art.This antigenic epitopes comprises that 1 (Pro) is to 6 (Asp) number amino-acid residue among (1) SEQ ID NO:3; (2) among the SEQ ID NO:3 26 (Ser) to 32 (Pro) number amino-acid residue; (3) among the SEQ ID NO:3 41 (Lys) to 47 (Asp) number amino-acid residue; (4) among the SEQ ID NO:3 49 (Val) to 62 (Cys) number amino-acid residue; (5) among the SEQ ID NO:3 41 (Lys) to 62 (Cys) number amino-acid residue; (6) among the SEQ ID NO:3 84 (Ala) to 97 (Ser) number amino-acid residue; (7) among the SEQ ID NO:3 103 (Thr) to 108 (Asp) number amino-acid residue; (8) among the SEQ ID NO:3 130 (Arg) to 135 (His) number amino-acid residue; (9) among the SEQ IDNO:3 164 (Gly) to 166 (Lys) number amino-acid residue; (10) among the SEQ ID NO:3 175 (Tyr) to 179 (Glu) number amino-acid residue; (11) among the SEQ ID NO:3 193 (Lys) to 196 (Ala) number amino-acid residue; (12) among the SEQ ID NO:3 203 (Lys) to 209 (Thr) number amino-acid residue.The present invention relates to use any generation among the antigenic polypeptide 1-12 at the antibody of IL-22RA, perhaps be used for screening or identify neutralizing monoclonal antibody of the present invention as instrument.The invention still further relates to and comprise polypeptide at least a among the antigenic peptide 1-10.The present invention relates to use any antigenic peptide described herein or epi-position to produce antibody at IL-22RA, and identify and screen anti-IL-22RA mono-clonal neutrality antibody, described antibody can in conjunction with, blocking-up, inhibition, reduction, antagonism or in and the activity of IL-22 and IL-20 (separately or together).

And, suitable antigen also comprises comprise top disclosed IL-22RA cytokine binding domains or extracellular domain and another I class or the II type cytokines extracellular domain IL-22RA polypeptide of--forming assorted dimerization of solubility IL-22RA or multimer polypeptide as those--, IL-22RA/CRF2-4 as solubility, IL-22RA/zcytor11, IL-22RA/zcytor7 or the like.

Can use the well-known method preparation of those skilled in that art at recombinant il-2 2RA albumen or from the polyclonal antibody of the isolating IL-22RA of natural origin.Referring to for example Green et al., " Production of Polyclonal Antisera; " inImmunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press1992), and Williams et al., " Expression of foreign proteinsin E.coli using plasmid vectors and purification of specificpolyclonal antibodies; " in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford UniversityPress 1995).The immunogenicity of IL-22RA polypeptide can by use adjuvant for example aluminium (aluminium hydroxide) or Fu Shi fully or Freund improve.The polypeptide useful for immunity also comprises fusion polypeptide, for example IL-22RA or its part and immunoglobulin polypeptides or the fusion polypeptide that forms with maltose binding protein.Polypeptide immunogen can be the molecule of total length or be its part.If polypeptide portion is " a haptens sample ", then such part can linking together be used for immunity with macromolecular carrier (for example key hole  hemocyanin KLH, bovine serum albumin BSA or Toxoid,tetanus) expediently.

Although prepare polyclonal antibody usually in animal such as horse, ox, dog, chicken, rat, mouse, rabbit, cavy, goat or sheep, anti-IL-22RA antibody of the present invention can also be derived from class people's primate antibody.In baboon preparation diagnosis with and treatment on the general technology of useful antibody can be at for example Goldenberg et al., international monopoly publication numbering WO91/11465, and at Losman et al., Int.J.Cancer 46:310 finds in (1990).

Perhaps, can produce monoclonal anti-IL-22RA antibody.Can obtain rodents monoclonal antibody (referring to for example Kohler et al. by the known method of those skilled in that art at specific antigen, Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols in Immunology, Vol.1, pages 2.5.1-2.6.7 (John Wiley ﹠amp; Sons 1991) [" Coligan "], Picksley et al., " Production of monoclonal antibodies against proteinsexpressed in E.coli; " DNA Cloning 2:Expression Systems, 2ndEdition, Glover et al. (eds.), page 93 (Oxford University Press1995)).

In brief, can obtain monoclonal antibody by following steps: to injected in mice comprise the IL-22RA gene product composition, confirm production of antibodies by taking out serum sample, take out spleen obtain bone-marrow-derived lymphocyte, bone-marrow-derived lymphocyte is merged with the myeloma cell produce hybridoma, clone hybridization knurl, select generation at the positive colony of this antigenic antibody, cultivate generation at the clone of this antigenic antibody and from the hybridoma culture separation antibody.

In addition, anti-IL-22RA antibody of the present invention can the derived from human monoclonal antibody.Human monoclonal antibodies can obtain from attacking the transgenic mice that produces human antibodies specific by genetically engineered operation and in response to antigen.In this technology, the element of people's heavy chain and light chain gene seat is introduced in by in the embryonic stem cell line deutero-mouse species, and the endogenous heavy chain and the light chain gene seat of described embryonic stem cell line are destroyed by target.This transgenic mice can synthesize special people's antibody for the human antigen, can produce the hybridoma of secretion people antibody with described mouse.From transgenic mice, obtain the method for people's antibody at for example Green et al., Nature Genet.7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Tayloret al., describe to some extent among the Int.Immun.6:579 (1994).

Can from the hybridoma culture, separate and be purified into monoclonal antibody by multiple proven technique method.This isolation technique comprises uses affinity chromatography, size exclusion chromatography and ion exchange chromatography that a-protein Sepharose carries out (referring to for example Coliganpp2.7.1-2.7.12 and pp2.9.1-2.9.3; Baines et al., " Purificationof Immunoglobulin G (IgG), " Methods in Molecular Biology, Vol.10, pages 79-104 (The Humana Press, Inc.1992)).

For special purposes, may need to prepare the fragment of anti-IL-22RA antibody.Such antibody fragment can obtain by the proteolysis of for example antibody.Can obtain antibody fragment with stomach en-or the whole antibody of papain digestion by ordinary method.As illustration, can obtain antibody fragment by using the stomach en-enzyme to cut, produce the fragment of 5S, be called F (ab ') ₂This fragment can produce the Fab ' unit price fragment of 3.5S further with the cutting of sulphur reductive agent.Alternatively, cleavage reaction also can use the blocking groups of sulfydryl to carry out, and described sulfydryl is because the cut-out of disulfide linkage produces.Perhaps, use the stomach en-cutting directly to produce two monovalent Fab fragments and Fc fragment.These methods are at for example Goldenberg, United States Patent (USP) 4,331,647, Nisonoff et al., Arch Biochem.Biophys.89:230 (1960), Porter, Biochem.J.73:119 (1959), Edelman et al., inMethods in Enzymology Vol.1, page 422 (Academic Press 1967), and Coligan, describe to some extent among pp 2.8.1-2.8.10 and the pp 2.10.-2.10.4.

As long as fragment can combine with the antigen of being discerned by complete antibody, just can use the method for other cutting antibody, for example with heavy chain separately with form monovalent gently-heavy chain fragment, segmental further cutting and other enzymatics, chemistry or genetic technique.

For example, the Fv fragment comprises V _HAnd V _LThe combination of chain.This combination can be non-covalent, as Inbar et al., described in the Proc.Nat ' l Acad.Sci.USA 69:2659 (1972).Perhaps, can make the variable region chain couple together (referring to for example, Sandhu, Crit.Rev.Biotech.l2:437 (1992)) by intermolecular disulfide bond or by chemical substance such as glutaraldehyde crosslinked.

The Fv fragment may comprise the V that is connected by peptide linker _HAnd V _LChain.The antigen-binding proteins of these strands (scFv) is to comprise coding V by structure _HAnd V _LThe structure gene of the dna sequence dna of structural domain prepares, this V _HAnd V _LConnect by oligonucleotide between the structural domain encoding sequence.Structure gene is inserted in the expression vector, then expression vector is incorporated into host cell, for example in the intestinal bacteria.The synthetic single chain polypeptide of the host cell of reorganization with linking peptide (between two V structural domains, putting up a bridge).The method for preparing scFvs is at for example Whitlow et al., Methods:A Companion to Methods in Enzymology 2:97 (1991) (also sees also Bird et al., Science 242:423 (1988), Ladner et al., United States Patent (USP) numbering 4,946,778, Pack et al., Bio/Technology 11:1271 (1993), and Sandhu, see before) in describe to some extent.

As illustration, make lymphocyte at the external IL-22RA of being exposed to polypeptide, the antibody display libraries in screening phage or the similar substrates (for example by using the IL-22RA protein or the peptide of immobilized or mark) can obtain scFV.Can be illustrated in the encoding gene that random peptide library on (phage display) on the phage or bacterium such as the intestinal bacteria obtains having the polypeptide of potential IL-22RA polypeptide binding domains by screening.The nucleotide sequence of these polypeptide of encoding can obtain with several different methods, for example by random mutagenesis with polynucleotide are synthetic at random.Can use these interactional peptides of peptide display libraries screening and known target (can be protein or polypeptide, for example aglucon or acceptor, biology or synthetic macromolecule, organic or inorganic substance) at random.Preparation and the technology of screening this display libraries of peptide at random are known (Ladner et al., United States Patent (USP) numbering 5,223 in this area, 409, Ladner et al., United States Patent (USP) numbering 4,946,778, Ladner et al., United States Patent (USP) numbering 5,403,484, Ladner et al., United States Patent (USP) numbering 5,571,698, and Kay et al., PhageDisplay of Peptides and Proteins (Academic Press, Inc.1996)), and the test kit that commercial random peptide library is arranged and be used to screen this library for example can be from Clontech Laboratories, Inc. (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ) etc. producer obtains.Can use IL-22RA sequence screening disclosed herein peptide display libraries at random, to identify and IL-22RA bonded protein.

The antibody fragment of another form is the peptide of the single complementary determining region of coding (CDR).CDR peptide (" atom ") can be by making up the coding target antibody the gene of CDR obtain.For example, by using the RNA synthetic variable region of polymerase chain reaction from antibody produced cell, can prepare such gene (referring to for example Larrick et al., Methods:ACompanion to Methods in Enzymology 2:106 (1991), Courtenay-Luck, " Genetic Manipulation of Monoclonal Antibodies; " MonoclonalAntibodies:Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995), and Ward et al., " Genetic Manipulation and Expression ofAntibodies; " Monoclonal Antibodies:Principles andApplications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc.1995)).

Perhaps, can obtain anti-IL-22RA antibody from " peopleization " monoclonal antibody.By the complementary determining region of mouse immuning ball protein light chain and variable region of heavy chain being transferred in people's the variable domains, can obtain the monoclonal antibody of peopleization.In the framework region of the corresponding antibody of mouse, replace typical people's antibody residue then.Use the antibody component of derived from human monoclonal antibody to avoid the possible problem relevant with the immunogenicity of mouse constant region.The general technology of clone's mouse immuning ball protein variable domains is described among the Proc.Nat ' l Acad.Sci.USA 86:3833 (1989) to some extent at for example Orlandi et al..The technology of preparation peopleization monoclonal antibody is at for example Jones et al., Nature 321:522 (1986), Carter et al., Proc.Nat ' l Acad.Sci.USA 89:4285 (1992), Sandhu, Crit.Rev.Biotech.12:437 (1992), Singer et al., J.Immun.150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols (Humana Press, Inc.1995), Kelley, " Engineering Therapeutic Antibodies; " ProteinEngineering:Principles and Practice, Cleland et al. (eds.), pages 399-434 (John Wiley ﹠amp; Sons, Inc.1996), and Queen et al., U.S.Patent No.5 describes in 693,762 (1997) to some extent.

And anti-IL-22RA antibody of the present invention or antibody fragment can use the method and the method PEGization described herein of this area.

Can with anti-IL-22RA antibody or antibody fragment immune animal, prepare the polyclone antiidiotypic antibody thus by using standard techniques.Referring to for example Green et al., " Production of Polyclonal Antisera, " Methods In MolecularBiology:Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana Press 1992).Also can be referring to Coligan pp2.4.1-2.4.7.Perhaps, can be as mentioned above with anti-IL-22RA antibody or antibody fragment as immunogen preparing monoclonal anti idiotype antibody.Alternatively, can use technology described above to prepare the antiidiotypic antibody of peopleization or class people's primates antiidiotypic antibody.The method for preparing antiidiotypic antibody is at for example Irie, and the U.S. United States Patent (USP) numbers 5,208,146, Greene, et.al., United States Patent (USP) numbering 5,637,677, and Varthakavi and Minocha, describe to some extent among the J.Gen.Virol.77:1875 (1996).

Can will resist IL-22RA antibody and detectable label coupling to form anti-IL-22RA immunoconjugates.Suitable detectable label comprises, for example, and radio isotope, fluorescent mark, chemiluminescent labeling, enzyme labelling, bioluminescence marker or Radioactive colloidal gold.Preparation and the method that detects the immunoconjugates of this detectable label are well-known for those of ordinary skills, and more detailed description is arranged below.

Detectable label can be can be by the radio isotope of autography detection.Have for the useful especially radio isotope of purpose of the present invention ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-IL-22RA immunoconjugates can also be used the fluorescent chemicals mark.By light that immunoconjugates is exposed to suitable wavelength and the fluorescence that detects generation, can detect the existence of fluorescent-labeled antibody.The fluorescent mark compound comprises fluorescein isothiocyanate, rhodamine, phycoerythrobilin, algocyan, allophycocyanin, o-phthalaldehyde(OPA) and fluorescamine.

Perhaps, can carry out detectable mark by antibody component and chemiluminescence compound coupling are resisted the IL-22RA immunoconjugates.By detecting the luminous existence that can detect the immunoconjugates of chemoluminescence label that produces in the chemical reaction process.The example of chemiluminescent labeling compound comprises luminol,3-aminophthalic acid cyclic hydrazide, different luminol,3-aminophthalic acid cyclic hydrazide, fragrant acridinium ester, imidazoles, acridine  salt and barkite.

Similarly, can use bioluminescent compound mark anti-IL-22RA immunoconjugates of the present invention.Noclilucence is a class chemoluminescence of finding in biosystem, and wherein catalytic protein has improved the efficient of chemiluminescence reaction.Determine the existence of bioluminescent protein by detecting luminous existence.The bioluminescent compound that can be used for mark comprises fluorescein, luciferase and aequorin.

Perhaps, can will resist the IL-22RA antibody component to link to each other and resist the IL-22RA immunoconjugates and carry out detectable mark with enzyme.When anti-IL22RA-enzyme conjugate was hatched in the presence of suitable substrate, the enzyme part reacted with substrate, the chemical part that generation can detect by the method for for example spectrophotometry, fluorescent method or range estimation.The example that can be used for the polyspecific immunoconjugates is carried out the enzyme of detectable label comprises beta-galactosidase enzymes, glucose oxidase, peroxysome and alkaline phosphatase.

Those skilled in the art know to have and can be used for other suitable marks of the present invention.Mark part can use standard technique known in the art to finish with combining of anti-IL-22RA antibody.The typical method of this respect is learned at Kennedy et al., Clin.Chim.Acta 70:1 (1976), Schurs et al., Clin.Chim.Acta 81:1 (1977), Shih etal., Int ' l J.Cancer 46:1101 (1990), Stein et al., Cancer Res.50:1330 (1990), and describe to some extent among the Coligan (ditto).

And, can use and avidin, accessibility that the immunochemistry that improves the anti-IL-22RA antibody of streptavidin and vitamin H link coupled detects and versatility are (referring to for example Wilchek et al. (eds.), " Avidin-Biotin Technology; " MethodsIn Enzymology, Vol.184 (Academic Press 1990), and Bayer et al., " Immunochemical Applications of Avidin-Biotin Technology; " Methods In Molecular Biology, Vol.10, Manson (ed.), and pages149-162 (The Humana Press, Inc.1992).

The method of carrying out immunodetection is very sophisticated.Referring to for example Cook and Self, " Monoclonal Antibodies in Diagnostic Immunoassays; " Monoclonal Antibodies:Production, Engineering, and ClinicalApplication, Ritter and Ladyman (eds.), pages 180-208, (Cambridge University Press, 1995), Perry, " The Role ofMonoclonal Antibodies in the Advancement of ImmunoassayTechnology; " Monoclonal Antibodies:Principles andApplications, Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc.1995), and Diamandis, and Immunoassay (AcademicPress, Inc.1996).

The invention still further relates to for IL-22RA genetic expression and carry out the test kit that immunodiagnosis detects.This test kit comprises at least one container, wherein comprises anti-IL-22RA antibody or antibody fragment.Can also comprise another container in the test kit, wherein comprise the reagent that one or more can indicate IL-22RA antibody or antibody fragment to exist.The example of this indicator comprises detectable mark, for example radio isotope, fluorescent mark, chemiluminescent labeling, enzyme labelling, bioluminescence marker or Radioactive colloidal gold or the like.It is the approach of IL-22RA antibody or antibody fragment that test kit can also comprise what inform that the user detects that IL-22RA albumen uses.For example, can illustrate that antibody or antibody fragment that test kit comprises can be used to detect IL-22RA in the written specification sheets.This written material can be printed directly on the container, and perhaps the form with package insert provides.

9. use anti-IL-22RA antibody antagonism IL-22RA and IL-22 combine or with the two combine of IL-20 and IL-22

The other technology that produces or be chosen in useful antibody herein comprises makes lymphocyte in external solubility IL-22RA receptor polypeptides or its fragment (for example antigenic epitopes) of being exposed to, and the antibody display libraries in screening phage or the similar substrates (for example by using solubility IL-22RA receptor polypeptides or its fragment, for example antigenic epitopes of immobilized or mark).Can by show (phage display) on the screening phage or bacterium such as intestinal bacteria on the random peptide library showed obtain having the encoding gene of the polypeptide (for example solubility IL-22RA receptor polypeptides or its fragment, for example antigenic epitopes) of potential binding domains.The nucleotide sequence of this polypeptide of encoding can obtain in several ways, for example by random mutagenesis with polynucleotide are synthetic at random.Can use these interactional peptides of peptide display libraries screening and known target (can be protein or polypeptide, for example aglucon or acceptor, biology or synthetic macromolecule, organic or inorganic substance) at random.Preparation and the technology of screening this display libraries of peptide at random are known (Ladner et al., United States Patent (USP) numbering 5,223 in this area, 409, Ladner et al., United States Patent (USP) numbering 4,946,778, Ladner etal., United States Patent (USP) numbering 5,403,484, Ladner et al., United States Patent (USP) numbering 5,571,698), and commercial random peptide library is arranged and be used to screen the test kit in this library can be for utilizing, for example can be from Clontech Laboratories, Inc. (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, and Inc. (Beverly, MA), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ) etc. producer obtains.Can use solubility IL-22RA receptor polypeptides or its fragment (antigenic epitopes peptide sequence for example disclosed herein) to screen peptide display libraries at random, with identify can with the receptor polypeptides bonded protein that comprises IL-22RA.These can be used for adding label, being used for separating the homologue polypeptide by affinity purification to cell with the soluble receptors polypeptide that comprises IL-22RA interactional " in conjunction with polypeptide "; They can be directly or indirectly and medicine, toxin, radionuclide or the like coupling.These can also be used in some analytical procedures in conjunction with polypeptide, for example be used for screening expression library and neutralization active (for example combination, blocking-up, inhibition, reduction, antagonism or and IL-22 aglucon and acceptor between interaction, the combining of perhaps viral and acceptor) analytical procedure.Can also be used in the diagnostic detection in conjunction with polypeptide, be used to measure the cyclical level of the soluble receptors polypeptide that comprises IL-22RA; Be used to detect or quantitatively as the solubility of potential pathology or stigmata or the acceptor of the insoluble IL-22RA of comprising.These can also be used for blocking-up in the external and body or suppress solubility or combination of film mating type IL-22RA monomeric acceptor or the equal dimerization of IL-22RA, assorted dimerization or multimer polypeptide (for example with aglucon combine) and signal transduction in conjunction with polypeptide as " antagonist ".Equally, these can be used as anti-IL-22RA monomeric acceptor or the equal dimerization of anti-IL-22RA, assorted dimerization or multimer polypeptide in conjunction with polypeptide, are used to suppress the two activity of IL-22 or IL-20 and IL-22, and receptor active or and combination of proteins.Antibody at natural receptor complex body generation of the present invention, and in conjunction with the antibody of IL-22RA-epi-position, with anti-IL-22RA neutralizing monoclonal antibody can be embodiment preferred, because they can act on IL-22RA more specifically, and can suppress IL-22 or IL-20 and IL-22 the two.And, the antagonism of antibody of the present invention and can under the condition that IL-20 or IL-22 exist and comprise the soluble receptors of IL-22RA to exist respectively, be used for IL-20 or IL-22 propagation, signal trapping, luciferase or in conjunction with testing in conjunction with active, and in other biological described herein or the biochemical measurement.

Can be used for suppressing in the body the two inflammatory effect of IL-20, IL-22 or IL-20 and IL-22 at the antibody of solubility IL-22RA receptor polypeptides (for example at SEQ ID NO:3 acceptor) or its fragment (for example antigenic epitopes), be used for the treatment of the inflammatory conditions that brings out by IL-20 and IL-22 at psoriasis, hereditary atopic dermatitis, inflammatory skin disease, endotoxemia, sacroiliitis, asthma, IBD, colitis, arthritic psoriasis, rheumatoid arthritis or other; Be used for adding label to the cell of expressing the IL-22RA acceptor; Be used for separating the receptor polypeptides that comprises IL-22RA of solubility by affinity purification; Be used for measuring the cyclical level of the soluble receptors polypeptide that comprises IL-22RA in diagnostic detection; Be used to detect or the quantitative soluble receptors that comprises IL-22RA as potential pathology or stigmata; Be used in the analytical procedure of using FACS; Be used to screen expression library; Be used to produce the antiidiotypic antibody that can be used as IL-22 or IL-20 agonist; And as neutrality antibody or antagonist, with the function of combination, blocking-up, inhibition, reduction or antagonism IL-22RA acceptor, perhaps combination in vitro and in vivo, blocking-up, inhibition, reduction, antagonism or in and the activity of IL-22 and/or I1-20 (perhaps separately or together).Suitable direct label or tag comprises radionuclide, enzyme, substrate, cofactor, vitamin H, inhibitor, fluorescent mark, chemiluminescent labeling, magnetic-particle or the like; Indirect label or mark may relate to use vitamin H-avidin or other complement/anti-complement to as media.Can also be directly or indirectly antibody of the present invention and medicine, toxin, radionuclide or the like be carried out coupling, and these conjugates are used for intravital diagnosis or therapeutic is used.And; can be in antibody or its fragment of external use, so that (for example Western trace or other mensuration known in the art) detects sex change or unmodified receptor polypeptides or its fragment that comprises IL-22RA in mensuration at the soluble receptors polypeptide that comprises IL-22RA.

Antibody at solubility IL-22RA acceptor or the equal dimerization of solubility IL-22RA, assorted dimerization or poly receptor polypeptides can be used for adding label and detecting these receptor expression levels to the cell of expressing corresponding acceptor, be used for affinity purification, in diagnostic detection, be used to measure the cyclical level of receptor polypeptides, be used for using the analytical procedure of fluorescence activated cell letter sorting art.And, can use bivalent antibody or antiidiotypic antibody as agonist, the effect of Simulation with I L-22RA aglucon, IL-22 or IL-20.

Antibody of the present invention directly or indirectly can also be carried out coupling with medicine, toxin, radionuclide or the like, these conjugates can be used for intravital diagnosis or treatment application.For example, the antibody of identification solubility IL-22RA acceptor or the equal dimerization of solubility IL-22RA, assorted dimerization or poly receptor polypeptides or can be used for identifying or handling the tissue or the organ of expressing corresponding anti-complement molecule (solubility or the film mating type acceptor that promptly comprise IL-22RA) in conjunction with polypeptide.More specifically, at the antibody of the soluble receptors polypeptide that comprises IL-22RA or its bioactive fragment or part can with detectable or cytotoxicity molecule coupling, and be delivered in the mammalian body with cell, tissue or the organ of expressing the acceptor (for example expressing the cancer of IL-22RA) that comprises IL-22RA.

But suitable detection molecules can be directly or indirectly with can adhere to for example " in conjunction with polypeptide " (comprising top disclosed binding peptide), antibody or its bioactive fragment or part in conjunction with the polypeptide of the receptor polypeptides that comprises IL-22RA mutually.Suitable can comprise radionuclide, enzyme, substrate, cofactor, vitamin H, inhibitor, fluorescent mark, chemiluminescent labeling, magnetic-particle or the like by detected molecule.Suitable cytotoxicity molecule can directly or indirectly link to each other with polypeptide or antibody, and comprise bacterium or plant poison (for example diphtheria toxin, Pseudomonas exotoxin, ricin, abrin or the like), and therapeutic radiation nucleic such as iodine 131, RE 188 or yttrium 90 (perhaps directly be connected, perhaps with indirect mode as being connected) by chelating moiety with polypeptide or antibody.Binding polypeptides or antibody can also with cytotoxic drug such as Zorubicin coupling.Indirect connection for detectable or cytotoxicity molecule, can be with detectable or cytotoxicity molecule and right member's coupling of complement/anti-complement, another member that complement/anti-complement is right is combined on this binding polypeptides or the antibody moiety.Vitamin H/streptavidin is that a kind of exemplary complement/anti-complement that is used for these purposes is right.

In the another one embodiment, binding polypeptides-toxin fusion rotein or antibody-toxin fusion rotein can be used for the target sexual cell or tissue suppresses or elimination (for example being used for the managing cancer cell or tissue).Perhaps, if binding polypeptides has a plurality of functional domains (promptly, activation domain or aglucon binding domains, add the target structural domain), the fusion rotein that then only comprises the target structural domain may just suitable molecule, cytotoxicity molecule or complement molecular guide target cell or types of organization that can be detected.Divide the period of the day from 11 p.m. to 1 a.m when the fusion rotein that only comprises the single structure territory comprises complement, can will resist complement molecule and detectable or cytotoxicity molecule coupling.Like this, this structural domain-complement molecule fusion protein has been represented a class targeting vector, and the cell/tissue specificity that can be used for general anti-complement-can detect/cytotoxicity molecular conjugate is delivered.

In another embodiment, if IL-22RA binding polypeptides-cytokine or anti-IL-22RA receptor antibody target excessive proliferated cell, then this IL-22RA binding polypeptides-cytokine or antibody-cytokine fusion protein can be used for strengthening killing and wounding (usually referring to Hornick et al. target tissue (for example spleen, pancreas, blood, lymph, colon and marrow cancer) in vivo Blood 89: 4437-47,1997).Described fusion rotein can make cytokine be targeted to required action site, and partial cytokine concentration is improved.But suitable anti-IL-22RA monomer, equal dimer, heterodimer or undesirable cell of polymer antibody target or tissue (being tumour or leukemia), the cytokine that is merged can mediate the effector cell target cell cracked is strengthened.The suitable cytokine that is used for this purpose comprises for example interleukin-22 and granulocyte-macrophage colony stimutaing factor (GM-CSF).

Perhaps, can use IL-22RA receptors bind polypeptide described herein or antibody fusion protein, the apoptosis pathway of regulating by direct stimulation IL-22RA acceptor improves the killing of body inner target tissue, and causes expressing the death of the excessive proliferated cell of the acceptor that comprises IL-22RA.

10. has the active polypeptide of IL-22RA or at the therepic use of the antibody of IL-22RA

Have the active aminoacid sequence of solubility IL-22RA and can be used for stoping IL-22RA aglucon and endogenous IL-22RA receptors bind by in conjunction with IL-22RA aglucon IL-20 and IL-22 (perhaps separately or together), thus the adjusting immunity system.Can also use IL-22RA antagonist (for example anti-IL-22RA antibody), regulate immunity system with combining of endogenous IL-22RA acceptor by suppressing the IL-22RA aglucon.Therefore, present invention includes study subject to the IL-22RA polypeptide that lacks capacity or excessive generation IL-22RA aglucon uses and has the active albumen of IL-22RA, polypeptide and peptide (for example solubility IL-22RA polypeptide, IL-22RA polypeptide fragment, IL-22RA analogue (for example anti-IL-22RA antiidiotypic antibody) and IL-22RA fusion rotein).Can also use the study subject of IL-22RA antagonist (for example anti-IL-22RA antibody) treatment excessive generation IL-22RA aglucon or IL-22RA.Suitable study subject comprises Mammals, for example the people.For example, this IL-22RA polypeptide and anti-IL-22RA antibody can be used for combination, blocking-up, inhibition, reduction, antagonism or and IL-20 and IL-22 (perhaps separately or together) in the treatment of psoriasis, hereditary atopic dermatitis, inflammatory skin disease, arthritic psoriasis, sacroiliitis, endotoxemia, asthma, inflammatory bowel (IBD), colitis and other inflammatory conditions disclosed herein.

And, but we have shown that the IL-22RA acceptor can be in conjunction with being called the T cell induction type factor (IL-22) (SEQ ID NO:6; Dumoutier, L.et al., Proc.Nat ' l. Acad.Sci. 97: 10144-10149,2000; Mouse IL-22 sequence is at Dumontier etal., J.Immunol. 164: 1814-1819 shows in 2000) aglucon.In addition, zcytor11 that owns together (IL-22RA) (United States Patent (USP) numbering 5,965,704) and CRF2-4 acceptor also combine with IL-22 (referring to WIPO publication WO00/24758 with the heterodimer form; Dumontier et al., J.Immunol. 164: 1814-1819,2000; SDencer, SD et al., J.Exp.Med.187:571-578,1998; Gibbs, VCand Pennica Gene 186:97-101,1997 (CRF2-4 cDNA); Xie, MH et al., J.Biol.Chem. 275: 31335-31339,2000; And Kotenko, SV et al., J.Biol.Chem. 276: 2725-2732,2001).And the IL-10 beta receptor may work as the acceptor of IL-22, think it and CRF2-4 be identical (Dumoutier, L.et al., Proc.Nat ' l.Acad.Sci. 97: 10144-10149,2000; LiuY et al, J Immunol. 1521821-1829,1994 (IL-10R cDNA).And we have shown IL-22RA acceptor and aglucon (the SEQ ID NO:8 that is called IL-20; WIPO publication numbering WO 99/27103) combination.In preferred embodiments, the soluble receptors form of IL-22RA (SEQ ID NO:3) be in vivo can in conjunction with, blocking-up, inhibition, reduction, antagonism or in and the monomer of IL-22 and IL-20, all dimer, heterodimer or polymer.At this IL-22RA monomer, all dimer, heterodimer or polymeric antibody and also can be used as the active antagonist of IL-22RA in conjunction with polypeptide, and the antagonist of IL-20 and IL-22 (separately or together), as described herein.

In addition, we described in this article and confirmed polyclone and the anti-IL-22 antibody of mono-clonal neutrality can combination in based on the neutralization test of cell, blocking-up, inhibition, reduction, antagonism or in and the activity of IL-22 and IL-20.

Shown that I1-22 is induced in the presence of IL-9, and suspected that it participates in promoting the immunne response and the inflammation of Th1 type.IL-9 promotes propagation, activation, differentiation and/or induction of immunity function in many ways, and relates to asthma, lung's mastocytosis and other diseases, and can activate the STAT approach.The antagonist of IL-22 or IL-9 function to this human diseases can be useful purposes.The invention provides this novel IL-22 antagonist.

Shown that IL-22 participates in raising the generation of acute phase reactant, for example serum amyloid sample substance A (SAA), α 1 chymotrypsin inhibitor, serum haptoglobin, and inject lipopolysaccharides (LPS) back IL-22 in vivo and express rising, this prompting IL-22 participates in inflammatory response (Dumoutier, L.et al. Proc.Nat ' l.Acad.Sci. 97: 10144-10149,2000).The generation of acute phase protein such as SAA is considered to the short-term survival mechanism, and wherein inflammation is useful; But the long term maintenance of acute phase protein can cause chronic inflammatory diseases, and may be harmful to people's health.Summary sees also Uhlar, CM and Whitehead, and AS, Eur.J. Biochem. 265: 501-523,1999, and Baumann H.and Gauldie, J. Immunology Today 15: 74-80,1994.And, acute phase protein SAA relates to the pathogenesis of many chronic inflammatory diseases, and also participates in arteriosclerosis and rheumatoid arthritis, and it is the precursor (Uhlar of sedimentary amyloid A protein in amyloidosis still, CM and Whitehead see before).Like this, because IL-22 serves as short scorching molecule and induces SAA to produce, antagonist can be used for the treatment of inflammatory disease with other and by IL-22 inductive and acute phase reactive protein diseases associated.This antagonist is by the invention provides.For example, the method that reduces IL-22 inductive or IL-9 inductive inflammation comprises the composition that gives the soluble receptors of a certain amount of IL-22RA of comprising to the Mammals with inflammation, presents in an amount at least sufficient to reduce inflammation.And the method that suppresses to have inflammatory reaction in the Mammals of inflammation can comprise: (1) measures the level of serum amyloid A protein; (2) use the composition that comprises solubility IL-22RA cytokine receptor polypeptide described herein, be included in acceptable carrier on the medicine in the described composition; (3) level of the serum amyloid A protein after the mensuration administration; (4) the serum amyloid A protein level in serum amyloid A protein level and the step (3) in the comparison step (1), wherein the serum amyloid A protein level does not raise or has reduction just to show that inflammatory reaction is suppressed.Experimental evidence described herein shows the IL-22 antagonist, and for example soluble receptors and antibody have reduced the level of SAA really in vivo in the inflammatory disease model, show combination, blocking-up, inhibition, reduction, antagonism or in and IL-22 have anti-inflammatory action.

Evidence suggests that IL-20 and acceptor thereof work in psoriasis.The dermopathic feature of this polygene is the increase of keratinocyte propagation, the change and the infiltration of immunocyte in skin of keratinocyte differentiation.It is the epidermis of observing hyperkeratosis and thickening in the unusual transgenic mice of simulating human psoriasis that IL-20 participates in psoriasic best evidence.The decline of tension force silk number (being considered to relevant with the keratinization of defective) is human psoriasic distinguishing feature.In mouse, all found inclusion body in the plastosome in skin hyperplasia situation chemical induction and natural appearance.The reason of inclusion body and the influence (if any) of mitochondrial function also do not known.The IL-20 transgenic mice show many in people's parapsoriasis observed characteristics.

And, to compare with normal skin, IL-20 receptor mrna (IL-20RA and IL-20RBmRNA) significantly raises in people's parapsoriasis skin, and this further points out IL-20 to work in psoriasis.Two subunits of IL-20 acceptor all have expression in the keratinocyte of whole epidermis, and also express in immunity partly and endotheliocyte.We infer that the IL-20 receptor expression that is activated improves the interaction that may change between endotheliocyte, immunocyte and the keratinocyte, causes the unusual adjusting of keratinocyte propagation and differentiation.In addition, mouse described herein knocks out data (wherein the IL-22RA acceptor is knocked out) and shows that IL-22RA is essential for IL-20 inductive inflammatory effect in the skin in the transgenic animal.These results have confirmed the activity of blocking IL-22RA effectively, for example by the IL-22RA gene knockout, perhaps similarly by anti-IL-22RA neutralizing monoclonal antibody of the present invention, can reduce IL-20 inductive skin and IL-22 inductive skin similarly, for example psoriasis, IBD, colitis or other are by IL-20 or IL-22 inductive inflammatory disease, comprise IBD, sacroiliitis, asthma, arthritic psoriasis, colitis, inflammatory skin is sick and hereditary atopic dermatitis.

And IL-20 stimulates the signal transduction in the human keratinized cell HaCaT clone, has proved the direct effect of this novel aglucon in skin.In addition, known have activity and participate in proliferative and the protein IL-1 β that urgees scorching signal in the skin in keratinocyte, and EGF and TNF-α all can improve replying for IL-20.In the HaCaT and bhk cell that express the IL-20 acceptor, IL-20 is by the STAT3 transduction signal.

Shown with following embodiment just as discussed above, IL-20 has participated in psoriasic pathologic process.The present invention be more particularly directed to a kind of by use combination, blocking-up, inhibition, reduction, antagonism or in and the medicament of IL-20 treat psoriasic method.At the antagonist of IL-20 can be in conjunction with IL-20 soluble receptors (as solubility IL-22RA) or in conjunction with the fragment of antibody, single-chain antibody or the antibody of IL-20 or IL-20 acceptor, as anti-IL-22RA antibody.Antagonist can stop the activation of IL-20 acceptor like this.And, since IL-20 and IL-22 with IL-22RA as the common acceptor, antagonist such as solubility IL-22RA or can be used for combination simultaneously, blocking-up, inhibition, reduction, antagonism or and the two activity of IL-22 or IL-20 and IL-22 in conjunction with antibody, single-chain antibody or the antibody fragment of IL-22RA acceptor.

Psoriasis is a kind of modal dermatosis, has influence on the nearly 1-2% of world population.It is a kind of chronic inflammation skin disorder, the erythema that characteristics are covered by silver color mica shape scale, the papule with remarkable boundary and circular patch.Not necessarily itch in psoriasic skin injury place.The psoriasis breakage often appears in site of injury.In addition, other extraneous factor also may increase the weight of psoriasis, comprises infection, pressure and medication for example lithium, Beta receptor blockers and anti-malaria medicaments.

Modal psoriasis kind is called the patch type.Have the psoriasic patient of patch type patch stable and that slowly grow is arranged, patch remains unchanged in long-time substantially.The patch psoriasis zone of normal appearance is ancon, knee, gluteal fold and scalp.Many symmetry morbidities.The inverse psoriasis influence comprises armpit, inguinal region to grinding the zone, submammary zone and navel, and it also can have influence on scalp, palm and sole.Independent breakage is the patch of obviously demarcating, but they also may be moistening owing to damaged residing position.The general slower development of the psoriasis of patch type, and be painless.It seldom spontaneously recurs.

Fulminant psoriasis (guttate psoriasis) is the most common in child and Young Adults.It does not suffer from acute attack among the psoriasic people of chronic patch type in having psoriasic individuality or at those.Patient many little erythema, flakey papules occur, is common in because after the upper respiratory tract infection that beta hemolytic streptococcus causes.Suffer from psoriasic patient and also can produce the breakage of purulence bubble.These breakages may be positioned at palm and sole place, perhaps can be generalizations, and accompany with fever, discomfort, diarrhoea and arthrodynia.

Only about half of suffer from psoriasic patient and have nail influence, that the first outward appearance presents is uneven, first thickens or first under excessive keratinization.Approximately the psoriasic patient that suffers from of 5-10% has the joint discomfort of following, and these are most commonly among the patient with nail influence.Although typical rheumatoid arthritis appears in some patients by chance, the joint disease that many patients suffer from belongs to one of following five kinds of types relevant with psoriasis: (1) is only limited to the disease (70% case) in one or several joints; (2) seronegative rheumatoid arthritis sample disease; (3) far-end refers to that the joint of (toe) internode involves; The disease that " arthritis mutilans " and (5) is only limited to backbone appears in (4) serious destructive sacroiliitis.

Can by use combination, blocking-up, inhibition, reduction, antagonism or in and the two pharmaceutical treatment psoriasis of IL-22, IL-20 or IL-20 and IL-22.Preferred antagonist is the soluble receptors of IL-20 and IL-22, IL-22RA (SEQ ID NO:3) for example, and perhaps in conjunction with antibody, antibody fragment or the single-chain antibody of IL-22RA acceptor, neutralizing antibody for example of the present invention.This antagonist can give separately or with other sophisticated therapies, for example methotrexate, psoralene ultraviolet therapy (PUVA), Yinxieling, isotretinoin, ciclosporin and partial vitamin D 3-derivatives calcipotriol are used in combination as lubricant, exfoliator, topical steroids, local vitamin D-derivatives, Dithranol, whole body metabolic antagonist.And this antagonist can be by subcutaneous, intravenous administration or with emulsifiable paste that contains antagonist or transdermal patches percutaneous dosing.If through subcutaneous administration, then antagonist can be injected and enter in one or the more a plurality of psoriasis patch.If percutaneous dosing, then antagonist can directly give on patch with forms such as the emulsifiable paste that contains antagonist, ointment, ointment or solution.

The antagonist of IL-20 or IL-22 can be given to the people who suffers from asthma, bronchitis or cystic fibrosis or other struvite pulmonary disorders and be treated these diseases.Antagonist can be by any suitable method, comprises that intravenously, subcutaneous, bronchial lavage and use contain the method administrations such as inhalation of antagonist.

Corresponding to the analysis revealed of the tissue distribution of the mRNA of IL-22RA cDNA, the mRNA level is the highest in placenta and spleen, and participates in inducing inflammatory reaction with IL-22RA bonded aglucon (IL-22), comprise induce acute phase reply (Dumoutier, L.et al., Proc.Nat ' L.Acad.Sci. 97: 10144-10149,2000).Therefore, particular embodiment of the present invention at be to use solubility IL-22RA and anti-IL-22RA antibody be used for inflammation and Immunological diseases or illness as antagonist, as psoriasis, arthritic psoriasis, hereditary atopic dermatitis, the inflammatory skin patient's condition, rheumatoid arthritis, inflammatory bowel (IBD), Crohn disease (Crohn disease), diverticulosis, asthma, pancreatitis, type i diabetes (IDDM), carcinoma of the pancreas, Graves disease, colon and carcinoma of small intestine, autoimmune disease, septicemia, organ or bone marrow transplantation; Because the inflammation that endotoxemia, wound, operation or infection cause; Amyloidosis; Splenomegaly; Graft is to versus-host disease; And need inflammation-inhibiting, immunosuppression, reduce hematopoietic cell, immunity, inflammation or lymphocyte, scavenger cell, T cell (comprising Th1 and Th2 cell) propagation, for the inhibition of cause of disease or antigenic immunne response, perhaps other need suppress the situation of IL-22 or IL-20 cytokine.

And, described herein and IL-22RA polypeptide bonded antibody or combine polypeptide and IL-22RA polypeptide self can be used for:

1) at the treatment acute inflammation, because the inflammation that wound, tissue injury, operation, septicemia or infection cause, and chronic inflammation disease such as asthma, inflammatory bowel (IBD), chronic colitis, splenomegaly, rheumatoid arthritis, recurrent acute inflammation outbreak (as tuberculosis), and amyloidosis and arteriosclerosis, Castleman disease, asthma and other and acute phase reply in the treatment of inducing diseases associated, blocking-up, inhibition, reduction, antagonism or neutralization are by IL-20 or the receptor-mediated signal of IL-22.

2) blocking-up in treatment autoimmune disease such as IDDM, multiple sclerosis (MS), systemic lupus erythematosus (SLE), myasthenia gravis, rheumatoid arthritis and IBD, inhibition, reduction, antagonism or neutralization are by IL-20 or the receptor-mediated signal of IL-22, to stop or to suppress signal transduction (the Hughes C et al. of immunocyte (for example lymphocyte, monocyte, white corpuscle) by IL-22RA J.Immunol 153: 3319-3325,1994).Perhaps, also can use at the antibody of the acceptor that comprises IL-22RA for example monoclonal antibody (mAb) remove unwanted immunocyte as antagonist, with the treatment autoimmune disease.Can use monoclonal antibody treatment asthma, allergy or other atopic allergologies, to suppress immunne response or to remove the cell of invading at for example solubility IL-22RA acceptor.Using polypeptide of the present invention and antibody blocking, inhibition, reduction or the antagonism signal by the IL-22RA mediation, may also be useful to the disease of pancreas, kidney, hypophysis and neuronal cell etc.IDDM, NIDDM, pancreatitis and pancreatic neoplasm also may be benefited.IL-22RA can be used as the target of cancer monoclonal antibody therapy, and wherein the monoclonal antibody of antagonism suppresses the growth of cancer, and with immune-mediated killing as target.(Holliger P，and Hoogenboom，H： Nature Biotech. 16：1015-1016，1998)。Monoclonal antibody at solubility IL-22RA can also be used for the treatment of kidney disease, for example the glomerulonephritis of glomerulosclerosis, membranaceous DPN, amyloidosis (also influencing tissues such as kidney), renal arteriosclerosis, different sources, kidney fibroplasia sick and and SLE, IDDM, type ii diabetes (NIDDM), the kidney disorder that tumor of kidney is relevant with other diseases.

3) treating for example IDDM of autoimmune disease, MS, synergy among SLE, myasthenia gravis, rheumatoid arthritis and the IBD, raising, increase or startup are by the signal of IL-20 or I1-22 acceptor.Anti-IL-22RA neutralizing monoclonal antibody can transmit signal to lymphocyte or other immunocytes, and the cell surface protein that makes cytokine or other improve autoimmunity power breaks up, changes its propagation or change its generation.Especially, t helper cell is replied the cytokine secretion of changing into another pattern, can make autoimmunity reply alienation, make disease alleviate (Smith JA et al., J.Immunol. 160: 4841-4849,1998).Similarly, the anti-solubility IL-22RA antibody of excitability, anti-solubility IL-22RA/CRF2-4 heterodimer and polymer monoclonal antibody can be used for sending signal or with its removing and alienation to the immunocyte that participates in asthma, allergy and atopic allergology disease.Signal conduction by IL-22RA also may be useful to pancreas, kidney, hypophysis and neuronal cell disease.IDDM, NIDDM, pancreatitis and carcinoma of the pancreas also may be benefited.IL-22RA can be used as the target of carcinoma of the pancreas monoclonal antibody therapy, and wherein signal conductivity monoclonal antibody suppresses the growth of cancer, and with immune-mediated kill as target (Tutt, AL et al., J Immunol. 161: 3175-3185,1998).Similarly, can use mab treatment renal cell carcinoma at the soluble receptors that comprises IL-22RA among the present invention.

Be used for combination, blocking-up, inhibition, reduction, antagonism or and the activity of IL-22 or IL-20 (separately or together) in the above in the treatment of autoimmune disease, atopic disorder, NIDDM, pancreatitis and renal tubal dysfunction that solubility IL-22RA polypeptide described herein can be described.The soluble form of IL-22RA can be used to promote cell-mediated antibody response and/or promotion lymphocyte or other immunocytes generation IL-4 or other cytokines by Th.

The soluble receptors that comprises IL-22RA among the present invention can be used as the antagonist of IL-20 or IL-22 cytokine.This antagonistic action can realize by direct neutralization or in conjunction with IL-20 or I1-22.Except the purposes of antagonism, soluble receptors of the present invention can also be in conjunction with IL-22, and as the carrier proteins of IL-20 or IL-22 cytokine, purpose is that aglucon is transported to tissues different in the body, organ and cell place.Like this, soluble receptors of the present invention can merge or coupling with the molecule, polypeptide or the chemical ingredients that instruct soluble receptors-aglucon complex body to arrive special position (for example tissue, special immunocyte or tumour).For example, induce inflammation and the local acute phase reactive protein that produces, acute infection or certain cancers are benefited by the effect of IL-22.Therefore, soluble receptors of the present invention can be used for instructing specifically the effect of IL-20 or IL-22.Referring to Cosman, D.Cvtokine 5:95-106,1993; And Fernandez-Botran, R.Exp.Opin. Invest.Drugs 9: 497-513,2000.

And, soluble receptors of the present invention can be used for stablizing IL-22 or IL-20, make it that degraded or removing not take place by stablizing aglucon, perhaps by aglucon being targeted to biological availability, treatment life time and/or the effectiveness that intravital action site improves aglucon.For example, naturally occurring IL-6/ solubility IL-6 complex body can make IL-6 stable, and can pass through gp30 acceptor transduction signal.Referring to Cosman, D. sees before, and Fernandez-Botran, and R. sees before.And, IL-22RA can with the homologous aglucon for example IL-22 combine, form aglucon/soluble receptors complex body.This species complex can be used for stimulating the cell generation of presenting companion's receptor subunit (for example pDIRSl (IL-20RB) or CRF2-4 (IL-10RB)) to reply.The cell-specific of IL-22RA/ aglucon complex body may be different with observed cell-specific when aglucon is individually dosed.And complex body may have unique pharmacokinetic property, for example influences half life, dosage/reaction and organ or tissue's specificity.Therefore, IL-22RA/IL-22 or IL-22RA/IL-20 complex body may have the activity of agonist, can enhancing immunity reply or stimulate mesangial cell or cell cultured supernatant.Perhaps, have only express with organizing ability that the signal of the assorted dimerization of complex body conducts subunit be subjected to at the similar influence of replying of IL6/IL6R complex body (Hirota H.et al., Proc.Nat ' L.Acad.Sci. 92: 4862-4866,1995; Hirano.T.in Thomason, A. (Ed.) " The Cytokine Handbook ", 3 ^RdEd., p.208-209).The soluble receptors of IL12 and CNTF/cytokine complex body has shown similar activity.

And inflammation is the protective response of organism, to keep out the factor of infringement.Inflammation is the process of a cascade, and it relates to the amboceptor of many cells and body fluid.On the one hand, the inhibition to inflammatory response can make the host can not produce normal immune response; But, if do not limited, then inflammation can cause severe complications, comprises chronic inflammatory diseases (for example psoriasis, sacroiliitis, rheumatoid arthritis, multiple sclerosis, inflammatory enteritis or the like), septic shock and multiple organ failure.Importantly, these different morbid states have the struvite amboceptor of common.These diseases with inflammation feature have very big influence for people's morbidity and mortality ratio.Therefore, it is apparent that anti-inflammatory albumen for example IL-22RA and anti-IL-22RA antibody may from asthma and irritated to autoimmune and septic shock, all have extremely important treatment potentiality to a large amount of human and animal's diseases.

1. sacroiliitis

Sacroiliitis comprises that because osteoarthritis, rheumatoid arthritis, arthritis knuckle due to the damage or the like is common inflammation venereal disease disease, they can be benefited from the therapeutic of anti-inflammatory albumen such as IL-22RA polypeptide of the present invention and use.For example, rheumatoid arthritis (RA) is a kind of general disease, and it influences whole machine body, and is a kind of sacroiliitis of most common form.It is characterized in that along the synovium of joint inflammation this causes pain, stiff, warm, rubescent and enlargement.The enzyme that struvite cell discharges can digest bone and cartilage.The result of rheumatoid arthritis is that bone and cartilage can be invaded and destroy to inflamed joints inner membrance-synovial membrane, causes joint wearing and tearing and violent pain and other physiological situations.Its shape and arrangement can be lost in ill joint, cause the forfeiture of pain and motion.

Rheumatoid arthritis (RA) is a kind of immune-mediated disease, and its feature especially is inflammation and tissue damage subsequently, causes the increase of handicap and mortality ratio.Produced the various kinds of cell factor in the rheumatoid joint of trouble part.Many studies show that, the pro-inflammatory cytokine-IL-1 of two kinds of prototypes and TNF α play important effect in the mechanism that relates to synovial membrane inflammation and carrying out property destruction of joint.Really in the patient who suffers from RA, give the remarkable improvement that TNF α and IL-1 supressor have caused the clinical of inflammation and biology sign, and the radiation sexology sign of bone erosion and cartilage destruction descends.But, although these challenging results are arranged, have the patient of significant percentage not produce reaction for these medicaments, this prompting also have other amboceptor also participate in arthritic physiopathology (Gabay, Expert.Opin.Biol.Ther. 2 (2): 135-149,2002).A kind of in these amboceptors may be exactly IL-20 or IL-22, if like this, in conjunction with or suppress IL-22 or the active molecule of IL-20, can become useful medicine as IL-22RA polypeptide or anti-IL-22RA antibody or binding partners and alleviate inflammation in rheumatoid arthritis and other joint property diseases.

A plurality of Animal Model of Rheumatoid Arthritis are arranged in the art.For example, in collagen-induced sacroiliitis (CIA) model, mouse produces chronic inflammatory arthritis, and it is the anthropoid rheumatoid arthritis of class very.Because CIA has similar immunology and pathological characteristics to RA, it becomes the ideal model of screening potential people anti-inflammatory compound.The CIA model is a well-known model in the mouse, its appearance depend on immunne response and inflammatory reaction the two.Immunne response comprises the B cell of the collagen reaction generation that gives as antigen and the interaction between the CD4+T cell, causes the generation of anticol original antibody.Inflammation phase is the result who is replied by the tissue that mediator of inflammation produces, and is some and the natural collagen generation cross reaction of mouse and the result of activating complement cascade in these antibody.Using an advantage of CIA model is that morbific fundamental mechanism is known.On II class collagen, identified relevant T cell and B cell epitope, and relevant amynologic parameter of various with immune-mediated sacroiliitis (for example delayed hypersensitivity and anticol original antibody) and inflammatory parameters (for example cytokine, chemokines and extracellular matrix degrading enzyme) have been determined, these parameters can be used for assessing the effectiveness (Wooley of compound to be checked at the CIA model Curr.Opin.Rheum. 3: 407-20,1999; Williams et al., Immunol. 89: 9784-788,1992; Myers et al., Life Sci. 63: 1861-78,1997; And Wang et al., Immunol. 92: 8955-959,1995).

The soluble polypeptide (zcytorl6) that comprises IL-22RA2 to these CIA model mices, for example zcytor16-Fc4 or other IL-22RA2 solubility and fusion rotein are estimated the effect of using IL-22RA2 to be used for the relief of symptoms and the change course of disease as the antagonist of IL-22.And for example IL-22RA or its antibody also can be used for relief of symptoms and change this viewpoint of the course of disease evidence is provided to the result that IL-22 is suppressed by IL-22RA2 for other IL-22 antagonists.Because the generation that the aglucon IL-22 of IL-22RA2 induces SAA, and SAA has participated in the morbidity of rheumatoid arthritis, and IL-22RA2 is proved to be able to suppress in vitro and in vivo the activity of IL-22 and SAA, thus the polypeptide that whole body or topical administration comprise IL-22RA2 for example zcytor16-Fc4 or other IL-22 soluble receptorss (as IL-22RA; SEQID NO:3) and anti-IL-22RA antibody and fusion rotein can suppress inflammatory reaction among the RA potentially.Inject 10 microgram zcytor16-Fc (around a Wednesday time injection) and significantly reduced disease score (pawl scoring, the sickness rate of inflammation or disease).Other potential therapeutical agents comprise IL-22RA polypeptide, anti-IL-22RA antibody or anti-IL-22 antibody or binding partners or the like.

Therefore a group studies show that, with respect to control mice, anti-mouse IL-22 antibody can alleviate the symptom of CIA model mice, shows that solubility IL-22RA polypeptide and IL-22RA neutralizing antibody have the treatment of benefiting human diseases in theory.Give a kind of mouse IL-22 specificity rat monoclonal antibody (P3/1) as prevention administration or administration after the CIA model is induced sacroiliitis, can alleviate arthritic symptom (the WIPO publication 02/068476 in the animal; 2002-09-09 publishes).Therefore, solubility IL-22RA polypeptide of the present invention and anti-IL-22RA antibody, comprise anti-human il-22 RA neutralizing antibody of the present invention, can when the special human diseases of treatment such as psoriasis, arthritic psoriasis, sacroiliitis, endotoxemia, inflammatory enteritis (IBD), colitis and other inflammatory conditions disclosed herein, be used for and IL-22 and IL-20.

2. endotoxemia

Endotoxemia is a kind of serious illness, usually because infectious factor such as bacterium and other infectious diseases factors, septicemia, toxic shock syndrome causes or in the patient of non-responsiveness owing to opportunistic infection occurs.Useful anti-inflammatory albumen in the treatment as IL-22RA polypeptide of the present invention and antibody, can help to stop and treat the endotoxemia of humans and animals.IL-22RA polypeptide, anti-IL-22RA antibody or anti-IL-22 antibody or binding partners can be used as inflammation and pathological effect that valuable therapeutical agent is used for alleviating endotoxemia.

Lipopolysaccharides (LPS) inductive endotoxemia has related to many short scorching amboceptors, they produce pathological effect in infectious diseases, LPS inductive endotoxemia is a kind of being extensive use of and the received model that is used to study short inflammation of potential or immune-regulating factor pharmacological action in rodent.The LPS that in gram negative bacterium, produces be in the septic shock pathogenesis main virulence factor (Glausner et al., Lancet 338: 732,1991).Shock sample state can be induced by experimentizing to animal single injection LPS really.The molecule that cell response LPS produces is the target cause of disease directly or indirectly.Although these biological response protection hosts are not invaded the invasion and attack of cause of disease, they also can cause injury.Therefore, the extensive stimulation of the innate immune power that is caused by serious gram-negative bacterial infections has caused the excessive generation of cytokine and other molecules, and fatal syndrome appears--septic shock syndrome is characterized in fever, hypopiesia, disseminated inravascular coagulation and multiple organ failure (Dumitru et al. Cell103:1071-1083,2000).

These toxic action great majority of LPS are activated, cause multiple mediator of inflammation to discharge with scavenger cell relevant.In these amboceptors, TNF has seemed important role, this point can by prevented by the neutralizing antibody that gives anti-TNF the toxicity of LPS show (Beutler et al., Science229:869,1985).Very clear and definite is to inject 1 microgram Escherichia coli LPS in C57B1/6 mouse body, the obvious increase of IL-6, TNF-α, IL-2 and acute phase protein (for example SAA) in can causing after about 2 hours circulating.The toxicity of LPS looks like by these cytokine mediated because to the passive immunization of these amboceptors can make mortality ratio descend (Beutler et al., Science229:869,1985).The potential immunologic intervention strategy that prevents and/or treats septic shock comprises anti-TNF monoclonal antibody, IL-1 receptor antagonist, LIF, IL-10 and G-CSF.

Comprise soluble polypeptide (for example Zcytor16-Fc4) or other IL-22RA solubility and the fusion rotein of IL-22RA2 to these LPS inductive models, be used to estimate and use the IL-22RA2 mitigation symptoms and change the effect that LPS induces disease course.And for example IL-22RA or its antibody also can be used for alleviating the symptom in the LPS inductive model and change this viewpoint of disease course evidence is provided IL-22 for other IL-22 antagonists by result that IL-22RA2 suppressed.This models show lps injection induced IL-22 and IL-22RA2 polypeptide may treat to disease.Because inducing, LPS produces short inflammatory IL-22, SAA or other proinflammatory factors that may in the endotoxemia pathology, work, so use in the antagonist IL-22RA2 polypeptide and activity, SAA or other proinflammatory factors of IL-22, can alleviate the symptom of endotoxemia, as seen in endotoxin shock, arriving.Other potential therapeutical agents comprise IL-22RA polypeptide, anti-IL-22RA antibody or anti-IL-22 antibody or binding partners or the like.

3. inflammatory bowel, IBD

The U.S. roughly 500000 people suffer from inflammatory bowel (IBD), it can influence colon and rectum (ulcerative colitis) or small intestine and large intestine the two (Crohn disease).The pathomechanism of these diseases is not clear, but they relate to infected chronic inflammation of tissue.IL-22RA polypeptide, anti-IL-22RA antibody or anti-IL-22 antibody or binding partners can be used as inflammation and the pathological effect that valuable therapeutical agent is used for alleviating IBD and relative disease.

Ulcerative colitis (UC) is the inflammatory disease of a kind of large intestine (being commonly referred to colon), it is characterized in that the inflammation and the ulcer of the mucous membrane or the innermost layer of colon.This inflammation causes the frequent emptying of colon, produces diarrhoea.Symptom comprises lax ight soil and abdominal colic, fever and the weight loss followed.Although the definite reason of UC also do not know, the natural defence of nearest research prompting body is to being thought the protein of external source work (a kind of " autoimmune reaction ") by body in that machine is intravital.May be because they resemble the interior bacterioprotein of enteron aisle, these albumen may evoke or stimulate inflammatory process, begin to destroy the colon inner membrance.Along with the colon inner membrance is destroyed, form ulcer, discharge mucus, purulence and blood.This disease finally can be spread to whole large intestine usually from rectal area.The outbreak repeatedly of inflammation causes on intestines and the rectal wall owing to scar tissue thickens.The death of colon or septicemia can occur in serious disease.Ulcerative colitis is different according to its symptom of difference of seriousness, their outbreak can be progressively or unexpected.Seizure of disease may cause by many factors, comprise respiratory system infection or pressure.

Can be although also do not cure the method for UC now for utilizing, treatment concentrates on and suppresses unusual inflammatory process in the colon inner membrance.Methods of treatment comprises reflunomide immunosuppressor (for example azathioprine, purinethol and methotrexate), and para-aminosalicylic acid also can be treated this disease.But life-time service immunosuppressor for example cortin and azathioprine can cause serious side reaction, comprises that bone attenuates, cataract, infection and to the influence of liver and marrow.For patient that can not be successful in current treatment, operation is a kind of selection.Operation comprises removes whole colon and rectum.

There are many animal models can partly simulate chronic ulcerative colitis.The most widely used model is 2,4,6 trinitro-benzene-sulfonic acids/ethanol (TNBS) inductive colitis model, and it induces chronic inflammatory diseases and ulcer in colon.In the time of when TNBS instils the colon that is introduced into responsive mouse by internal rectum in, the immunne response of its inducing T cell mediation in the mucous membrane of colon, cause large-scale mucosal inflammation in this case, be characterized in T cell and macrophages infiltration intensive in whole large intestine wall.And, this histopathology sight also be accompanied by clinically carry out gonosome heavily descend (becoming thin), bloody diarrhea, proctoptosis and large intestine wall thickening (Neurath etal. Intern.Rev.Immunol. 19: 51-62,2000).

The another one colitis model is used dextran sulfate sodium (DSS), and it has induced acute colitis, and it shows as bloody diarrhea, weight loss, colon shortens and have the mucosal ulcer that neutrophilic granulocyte soaks into.The histological characteristic of DDS inductive colitis is that inflammatory cell damages and epithelium ulcer to lamina propria infiltration, lymphoid hyperplasia, local folliculus.The appearance of these changes is considered to because toxic action, the phagolysis of lamina propria cell and the generation of TNF α and IFN γ of DSS on epithelial cell.Although because the generally use of DSS model, the many problems that relate to about the DSS mechanism of human diseases dependency are still waiting to solve.DSS is considered to a model that does not rely on the T cell, because for example also observed this model in the SCID mouse the animal of T cell disappearance.

Comprise soluble polypeptide (for example Zcytor16-Fc4) or other IL-22RA solubility and the fusion rotein of IL-22RA2 to these TNBS or DSS model, can be used to estimate the effect of using the IL-22RA2 mitigation symptoms and changing the gastrointestinal tract disease course of disease.And for example IL-22RA or its antibody also can be used for alleviating the symptom in colitis/IBD model and change this viewpoint of disease course evidence is provided the result that IL-22 is suppressed by IL-22RA2 for other IL-22 antagonists.We are by the synergistic activity that RT-PCR and IL-22 and IL-1 β fasten at intestinal cells, and the expression of observing IL-22 in the colon of DSS mouse increases.It shows that IL-22 may work in the inflammatory reaction of colitis, and by giving in the IL-22RA2 polypeptide and the activity of IL-22 is a kind of potential IBD methods of treatment.Other potential therapeutical agents comprise IL-22RA polypeptide, anti-IL-22RA antibody or anti-IL-22 antibody or binding partners or the like.

4. psoriasis

Psoriasis is a kind of chronic skin sufferer, and it has influence on the American more than 7,000,000.Psoriasis occurs when new skin cells growth failure, cause chafing, swelling and lepidiod spot, wherein old skin can not come off rapidly.Patch type psoriasis is modal form, is characterized in that spot (" damage ") red and swollen on the skin is coated with argenteous scale.Psoriasis can be only limited to the minority patch, perhaps involves the skin moderate to most of zone, and the most often the position that occurs is scalp, knee, ancon and trunk.Although psoriasis is not a kind of communicable disease obviously as seen.The pathogenesis of this disease relates to the chronic inflammatory diseases of incidence tissue.IL-22RA polypeptide, anti-IL-22RA antibody or anti-IL-22 and anti-IL-20 antibody or binding partners can be used as valuable therapeutical agent, reduce inflammation and pathological effect in psoriasis, other inflammatory skin diseases, skin and mucous membrane irritability and relative disease.

Psoriasis is the cell-mediated skin inflammation venereal disease disease of a kind of T, and it can cause the uncomfortable of certain degree.The also readily good curing method of this disease influences the people of institute's has age.2% of about Europe of psoriasis influence and north American population.Can use topical agent to control its disease although have slight psoriasic individuality, have in the world to surpass 1,000,000 needs of patients ultraviolet ray or general immunosuppressant therapy.But unfortunately, the toxicity of the inconvenience of ultraviolet radiation and risk and many methods of treatment has limited their life-time service.And soon, patient can be recurred psoriasis usually after stopping immunosuppressant therapy, can rebound under some situation.

IL-20 is a kind of novel IL-10 homologue, shows that it can cause the newborn infant to cause death in the IL-20 transgenic mice and skin unusual, comprise the odd-shaped epidermal differentiation (Blumberg H et al., Cell 104: 9-19,2001).The IL-20 acceptor sharply raises in psoriasic skin.Possible IL-22 because IL-22 and IL-20 acceptor are shared receptor subunit (zcytor11) is arranged, and the IL-22 transgenic mice shows similar phenotype, so also participate in for example psoriasis of inflammatory skin diseases.By subcutaneous or topical administration IL-22RA polypeptide or anti-IL-22RA antibody antagonist, might reduce inflammation and symptom.Other possible healing potions comprise the IL-22RA polypeptide, solubility zcytor11/CRF2-4 receptor polypeptides, perhaps anti-IL-22 antibody or binding partners or the like.

And, in people's parapsoriasis damage, show the overexpression of IL-22 and IL-20, the same people's parapsoriasis that also relates to of prompting IL-22 with IL-20.And as described herein-in, IL-20 or the IL-22 overexpression in transgenic mice has shown that epidermis thickens with immunocyte and has involved that this is the indication of psoriasis phenotype; In addition, IL-22 is injected in normal mouse shown that epidermis thickens with immunocyte and involve (indication that this is the psoriasis phenotype) that this phenomenon can be passed through soluble receptors antagonist IL-22RA2 (zcytor16; WIPO publication numbering WO 01/40467) eliminates.Data further point out short scorching IL-22 to participate in psoriasis in this body.Similarly, the active antagonist of IL-22 and IL-20, for example IL-22RA soluble receptors and antibody thereof, comprise anti-human il-22 RA mono-clonal neutralizing antibody of the present invention, in the therapeutic treatment of diseases associated with inflammation, be useful, particularly in psoriasis treatment, can be used as the antagonist of IL-22 and IL-20 (separately or together).And, the active antagonist of IL-22, for example IL-22RA soluble receptors and antibody thereof, comprise anti-human il-22 RA mono-clonal neutralizing antibody of the present invention, in the therapeutic treatment of other diseases associated with inflammation, be useful, for example can be in the hereditary atopic dermatitis of treatment, IBD, colitis, endotoxemia, sacroiliitis, rheumatoid arthritis, arthritic psoriasis, adult respiratory disease (ARD), septic shock, multiple organ failure, struvite injury of lung is asthma or bronchitis for example, bacterial pneumonia, psoriasis, oedema, in heredity atopic dermatitis and contact dermatitis and inflammatory bowel such as ulcerative colitis and the Crohn disease, as combination, blocking-up, suppress, reduce, antagonism or in and the medicament of IL-22 and IL-20.

And, anti-IL-22RA antibody of the present invention can be used to prevent and treatment and the relevant weight loss of a series of diseases associated with inflammation described herein with the IL-22RA soluble receptors, and is used for prevention and treatment and cancer (for example chemotherapy and the emaciation) weight loss relevant with infectious diseases.For example, serious weight loss is the key point relevant with septicemia, MS, RA and tumor model.In addition, weight loss is the important parameter that many human diseasess comprise cancer, infectious diseases and inflammatory disease.The mouse of having injected IL-22 adenovirus described herein has shown weight loss.Can test anti-IL-22 antibody of the present invention and IL-22 antagonist for example solubility IL-22RA acceptor and antibody thereof, and zcytor16 (IL-22RA2) acceptor prevents and treats the ability of weight loss in the mouse of having injected IL-22 adenovirus described herein.The method of determining the preventative of this IL-22 antagonist or therapeutic scheme is known in this area, and can use method described herein to determine.

IL-22RA soluble receptors polypeptide and antibody thereof can also use in diagnositc system, are used to detect the cyclical level of IL-22 or IL-20 aglucon, and are used to detect the IL-22 relevant with the acute phase inflammatory reaction.In relevant embodiment, can use the medicament of antibody or other and IL-22RA soluble receptors specific combination of the present invention, detect the receptor polypeptides in the circulation; On the contrary, IL-22RA soluble receptors self can be used for detecting in the circulation or the IL-22 of local action or IL-20 polypeptide.The rising of aglucon or receptor polypeptides or to be subjected to the inhibition level can be the indication that pathological condition is comprised inflammation or cancer.Known IL-22 induces relevant acute phase inflammatory reaction.And, to acute phase protein or the molecule detection of IL-20 or the IL-22 chronic inflammatory condition that can indicate (for example psoriasis, rheumatoid arthritis, colitis, IBD) in some disease for example.The detection of this situation can help the diagnosis of disease and help the physician to select correct methods of treatment.

Can use the intrauterine administration of anti-IL-22 or anti-IL-20 neutralizing antibody, by reducing or eliminate in the IL-22 of overexpression IL-22 transgenosis young baby or the skin phenotype of in the IL-20 of overexpression IL-20 transgenosis young baby, finding, render a service thereby be presented in the body in the disease model.The precedent of using neutralizing monoclonal antibody to carry out the intrauterine treatment is arranged in this area.In an example, handle conceived female mice by using for the special monoclonal antibody of B cell-specific molecule CD19, the growth of the B1 subgroup of B cell is had a strong impact on (Krop I.Et al. for example, Eur.J.Immunol. 26 (1):238-42,1996).People such as Krop were dissolved in the rat anti-mouse CD19 monoclonal antibody (or rat is with contrast monoclonal antibody of I type coupling) of PBS by intraperitoneal injection 500 micrograms on the 9th day after gestation, next injection every other day is until childbirth.The young baby also injects 500 micrograms when 10 ages in days these antibody once.In the another one example, people such as Tanaka find to use the monoclonal antibody of IL-2 acceptor β chain to carry out the intrauterine processing, have stopped the growth of Thy-1+ dendron shape epidermic cell fully.Two different subunits of IL-2 acceptor, promptly α chain (IL-2R α) and β chain (IL-2R β) are expressed in embryo's thymus gland in a kind of mode of almost mutual exclusion.By giving neutrality monoclonal antibody blocking-up IL-2R β--the signal transduction component of IL-2R, caused the complete and selectivity of Thy1+ skin dendron shape epidermic cell to disappear at IL-2R β.The growth of any other T cell subsets is not affected.This show IL-2 in embryo V γ 5+ cell and descendant's thereof growth, rise important role (referring to Tanaka, T.et al., Int Immunol. 4 (4): 487-9,1992).In addition, people such as Schattemann GC uses uterine system to show that normal mouse core vascular development needs PDGF-A.A plurality of evidence promptings need Thr6 PDGF BB A chain (PDGF-A) in the normal cardiovascular growth of embryo.To resist the PDGF-A neutralizing antibody to introduce the mouse pregnancy uterine endometrium, cause selective destruction 18-24 hour to PDGF-A aglucon-acceptor interaction in the body, thereby whether definite cardiovascular growth needs PDGF-A and when needs (referring to denying Schattemann GC et al. Dev.Biol. 176 (1): 133-42,1996) other results of describing in these results and this area, proved the neutrality monoclonal antibody and can cause strong effect in uterus.Similarly, can provide show in disease model use in the monoclonal antibody in the body and reduce when IL-20 or IL-22 or the IL-20 that removes at overexpression IL-20 and IL-22 respectively and IL-22 transgenic mice young baby in the effectiveness of the skin phenotype found.Have " light " skin appearance during these transgenic mice births, this is at least in part because epidermis described herein thickens.The IL-20 transgenosis young baby who expresses the transgenic cell factor of lower level can recover, and can survive really and breed the offspring, but the IL-22 transgenic mice is dead soon after birth, generally before 5 ages in days.

For example, the neutralizing antibody of IL-20 comprise can in conjunction with the IL-20 antigenic epitopes and in and the active antibody of IL-20, as neutralizing monoclonal antibody.Therefore, carry the IL-20 peptide and the polypeptide of antigenic epitopes, can be used to prepare antibody, and be used for identifying and screening can be in conjunction with, blocking-up, inhibition, reduction, antagonism or and the anti-IL-20 monoclonal antibody of the active neutrality of IL-20 in conjunction with IL-20 polypeptide of the present invention.This neutralizing monoclonal antibody of the present invention can be in conjunction with the IL-20 antigenic epitopes.Epi-position in this SEQ ID NO:8 (is predicted by the Jameson-Wolf figure line, for example use DNASTAR Protean program (DNASTAR, Inc., Madison, WI)) can be used as preferred antigenic epitopes, and can determine by those skilled in the art.This antigenic epitopes comprises: 42 (Ile) of SEQ ID NO:8 are to 102 (Asp) number amino-acid residue; 42 (Ile) of SEQ ID NO:8 are to 60 (Ile) number amino-acid residue; 42 (Ile) of SEQID NO:8 are to 69 (Glu) number amino-acid residue; 42 (Ile) of SEQ ID NO:8 are to 81 (Cys) number amino-acid residue; 42 (Ile) of SEQ ID NO:8 are to 96 (Lys) number amino-acid residue; 42 (Ile) of SEQ ID NO:8 are to 102 (Asp) number amino-acid residue; 60 (Ile) of SEQ ID NO:8 are to 69 (Glu) number amino-acid residue; 60 (Ile) of SEQ ID NO:8 are to 81 (Cys) number amino-acid residue; 60 (Ile) of SEQ ID NO:8 are to 96 (Lys) number amino-acid residue; 60 (Ile) of SEQ ID NO:8 are to 102 (Asp) number amino-acid residue; 69 (Glu) of SEQ ID NO:8 are to 81 (Cys) number amino-acid residue; 69 (Glu) of SEQ ID NO:8 are to 96 (Lys) number amino-acid residue; 69 (Glu) of SEQ ID NO:8 are to 102 (Asp) number amino-acid residue; 81 (Cys) of SEQ ID NO:8 are to 96 (Lys) number amino-acid residue; 81 (Cys) of SEQ ID NO:8 are to 102 (Asp) number amino-acid residue; And 96 (Lys) of SEQ ID NO:8 are to 102 (Asp) number amino-acid residue.

Except other diseases model described herein, anti-IL-22RA antibody can use severe severe combined immunodeficiency (SCID) mouse model to carry out measuring in the body from the structural activity of inflammation of people's psoriasis damage.Developed several mouse models, wherein people's cell has been transplanted in the immunodeficient mouse body and (is referred to as heteroplastic transplantation model); Referring to for example Cattan AR, Douglas E, Leuk.Res. 18: 513-22,1994 and Flavell, DJ, Hematological Oncology 14: 67-82,1996.As an individual interior psoriasis heteroplastic transplantation model, people's psoriasis skin histology is transplanted in the SCID mouse model, attack with suitable antagonist.And other psoriasis animal models in this area also can be used to estimate IL-20 and IL-22 antagonist, for example people's psoriasis skin histology is transplanted in the AGR129 mouse model, with suitable antagonist attack (for example referring to Boyman, O.et al., J.Exp.Med.The online numbering #20031482 that publishes, 2004, quote as a reference here).Can in conjunction with, blocking-up, inhibition, reduction, antagonism or in and IL-22 or IL-20 and the two active anti-IL-22RA antibody of IL-22 be preferred antagonist, but anti-IL-20 and anti-IL-22 antibody (separately or combine), solubility IL-22RA and other IL-20 and IL-22 antagonist also can use in this model.Similarly, can be used for the SCID model from the tissue or the cell of people's colitis, IBD, sacroiliitis or other inflammation damnifications, to study the antiinflammatory property of IL-20 described herein and IL-22 antagonist.

Be the treatment plan of eliminating, delaying or reduce inflammation and design, use anti-IL-22RA antibody or its derivative, agonist, conjugate or variant, can have SCID mouse or other models described herein of human inflammation tissue (for example psoriasis damage or the like) by resisting IL-22RA antibody or solubility IL-22RA compound, test.Use well-known method in this area, measure effectiveness and antiphlogistic effects in time the relation of statistical evaluation handled in handled colony, increasing.Some exemplary methods comprise, but are not limited to, and measure number, the Parakeratotic degree of inflammatory cell in epidermal thickness in the psoriasis model for example, the upper strata corium.These methods are known in this area, and here describe to some extent.For example, referring to Zeigler, M.et al0. Lab Inyest 81: 1253,2001; Zollner, T.M.et al. J.Clin.Invest. 109: 671,2002; Yamanaka, N.et al. Microbio.l Immunol. 45: 507,2001; Raychaudhuri, S.P.et al. Br.J.Dermatol. 144: 931,2001; Boehncke, W.H et al.Ar Ch.Dermatol.Res. 291104,1999; Boehncke, W.H et al.. J.Invest.Dermatol. 116: 596,2001; Nickoloff, B.J.et al. Am.J.Pathol. 146: 580,1995; Boehncke, W.H et al. J.Cutan.Pathol. 24: 1,1997; Sugai, J., M.et al. J.Dermatol.Sci. 17: 85,1998; And Villadsen L.S.et al. J.Clin. Invest. 112: 1571,2003.Can also use well-known method to monitor inflammation in time, for example adopt the inflammation in flow cytometer (perhaps PCR) the quantitative assay sample or the number of damaging cells, mark (weight loss, diarrhoea, proctorrhagia, colon length) for IBD, carry out pawl disease score and inflammation scoring for CIA RA model.For example, the therapeutic strategy that is suitable for testing in this model comprises direct treatment, wherein use anti-IL-22RA antibody, other IL-20 and IL-22 antagonist (separately or together) or relevant conjugate or, perhaps use the methods of treatment based on cell of anti-IL-22RA antibody or derivatives thereof, agonist, conjugate or variant based on destructive interactional antagonist between anti-IL-22RA antibody and its aglucon IL-20 and the IL-22.

And psoriasis is a kind of chronic inflammatory diseases dermatosis, and it is relevant with the monocyte of outgrowth epidermal keratinocyte and infiltration, comprise CD4+ memory T cell, neutrophilic granulocyte and scavenger cell (Christophers, Int.Arch.Allergy Immunol., 110: 199,1996).Think that now environmental antigens plays an important role on the pathomechanism of initiation and promotion disease.But the forfeiture to the self antigen tolerance is considered to mediate psoriasic pathomechanism just.Dendritic cell and CD4+T cell are considered to play an important role in antigen presentation and identification, this present and discern mediated the immunne response that causes psoriasis pathology.We recently according to the CD4+CD45RB metastasis model developed the psoriasis model (Davenport et al., Internat.Immunopharmacol., 2: 653-672).Give anti-IL-20 antibody, anti-IL-22 antibody or at the antibody of IL20R and/or IL22R, anti-IL-22RA antibody for example of the present invention or solubility IL-22RA to mouse.Inhibition to disease score (skin injury, struvite cytokine) shows IL-20 and the validity of IL-22 antagonist in psoriasis, antibody or their acceptors of for example anti-IL20 of for example anti-IL-22RA antibody or IL-22RA soluble receptors or other antagonists and/or IL-22.

The heredity atopic dermatitis

Raising appears in hereditary atopic dermatitis (AD) patient's sample in IL-20 and IL-22.AD is a kind of common chronic inflammation disease, is characterized in belonging to the cytokine overactivity of helper cell subclass 2 (Th2).Although the definite etiology of AD does not know that also having known has a plurality of factors to participate in, and comprises Th2 immunne response, autoimmunity, infection, anaphylactogen and the hereditary inducement of overacfivity.The principal character of disease comprises the chronic dermatitis that eosinocyte rising, SERUM IgE and the IgG1 in axersis (xerosis cutis), scabies overworked (the skin scabies is overworked), conjunctivitis, inflammatory skin damage, infection of staphylococcus aureus, the blood raises and have T cell, mastocyte, scavenger cell and eosinocyte infiltration.Have realized that the colonyization of streptococcus aureus or infection can make AD worsen, and to make this dermatosis be permanent chronic disease.

AD finds in the patient with asthma and allergic rhinitis usually, and often is the initial representation of anaphylactic disease.In western countries, about 20% population is suffered from these anaphylactic diseases, and the sickness rate of AD is owing to ignorant reason rises in developed country.AD is usually in the Childhood, and usually lasts till adult from pubescence always.Treatment to AD at present comprises partial reflunomide, oral Ciclosporin A, non-reflunomide immunosuppressor for example Ta Kemosi (FK506, ointment form) and interferon-gamma.Although multiple AD therapeutic modality is arranged, many patients' symptom is not improved, and perhaps they have produced untoward reaction to medication, needs to seek other more effectively therapeutic medicaments.Solubility IL-22RA polypeptide of the present invention and anti-IL-22RA antibody, comprise anti-human il-22 RA neutralizing antibody, can in the special human diseases of treatment such as hereditary atopic dermatitis, inflammatory skin illness and other inflammatory conditions disclosed herein, be used for and IL-22 and IL-20.

For drug use, solubility IL-22RA of the present invention or anti-IL-22RA antibody are mixed with are used for gi tract external administration, particularly intravenously or subcutaneous administration according to conventional methods.Use bolus injection, sustained release to carry out intravenous administration, for example use micropump or other suitable technology, perhaps in common one to several hours, pour into.Usually, comprise the combination of acceptable carrier on hematopoietic protein and the medicine such as salts solution, buffer salt solution, 5% D-glucose aqueous solution etc. in the formula of medicine.Can also comprise in the prescription that a kind of or more kinds of vehicle, sanitas, solubilizing agent, buffer reagent, albumin cause damage on tube wall to prevent protein attachment.When using this combination treatment, cytokine can be combined in the prescription, perhaps the prescription administration to separate.The method of prescription is well-known in this area, and for example is disclosed in Remington ' s Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton PA in 1990, quotes as a reference here.The scope that therapeutic dosage is general is that every day 0.1 is to the 100mg/kg patient body weight, be preferably 0.5-20mg/kg every day, definite dosage determined according to the standard of accepting by the clinician, and considers the character and the seriousness of the patient's condition that will treat, hereditary property of patient or the like.Determining in this area in the ordinary skill level of dosage.Protein usually after chemotherapy or bone marrow transplantation after administration reach 28 days at most or reach greater than 20 000/mm until platelet count ³, be preferably greater than 50,000/mm ³More commonly, with protein administration in a week or shorter time, administration in one to three day time period usually.Usually, the solubility IL-22RA of the present invention of significant quantity or anti-IL-22RA antibody are to be enough to cause lymphocyte or the propagation of marrow progenitor cell and/or the amount that the differentiation generation significantly increases clinically clinically in the treatment, and this increase shows as the increase of mature cell (for example thrombocyte or neutrophilic granulocyte) cyclical level.To continue to platelet count to the treatment of thrombocyte deficiency disorder and reach at least 20,000/mm ³, be preferably 50,000/mm ³Solubility IL-22RA of the present invention or anti-IL-22RA antibody can also with other cytokine IL-3 for example ,-6 and-11, STEM CELL FACTOR, erythropoietin, G-CSF and GM-CSF combination medicine-feeding.In the scheme of combined therapy, dosage every day of other cytokines generally is: EPO, 150U/kg; GM-CSF, 5-15lg/kg; IL-3,1-5lg/kg; G-CSF, 1-25lg/kg.For example, the combination treatment of use EPO is used for the anaemia patient of low EPO level.

Usually, the dosage that gives of solubility IL-22RA (perhaps IL-22RA analogue or fusion rotein) or anti-IL-22RA antibody will be according to factors such as age of patient, body weight, height, sex, general physical condition and former treatment histories and different.Usually more satisfactory is to provide the solubility IL-22RA or the anti-IL-22RA antibody of dosage range in from about 1pg/kg to 10mg/kg (amount/patient's of medicament body weight) to acceptor, certainly according to circumstances, can give lower or high dosage more.

Solubility IL-22RA or anti-IL-22RA antibody to the experimenter give can be by intravenously, intra-arterial, intraperitoneal, intramuscular, subcutaneous, thoracic cavity in, intrathecal route, pour into or directly to the intralesional injection administration by partial conduit.When giving human cytokines by injection, administration can be the large bolus injection of successive perfusion or single or multiple.

That other route of administration comprises is oral, through mucous membrane, through lung and stride skin.Oral administration is suitable for based on the system of polyester microsphere, zein microballoon, proteinlike granule, polycyanoacrylate microballoon and lipid (referring to for example DiBase and Morrel, " Oral Deliveryof Microencapsulated Proteins; " Protein Delivery:PhysicalSystems, Sanders and Hendren (eds.), pages 255-288 (PlenumPress 1997)).By a kind of like this insulin administration mode example the convenience of intranasal administration (referring to for example Hinchcliffe and Illum, Adv.Drug Deliv.Rev.35:199 (1999)).Can prepare the drying or the liquid particle that comprise IL-22RA, and suck (for example, Pettit and Gombotz, TIBTECH 16:343 (1998) in the patient body by dry powder diverting device, liquid aersol producer or atomizer; Patton et al., Adv.Drug Deliv.Rev.35:235 (1999)).This method is illustrated by AERX diabetes treatment system, and this system is a kind of portable electronics sucker, the Regular Insulin of aerosol-type can be sent into lung.Studies show that big protein to 48000kDa all transports at the auxiliary skin of striding down of low-frequency ultrasonic waves, reach curative concentration, this has proved the feasibility (Mitragotri et al., Science 269:850 (1995)) of passing through percutaneous drug delivery.Use electroporation to provide another to have the mode (Potts et al., Pharm.Biotechnol.10:213 (1997)) of IL-22RA in conjunction with active molecule by percutaneous drug delivery.

The pharmaceutical composition that comprises solubility IL-22RA or anti-IL-22RA antibody can be prepared according to known method, and with useful composition on the preparation medicine, wherein the acceptable carrier combinations forms mixture on therapeutic protein and the medicine.Can tolerate giving of composition if accept the patient of administration, then claim this composition to be " acceptable carrier on the medicine ".Aseptic phosphate buffered saline buffer is an example of acceptable carrier on the medicine.Other appropriate carrier is well-known to those skilled in the art.Referring to for example Gennaro (ed.), Remington ' s Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For realizing therapeutic purpose, amount gives the patient to treat effectively with acceptable carrier on solubility IL-22RA or anti-IL-22RA antibody molecule and the medicine.If the dosage of the combination of acceptable carrier is significant on the physiology on therapeutic molecules of the present invention and the medicine, then claim this combination to give with " effectively measuring in the treatment ".If the existence of medicament cause accepting its patient occurs can detected physiological change, then this medicament is significant on the physiology.For example, if the existence of the medicament of treatment inflammation can be reacted by amelioration of inflammation, claim that then this medicament is significant on the physiology.

The pharmaceutical composition that comprises IL-22RA (perhaps IL-22RA analogue or fusion rotein) or anti-IL-22RA neutralizing antibody can provide with liquid form, aerosol form or solid form.The illustration of liquid form has injectable solution and oral suspension.The solid form of example comprises capsule, tablet and controlled release forms.The illustration of a kind of form in back has miniature infiltration press pump and implant (Bremer et al., Pharm.Biotechnol.10:239 (1997); Ranade, " Implants in Drug Delivery, " Drug Delivery Systems, Ranade andHollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al., " Protein Delivery with Infusion Pumps, " Protein Delivery:Physical Systems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997); Yewey et al., " Delivery of Proteins froma Controlled Release Injectable Implant; " Protein Delivery:Physical Systems, Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).

Liposome provide a kind of by in the intravenously, intraperitoneal, sheath, intramuscular, subcutaneous or by oral, suction or intranasal administration and to the mode of study subject delivery therapeutical peptide.Liposome is a microscopically visible vesica, it is made up of one or more a plurality of lipid bilayer, in the bilayer parcel be the water-based cell (referring to, usually, Bakker-Woudenberget al., Eur.J.Clin.Microbiol.Infect.Dis.12 (Suppl.1): S61 (1993), Kim, Drugs 46:618 (1993), and Ranade, " Site-SpecificDrug Delivery Using Liposomes as Carriers, " Drug DeliverySystems, Ranade and Hollinger (eds.), pages 3-24 (CRC Press1995)).Liposome is similar to cytolemma on forming, so liposome can give safely and can biological degradation.Decide according to the preparation method, liposome has individual layer or multiwalled, and the diameter range of liposome can be from 0.02 μ m to greater than 10 μ m.Various medicaments can be wrapped in the liposome.Hydrophobic medicament is dispensed in the bilayer, and hydrophilic medicament is dispensed on inner water-based space (referring to for example Machy et al., Liposomes In Cell BiologyAnd Pharmacology (John Libbey 1987), and Ostro et al., AmericanJ.Hosp.Pharm.46:1576 (1989)).And, can form and the electric charge of liposome and the treatment availability that surface properties is controlled the medicament that is wrapped by the size, the number of bilayer, the lipid that change liposome.

Liposome can be adsorbed onto on the cell of any kind of almost, slowly discharges the medicament that is wrapped up then.Perhaps, the liposome of absorption can be by the phagocytic cell cell endocytic.After the endocytosis, the lipid generation lyase vivo degradation of liposome discharges the medicament (Scherphof etal., Ann.N.Y.Acad.Sci.446:368 (1985)) that is wrapped up.Behind the intravenous administration, little (0.1 to 1.0 μ m) liposome mainly is positioned at the cellular uptake of the reticuloendothelial system of liver and spleen usually, and greater than the liposome of 3.0 μ m in pulmonary deposition.The cell of reticuloendothelial system is to being used to the medicament of chemotherapy is delivered to the tumor site of scavenger cell and liver than the preferential picked-up of small liposome.

Reticuloendothelial system can be evaded by many methods, comprises that to use heavy dose of liposome particles saturated or carry out optionally scavenger cell inactivation (Claassenet al., Biochim.Biophys.Acta 802:428 (1984)) by the pharmacology mode.In addition, shown glycolipid or polyoxyethylene glycol deutero-phosphatide is incorporated on the film of liposome and also can reduce liposome greatly by reticuloendothelial system picked-up (Allen et al., Biochim.Biophys.Acta 1068:133 (1991); Allen et al., Biochim.Biophys.Acta1150:9 (1993)).

Can also prepare the liposome of target special cells or organ by changing the phosphatide composition or in liposome, inserting acceptor or aglucon.For example, will be used for target liver (Hayakawa et al., JapanesePatent 04-244,018 with the liposome of high-content nonionogenic tenside preparation; Kato et al., Biol.Pharm.Bull.16:960 (1993)).Soy phosphatidylcholine, alpha-tocopherol and oxyethyl group hydrogenated castor oil (HCO-60) are mixed in the methyl alcohol, vacuum concentrated mixture, water restores mixture then, prepares these prescriptions.Use stearyl glucoside mixture (the soybean-derived sterylglucosidemixture in dipalmitoyl phosphatidylcholine (DPPC) and soybean source, SG) and the liposome prescription of cholesterol (Ch), but also be proved to be target liver (Shimizu et al., Biol.Pharm.Bull.20:881 (1997)).

Perhaps, different target ligands can be attached to the surface of liposome, for example antibody, antibody fragment, sugar, VITAMIN and translocator.For example, can use ramiform galactosyl lipid derivant modified liposome, so that target asialoglycoprotein (semi-lactosi) acceptor, this receptor surface expression (Kato and Sugiyama, Crit.Rev.Ther.Drug Carrier Syst.14:287 (1997) at liver cell; Murahashi et al., Biol.Pharm.Bull.20:259 (1997)).Similarly, Wu et al., Hepatology 27:772 (1998) shows with taking off sialic acid Pp63 glycophosphoproteins mark liposome the blood plasma half life of liposome is shortened, and significantly improves the picked-up of liver cell to the liposome that takes off sialic acid Pp63 glycophosphoproteins mark.On the other hand, the accumulation of liposome in liver that comprises ramiform galactosyl lipid derivant can be taken off the sialic acid Pp63 glycophosphoproteins by prior injection and suppressed (Murahashi et al., Biol.Pharm.Bull.20:259 (1997)).Poly rhizome of Chinese monkshood acidifying human serum albumin liposome provides another liposome to be targeted to the method (Kamps et al., Proc.Nat ' l Acad.Sci.USA 94:11681 (1997)) of liver cell.And, Geho, et al. United States Patent (USP) numbering 4,603,044 has been described a kind of liposome vesicle delivery system that is oriented to liver, and it has specificity to the hepatic duct acceptor relevant with liver specialization metabolism cell.

More general organizing in the targeted approach, use the biotinylated antibody labels targets cell special (Harasym et al., Adv.Drug Deliv.Rev.32:99 (1998)) in advance to the expressed aglucon of target cell.After free antibodies in the blood plasma is eliminated, puted together the liposome of streptavidin.In another method, targeting antibodies directly is attached to (Harasym et al., Adv.Drug Deliv.Rev.32:99 (1998)) on the liposome.

The little technique for packing of protein that can use standard is wrapping to polypeptide and antibody in the liposome (referring to for example, Anderson et al., Infect.Immun.31:1099 (1981), Anderson et al., Cancer Res.50:1853 (1990), Cohen et al., Biochim.Biophys.Acta 1063:95 (1991), Alving et al. " Preparation and Use of Liposomes in Immunological Studies; " Liposome Technology, 2nd Edition, Vol.III, Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al., Meth.Enzymol.149:124 (1987)).As noted above, liposome useful in the treatment can contain various ingredients.For example, liposome can comprise the lipid derivant (Allen et al., Biochim.Biophys.Acta 1150:9 (1993)) of polyoxyethylene glycol.

Designed the degradable polymer microsphere, to keep the general high level of human cytokines.From degradable polymer for example poly-(lactide-be total to-glycollide) (PLG), poly-acid anhydrides, poe, non-biodegradable ethyl vinyl acetate polymkeric substance prepare microsphere, wherein protein is enclosed in (Gombotz and Pettit, BioconjugateChem.6:332 (1995) in the polymkeric substance; Ranade, " Role of Polymers in DrugDelivery, " Drug Delivery Systems, Ranade and Hollinger (eds.), pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, " Degradable Controlled Release Systems Useful for ProteinDelivery; " Protein Delivery:Physical Systems, Sanders andHendren (eds.), pages 45-92 (Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney and Burke, Nature Biotechnology16:153 (1998); Putney, Curr.Opin.Chem.Biol.2:548 (1998)).The nanometer bead of polyoxyethylene glycol coating also can be used as the carrier (referring to for example Gref et al., Pharm.Biotechnol.10:167 (1997)) of human cytokines intravenous administration.

The invention still further relates to the active polypeptide that has of chemically modified in conjunction with IL-22RA, for example IL-22RA monomer, all dimerization, assorted dimerization or poly soluble receptors, and the IL-22RA antagonist, the anti-IL-22RA antibody that for example is connected with polymkeric substance as described above or in conjunction with polypeptide or anti-IL-22RA neutralizing antibody.

Those skilled in the art can design other dosage forms, Ansel andPopovich for example, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5 ^ThEdition (Lea ﹠amp; Febiger 1990), Gennaro (ed.), Remington ' sPharmaceutical Sciences, 19 ^ThEdition (Mack Publishing Company1995), and Ranade and Hollinger are shown in the Drug Delivery Systems (CRCPress 1996).

As illustration, pharmaceutical composition can be used as the test kit that comprises container and provides, comprise polypeptide in this container with IL-22RA extracellular domain, for example IL-22RA monomer, all dimerization, assorted dimerization or poly soluble receptors or IL-22RA antagonist (for example with IL-22RA polypeptide bonded antibody or antibody fragment, perhaps anti-IL-22RA neutralizing antibody).Therapeutical peptide can provide with the Injectable solution form, is used for the injection of list or multiple doses, perhaps provides as sterilized powder, can be before injection that powder is heavy molten.Perhaps, can comprise the dry powder decollator in this test kit, liquid aersol maker or atomizer, the administration of being used for the treatment of property polypeptide.This test kit can also comprise the written information about pharmaceutical composition indication and usage.And this information can comprise explanation, indicates the IL-22 composition and be the known patient's forbidding that IL-22RA is had an allergy.

Comprise the pharmaceutical composition of anti-IL-22RA antibody or binding partners (perhaps anti-IL-22RA antibody fragment, antibody fusion protein, humanized antibodies or the like) or IL-22RA soluble receptors, can provide with liquid form, aerosol form or solid form.The illustration of liquid form has injectable solution, aerosol, drops, topical solutions and oral suspension.Exemplary solid form comprises capsule, tablet and controlled release forms.The illustration of a kind of form in back has miniature infiltration press pump and implant (Bremer et al., Pharm.Biotechnol.10:239 (1997); Ranade, " Implants in Drug Delivery, " Drug DeliverySystems, Ranade and Hollinger (eds.), pages 95-123 (CRC Press1995); Bremer et al., " Protein Delivery with Infusion Pumps, " Protein Delivery:Physical Systems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997); Yewey et al., " Delivery ofProteins from a Controlled Release Injectable Implant; " Protein Delivery:Physical Systems, Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).Other solid forms comprise emulsifiable paste, paste, other topical application medicines or the like.

Liposome provide a kind of by in the intravenously, intraperitoneal, sheath, intramuscular, subcutaneous or oral, suction or the intranasal administration approach mode of delivering therapeutical peptide.Liposome is a microscopically visible vesica, it is made up of one or more a plurality of lipid bilayer, be wrapped in the water-based cell in the film (totally referring to Bakker-Woudenberg et al., Eur.J.Clin.Microbiol.Infect.Dis.12 (Suppl.1): S61 (1993), Kim, Drugs46:618 (1993), and Ranade, " Site-Specific Drug Delivery UsingLiposomes as Carriers; " Drug Delivery Systems, Ranade andHollinger (eds.), pages 3-24 (CRC Press 1995)).Liposome is similar to cytolemma on forming, so liposome can give safely and can biological degradation.Decide according to the preparation method, liposome has individual layer or multiwalled, and the diameter range of liposome can be from 0.02 μ m to greater than 10 μ m.Various medicaments can be wrapped in the liposome: the hydrophobicity medicament is dispensed in the lipid bilayer, and the wetting ability medicament is dispensed on inner water-based space (referring to for example Machy et al., Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and Ostro et al., American J.Hosp.Pharm.46:1576 (1989)).And, can form and the electric charge of liposome and the treatment availability that surface properties is controlled the medicament that is wrapped by the size, the number of lipid bilayer, the lipid that change liposome.

Liposome can be adsorbed onto on the cell of any kind of almost, slowly discharges the medicament that is wrapped up then.Perhaps, the liposome of absorption can be by the phagocytic cell cell endocytic.After the endocytosis, the lipid generation lyase vivo degradation of liposome discharges the medicament (Scherphof etal., Ann.N.Y.Acad.Sci.446:368 (1985)) that is wrapped up.Behind the intravenous administration, little (0.1 to 1.0 μ m) liposome mainly is positioned at the cellular uptake of the reticuloendothelial system of liver and spleen usually, and greater than the liposome of 3.0 μ m in pulmonary deposition.The cell of reticuloendothelial system is to being used to chemotherapeutics is delivered to the tumor site of scavenger cell and liver than the preferential picked-up of small liposome.

Reticuloendothelial system can be evaded by many methods, comprises that to use heavy dose of liposome particles saturated or carry out optionally scavenger cell inactivation (Claassenet al., Biochim.Biophys.Acta 802:428 (1984)) by the pharmacology mode.In addition, shown glycolipid or polyoxyethylene glycol deutero-phosphatide is incorporated on the film of liposome and also can reduce liposome greatly by reticuloendothelial system picked-up (Allen et al., Biochim.Biophys.Acta 1068:133 (1991); Allen et al., Biochim.Biophys.Acta1150:9 (1993)).

Can also prepare the liposome of target special cells or organ by changing the phosphatide composition or in liposome, inserting acceptor or aglucon.For example, will be used for target liver (Hayakawa et al., JapanesePatent 04-244,018 with the liposome of high-content nonionogenic tenside preparation; Kato et al., Biol.Pharm.Bull.16:960 (1993)).Soy phosphatidylcholine, alpha-tocopherol and oxyethyl group hydrogenated castor oil (HCO-60) are mixed in the methyl alcohol, vacuum concentrated mixture, water restores mixture then, prepares these prescriptions.Use the stearyl glucoside mixture (SG) in dipalmitoyl phosphatidylcholine (DPPC) and soybean source and the liposome prescription of cholesterol (Ch), but also be proved to be target liver (Shimizu et al., Biol.Pharm.Bull.20:881 (1997)).

Perhaps, different target ligands can be attached to the surface of liposome, for example antibody, antibody fragment, sugar, VITAMIN and translocator.For example, can use branching type galactosyl lipid derivant modified liposome, so that target asialoglycoprotein (semi-lactosi) acceptor, this receptor surface expression (Kato and Sugiyama, Crit.Rev.Ther.Drug Carrier Syst.14:287 (1997) at liver cell; Murahashi et al., Biol.Pharm.Bull.20:259 (1997)).Similarly, Wu et al., Hepatology 27:772 (1998) shows with taking off sialic acid Pp63 glycophosphoproteins mark liposome the blood plasma half life of liposome is shortened, and significantly improves the picked-up of liver cell to the liposome that takes off sialic acid Pp63 glycophosphoproteins mark.On the other hand, the accumulation of liposome in liver that comprises branching type galactolipid derivative can be taken off the sialic acid Pp63 glycophosphoproteins by prior injection and suppressed (Murahashi et al., Biol.Pharm.Bull.20:259 (1997)).Poly rhizome of Chinese monkshood acidifying human serum albumin liposome provides another liposome to be targeted to the method (Kamps et al., Proc.Nat ' lAcad.Sci.USA 94:11681 (1997)) of liver cell.And, Geho, et al. United States Patent (USP) numbering 4,603,044 has been described a kind of liposome vesicle delivery system that is oriented to liver, and it has specificity to the hepatic duct acceptor relevant with liver specialization metabolism cell.

More general organizing in the targeted approach, use the biotinylated antibody labels targets cell special (Harasym et al., Adv.Drug Deliv.Rev.32:99 (1998)) in advance to the expressed aglucon of target cell.After free antibodies in the blood plasma is eliminated, puted together the liposome of streptavidin.In another method, targeting antibodies directly is attached to (Harasym et al., Adv.Drug Deliv.Rev.32:99 (1998)) on the liposome.

Can use the little technique for packing of protein of standard will have IL-22 or IL-20 in conjunction with active anti-IL-22RA neutralizing antibody and binding partners, perhaps with the IL-22RA soluble receptors, be wrapping in the liposome (referring to for example, Anderson et al., Infect.Immun.31:1099 (1981), Anderson et al., Cancer Res.50:1853 (1990), Cohen et al., Biochim.Biophys.Acta 1063:95 (1991), Alving etal. " Preparation and Use of Liposomes in ImmunologicalStudies, " Liposome Technology, 2nd Edition, Vol.III, Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al., Meth.Enzymol.149:124 (1987)).As noted above, liposome useful in the treatment can contain various ingredients.For example, liposome can comprise the lipid derivant (Allen et al., Biochim.Biophys.Acta 1150:9 (1993)) of polyoxyethylene glycol.

Designed the degradable polymer microsphere, to keep the general high level of human cytokines.From degradable polymer for example poly-(lactide-be total to-glycollide) (PLG), poly-acid anhydrides, poe, non-biodegradable ethyl vinyl acetate polymkeric substance prepare microsphere, wherein protein is enclosed in (Gombotz and Pettit, BioconjugateChem.6:332 (1995) in the polymkeric substance; Ranade, " Role of Polymers in DrugDelivery, " Drug Delivery Systems, Ranade and Hollinger (eds.), pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, " Degradable Controlled Release Systems Useful for ProteinDelivery; " Protein Delivery:Physical Systems, Sanders andHendren (eds.), pages 45-92 (Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney and Burke, Nature Biotechnology16:153 (1998); Putney, Curr.Opin.Chem.Biol.2:548 (1998)).The nanometer bead of polyoxyethylene glycol (PEG) coating also can be used as the carrier (referring to for example Gref et al., Pharm.Biotechnol.10:167 (1997)) of human cytokines intravenous administration.

The invention still further relates to the anti-IL-22RA antibody or the binding partners of chemically modified, the anti-IL-22RA antibody or the IL-22RA soluble receptors that for example are connected with polymkeric substance as mentioned above.

Those skilled in the art can design other dosage forms, Ansel andPopovich for example, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5 ^ThEdition (Lea ﹠amp; Febiger 1990), Gennaro (ed.), Remington ' sPharmaceutical Sciences, 19 ^ThEdition (Mack Publishing Company1995), and Ranade and Hollinger are shown in the Drug Delivery Systems (CRCPress 1996).

The present invention relates to comprise the anti-IL-22 antibody compositions of antibody described herein, peptide or polypeptide, and method and therapeutic use.Can also comprise carrier in this composition.Carrier can be conventional organic or inorganic carrier.The example of carrier comprises water, buffered soln, alcohol, propylene glycol, macrogol, sesame oil, Semen Maydis oil or the like.

11. the preparation of transgenic mice

Shown the two overexpression of IL-22 and IL-20 in people's parapsoriasis damage, prompting IL-22 and IL-20 relate to human psoriasic morbidity.And as described herein-in, IL-20 and the IL-22 overexpression in transgenic mice has shown that epidermis thickens with immunocyte and has involved that this is the indication of psoriasis phenotype; In addition, IL-22 is injected in normal mouse shown that epidermis thickens with immunocyte and involve (indication that this is the psoriasis phenotype) that this can eliminate by soluble receptors antagonist zcytor16 (IL-22RA2).Data further point out short scorching IL-22 to participate in the psoriasis morbidity in this body.Like this, the active antagonist of IL-22, anti-human il-22 RA mono-clonal neutralizing antibody for example of the present invention, and solubility IL-22RA acceptor, in the therapeutic treatment of diseases associated with inflammation, be useful, particularly in psoriasis treatment as the antagonist of IL-22 and IL-20.And, in conjunction with, blocking-up, suppress, reduce, antagonism or in and IL-22 or IL-20 and the two active medicament of IL-22, anti-human il-22 RA mono-clonal neutralizing antibody for example of the present invention, and solubility IL-22RA acceptor, in the therapeutic treatment of other diseases associated with inflammation, be useful, for example in hereditary atopic dermatitis, IBD, colitis, endotoxemia, sacroiliitis, rheumatoid arthritis, arthritic psoriasis, adult respiratory disease (ARD), septic shock, multiple organ failure, struvite injury of lung is asthma or bronchitis for example, bacterial pneumonia, psoriasis, oedema, be used as the two antagonist of IL-22 or IL-20 and IL-22 in the treatment of heredity atopic dermatitis and contact dermatitis and inflammatory bowel such as ulcerative colitis and Crohn disease or the like.

In one aspect, the invention provides the method for a kind of preparation at the antibody of polypeptide, this method comprises: will be selected from peptide vaccination in following in animal: the polypeptide of being made up of to 102 (Asp) the amino-acid residue 42 (Ile) of SEQ ID NO:8; The polypeptide of forming to 60 (Ile) by the amino-acid residue 42 (Ile) of SEQ ID NO:8; The polypeptide of forming to 69 (Glu) by the amino-acid residue 42 (Ile) of SEQ ID NO:8; The polypeptide of forming to 81 (Cys) by the amino-acid residue 42 (Ile) of SEQ ID NO:8; The polypeptide of forming to 96 (Lys) by the amino-acid residue 42 (Ile) of SEQ ID NO:8; The polypeptide of forming to 102 (Asp) by the amino-acid residue 42 (Ile) of SEQ ID NO:8; The polypeptide of forming to 69 (Glu) by the amino-acid residue 60 (Ile) of SEQ ID NO:8; The polypeptide of forming to 81 (Cys) by the amino-acid residue 60 (Ile) of SEQ ID NO:8; The polypeptide of forming to 96 (Lys) by the amino-acid residue 60 (Ile) of SEQ ID NO:8; The polypeptide of forming to 102 (Asp) by the amino-acid residue 60 (Ile) of SEQ IDNO:8; The polypeptide of forming to 81 (Cys) by the amino-acid residue 69 (Glu) of SEQ ID NO:8; The polypeptide of forming to 96 (Lys) by the amino-acid residue 69 (Glu) of SEQ ID NO:8; The polypeptide of forming to 102 (Asp) by the amino-acid residue 69 (Glu) of SEQ ID NO:8; The polypeptide of forming to 96 (Lys) by the amino-acid residue 81 (Cys) of SEQ ID NO:8; The polypeptide of forming to 102 (Asp) by the amino-acid residue 81 (Cys) of SEQ ID NO:8; With the polypeptide of forming to 102 (Asp) by the amino-acid residue 96 (Lys) of SEQ ID NO:8; Wherein said polypeptide brings out immunne response in animal, produce described antibody; And from animal separation antibody; Wherein said antibody is specifically in conjunction with IL-20 polypeptide (SEQ ID NO:8).

In one embodiment, provide aforesaid method, wherein the described antibody that is generated by this method can suppress the short scorching active of IL-20 (SEQ ID NO:8).In another embodiment, provide aforesaid method, wherein said antibody can reduce the short scorching active of IL-20 (SEQ IDNO:8).In another embodiment, aforesaid method is provided, wherein said antibody can in and the interaction of IL-20 (SEQ ID NO:8) and IL-22RA (SEQ ID NO:2).Specifically, the neutralizing effect of described antibody is measured by in based on the cells in vitro neutralization test IL-20 (SEQ ID NO:8) being shown neutralization.

Aspect second, the invention provides the antibody that produces by aforesaid method, it with polypeptide shown in the SEQ IDNO:8 can in conjunction with.In one embodiment, the invention provides aforesaid antibody, wherein said antibody is selected from following group: (a) polyclonal antibody, (b) mouse monoclonal antibody, (c) derived from the humanized antibodies of (b), (d) antibody fragment and (e) human monoclonal antibodies.In one embodiment, described antibody also comprises radionuclide, enzyme, substrate, cofactor, fluorescent mark, chemiluminescent labeling, peptide tag, magnetic-particle or toxin.In another embodiment, described antibody also comprises PEGization.

Aspect the 3rd, the invention provides a kind of antibody or antibody fragment, it can combine with the polypeptide that comprises amino acid residue sequence shown in the SEQ ID NO:8, and reduces the short scorching active of IL-20 (SEQID NO:8).In one embodiment, the invention provides and to reduce IL-20 (SEQ ID NO:8) or active antibody of short inflammation or the antibody fragment of IL-22 (SEQ ID NO:6).In one embodiment, the invention provides aforesaid antibody, wherein said antibody is selected from down in the group: (a) polyclonal antibody, and (b) mouse monoclonal antibody, (c) derived from the humanized antibodies of (b), (d) antibody fragment and (e) human monoclonal antibodies.In one embodiment, described antibody also comprises radionuclide, enzyme, substrate, cofactor, fluorescent mark, chemiluminescent labeling, peptide tag, magnetic-particle or toxin.In another embodiment, described antibody also comprises PEGization.

Aspect the 4th, the invention provides a kind of be used to reduce or the suppress propagation of IL-20 inductive hematopoietic cell and hematopoiesis progenitor cell or the method for differentiation, this method comprises cultivates marrow or peripheral blood cells with the composition that comprises a certain amount of antibody as mentioned above, the marrow or the peripheral blood cells of cultivating during with this antibody are not compared, and the amount of described antibody is enough to reduce the propagation or the differentiation of the hematopoietic cell in marrow or the peripheral blood cells.In one embodiment, described hematopoietic cell and hematopoiesis progenitor cell are lymphoidocytes.In another embodiment, described lymphoidocyte is scavenger cell or T cell.

Aspect the 5th, the invention provides a kind of method of the IL-20 of reduction inductive inflammation, this method comprises the composition that gives a certain amount of antibody as mentioned above to the Mammals with inflammation, the amount of this antibody is enough to reduce inflammation.

Aspect the 6th, the invention provides the method for inflammatory reaction in the Mammals that a kind of inhibition has inflammation, this method comprises: (1) determines the proteic level of serum amyloid sample substance A; (2) be included in the composition of the described antibody of claim 3 in the acceptable drug carrier; (3) determine the proteic level of serum amyloid sample substance A after the administration; (4) the proteic level of serum amyloid sample substance A in comparison step (1) and the step (3); Wherein the proteic level of serum amyloid sample substance A does not raise or decline occurs and shows that inflammatory reaction is suppressed.

Aspect the 7th, the invention provides the method for inflammatory reaction in the Mammals that a kind of inhibition has inflammation, this method comprises: (1) determines the proteic level of serum amyloid sample substance A; (2) be included in the composition of the described antibody of claim 5 in the acceptable pharmaceutical carrier; (3) determine the proteic level of serum amyloid sample substance A after the administration; (4) the proteic level of serum amyloid sample substance A in comparison step (1) and the step (3); Wherein the proteic level of serum amyloid sample substance A does not raise or decline occurs and shows that inflammatory reaction is suppressed.

Aspect the 8th, the invention provides the method for inflammatory reaction in the Mammals that a kind of inhibition has inflammation, this method comprises: (1) determines the proteic level of serum amyloid sample substance A; (2) be included in the composition of the described antibody of claim 16 in the acceptable pharmaceutical carrier; (3) determine the proteic level of serum amyloid sample substance A after the administration; (4) the proteic level of serum amyloid sample substance A in comparison step (1) and the step (3); Wherein the proteic level of serum amyloid sample substance A does not raise or decline occurs and shows that inflammatory reaction is suppressed.

Aspect the 9th, the invention provides a kind of treatment and be subjected to the mammiferous method that inflammatory disease that IL-20 works therein torments, this methods of treatment comprises: the antagonist that gives IL-20 to Mammals, inflammation is alleviated, wherein said antagonist comprises with antibody, the antibody fragment of the polypeptide of IL-22RA (SEQID NO:3) or polypeptide fragment specific combination or combines polypeptide, or polypeptide or the polypeptide fragment of IL-22RA (SEQ ID NO:3); And wherein the inflammatory activity of IL-20 (SEQ ID NO:8) is lowered.In one embodiment, described disease is a chronic inflammatory diseases.In another embodiment, described chronic inflammatory diseases is selected from following group: inflammatory bowel, ulcerative colitis, Crohn disease, sacroiliitis, atopic dermatitis or psoriasis.In another embodiment, described disease is the acute inflammation disease.In one embodiment, described acute inflammation disease is selected from following group: endotoxemia, septicemia, toxic shock syndrome, TSS or infectious diseases.In another embodiment, the invention provides aforesaid method, wherein said antibody also comprises radionuclide, enzyme, substrate, cofactor, fluorescent mark, chemiluminescent labeling, peptide tag, magnetic-particle, medicine or toxin.

Aspect the tenth, the invention provides comprise can with human IL-2 0 (SEQ ID NO:8) in be selected from down the antibody of the monoclonal antibody that combines of epi-position of group: the polypeptide of forming to 102 (Asp) by the amino-acid residue 42 (Ile) of SEQ ID NO:8; The polypeptide of forming to 60 (Ile) by the amino-acid residue 42 (Ile) of SEQ ID NO:8; The polypeptide of forming to 69 (Glu) by the amino-acid residue 42 (Ile) of SEQ ID NO:8; The polypeptide of forming to 81 (Cys) by the amino-acid residue 42 (Ile) of SEQ ID NO:8; The polypeptide of forming to 96 (Lys) by the amino-acid residue 42 (Ile) of SEQ ID NO:8; The polypeptide of forming to 102 (Asp) by the amino-acid residue 42 (Ile) of SBQ ID NO:8; The polypeptide of forming to 69 (Glu) by the amino-acid residue 60 (Ile) of SEQ ID NO:8; The polypeptide of forming to 81 (Cys) by the amino-acid residue 60 (Ile) of SEQ ID NO:8; The polypeptide of forming to 96 (Lys) by the amino-acid residue 60 (Ile) of SEQ ID NO:8; The polypeptide of forming to 102 (Asp) by the amino-acid residue 60 (Ile) of SEQID NO:8; The polypeptide of forming to 81 (Cys) by the amino-acid residue 69 (Glu) of SEQ IDNO:8; The polypeptide of forming to 96 (Lys) by the amino-acid residue 69 (Glu) of SEQ ID NO:8; The polypeptide of forming to 102 (Asp) by the amino-acid residue 69 (Glu) of SEQ ID NO:8; The polypeptide of forming to 96 (Lys) by the amino-acid residue 81 (Cys) of SEQ ID NO:8; The polypeptide of forming to 102 (Asp) by the amino-acid residue 81 (Cys) of SEQ ID NO:8; With the polypeptide of forming to 102 (Asp) by the amino-acid residue 96 (Lys) of SEQ ID NO:8.In another embodiment, the invention provides aforesaid antibody, wherein said antibody can reduce or in and human IL-2's 0 (SEQ IDNO:8) activity.In another embodiment, described antibody is selected from down in the group: (a) mouse monoclonal antibody, and (b) derived from the humanized antibodies of (a), (c) antibody fragment and (d) human monoclonal antibodies.In another embodiment, described antibody also comprises PEGization.

Aspect the 11, the invention provides a kind of method for the treatment of pathological condition relevant in the study subject with the activity of IL-20, this method comprises the antibody as mentioned above that gives significant quantity, treats described pathological condition thus.In one embodiment, described pathological condition is the chronic inflammatory diseases patient's condition.In another embodiment, the wherein said chronic inflammatory diseases patient's condition be selected from following in: inflammatory bowel, ulcerative colitis, Crohn disease, sacroiliitis, atopic dermatitis or psoriasis.In another embodiment, described pathological condition is the acute inflammation patient's condition.In another embodiment, the described acute inflammation patient's condition be selected from following in: endotoxemia, septicemia, toxic shock syndrome, TSS or infectious diseases.

The present invention is also illustrated for example by following non-restrictive example.

Embodiment 1

Purifying IL-22RA2-Fc4 polypeptide from the BHK570 cell of transfection

Except that specifying, all operate in 4 ℃ and carry out.Use following step purifying at IL-22RA2 polypeptide (sophisticated soluble receptors polypeptide, the 23-231 residue of SEQ ID NO:13 of C end with people Fc4 (SEQ ID NO:14) fusion; Polynucleotide are seen SEQ ID NO:12), called after IL-22RA2-Fc4.Hang oneself the in the future degerming membrane filtration of about 16500 milliliters of conditioned mediums by 0.2 micron of BHK570 cell of IL-22RA2-Fc4 transfection, add proteinase inhibitor solution then, to final concentration be the bright M-band enzyme of 0.001mM peptide (Boerhinger-Mannheim, Indianapolis, IN), 0.001mM pepstatin (Boerhinger-Mannheim) and 0.4mM Pefabloc (Boerhinger-Mannheim).Dress Poros protein A50 chromatography column (20 milliliters of column volumes, Applied Biosystems) washs with 400 milliliters of PBS (Gibco/BRL).Added the conditioned medium upper prop of proteinase inhibitor, flow velocity is 15 milliliters of per minutes, uses 800 milliliters of PBS (Gibco/BRL) washing then.With 0.1M glycine pH 3.0 wash-out IL-22RA2-Fc4 from post, 5 milliliters of fractions are directly collected among 0.5 milliliter of 2M Tris pH 7.8, adjust the final pH to 7.4 of fraction.

By initial substratum and chromatography column effluent liquid being reduced the Western trace of SDS-PAGE gel, column performance is studied.Use anti-human IgG HRP (Amersham) antibody in the Western trace, be presented at that there is immunoreactive protein at 60000Da place in the initial substratum, no any composition in the effluent liquid is pointed out and is caught fully.The elutriated fraction of protein A50 is studied with reduced form SDS PAGE gel.Show that fraction 3 to 11 all has the very dark band of coomassie brilliant blue staining at the 60000Da place.Mixed fraction 3-11.

(15 ml volumes Millipore) are concentrated into 4 milliliters with Protein A 50 elution mixture from 44 milliliters to use the centrifugal evaporating pipe of 30,000 Da Ultrafree Biomax.With 350 milliliters of PBS (BRL/Gibco) washing Sephacryl S-300 gel permeation chromatography post (175 milliliters of bed volumes; Pharmacia).Enriched mixture is injected in the chromatography column, and flow velocity is 1.5ml/min, uses 225 milliliters of PBS (BRL/Gibco) washing then.Elution peak is collected according to 2 milliliters fraction.

Dye (Geno Technology) SDS-PAGE gel research elutriated fraction with reductibility and irreducibility silver.Reductibility silver dyes the SDS-PAGE gel and shows in the 14-31 fraction that the degree of depth painted band in 60000Da place is arranged, and irreducibility silver dyes in the SDS-PAGE gel demonstration 14-31 fraction degree of depth painted band in 160000Da place is arranged.Fraction 1-13 shows the band of a plurality of different sizes.Mixed 14-31 fraction, (15 ml volumes Millipore) are concentrated to mixture 22 milliliters to use the centrifugal evaporating pipe of 30,000 Da Ultrafree Biomax.Enriched material filters with 0.2 μ m Acrodisc degerming filter membrane (Pall Corporation).

According to our standard step, (Pierce, Rockford IL) measure the protein concn that concentrates mixed fraction, mixture are divided into aliquot ,-80 ℃ of storages by the BCA analysis.The concentration of mixed fraction is 1.50mg/ml.

Embodiment 2

The structure of the BaF3 cell (BaF3/CRF2-4 cell) of expression CRF2-4 acceptor and the BaF3 cell (BaF3/CRF2-4/IL-22RA that expresses CRF2-4 acceptor and IL-22RA acceptor

Cell) structure

Use 30 microgram CFR2-4 expression vectors, the BaF3 cell of construction expression total length CFR2-4 acceptor is described below.The BaF3 cell of expressing the CRF2-4 acceptor is named as the BaF3/CRF2-4 cell.These cells are used IL-22RA acceptor (United States Patent (USP) numbering 5,965, the 704) transfection of total length again with comparing, and are used for according to following description the IL-22 activity being screened.

A. express the structure of the BaF3 cell of CRF2-4 acceptor

From Daudi clone cDNA library, isolate the CRF2-4 (cDNA Genbank login numbering Z17227) of total length, be cloned into then among the expression vector pZP7P.

BaF3 be the preceding lymph sample system that depends on interleukin-13 (IL-3) that derives from mouse bone marrow cells (Palacios and Steinmetz, Cell 41: 727-734,1985; Mathey-Prevotet al., Mol.Cell.Biol. 6: 4133-4135,1986), adding 10% heat-inactivated fetal bovine serum, 2ng/ml mouse IL-3 (mIL-3) (R ﹠amp; D, Minneapolis, MN), 2mM L-glutaMax-1 ^TM(Gibco BRL), 1mM Sodium.alpha.-ketopropionate (Gibco BRL) and PSN microbiotic (GIBCO BRL)) perfect medium ((JRHBioscience Inc., Lenexa keep in KS) the RPMI substratum.Before electroporation,, use Qiagen Maxi Prep test kit (Qiagen) preparation and purifying CRF2-4/pZP7P plasmid according to the specification sheets of manufacturers.For electroporation, the RPMI substratum rinsing BaF3 cell of usefulness serum-free once is resuspended in the RPMI substratum of serum-free, and cell density is 10 ⁷Individual cells/ml.The CRF2-4/pZP7P plasmid DNA of 1 milliliter resuspended BaF3 cell and 30 micrograms is mixed, transfer among the independent disposable electroporation Xiao Chi (GIBCO BRL).After the incubated at room 15 minutes, pair cell carries out electric shock (800lFad/300V. continuously twice; 1180lFad/300V.), electric shock is by electroporation device (CELL-PORATOR; GIBCO BRL) provides.After the recovery in 5 minutes, in cell transfer to the 50 milliliter perfect medium with electroporation, placed incubator interior 15-24 hour (37 ℃, 5%CO ₂).Then with cell centrifugation, be resuspended in T-162 and shake in the perfect medium that in the bottle 50 milliliters contain 2 mcg/ml tetracyclines, separate anti-tetracycline cell mass.To the mixture (after this being called the BaF3/CRF2-4 cell) of the BaF3 cell of transfection according to following description detection signal transduction ability.And, these cells are further used the transfection of IL-22RA acceptor, as described below.

B. express the structure of the BaF3 cell of CRF2-4 acceptor and IL-22RA acceptor

The BaF3/CRF2-4 cell of expressing total length IL-22RA acceptor is according to as mentioned above, uses the IL-22RA expression vector establishment of 30 micrograms.After recovery, use 200 μ g/mlzeocin and 2 μ g/ml tetracyclines screening transfectant.The BaF3/CRF2-4 cell of expressing the IL-22RA acceptor is named as the BaF3/CRF2-4/IL-22RA cell.These cells are used to screen IL-22 activity described herein and IL-22RA antagonistic activity.

Embodiment 3

Use Alamar Blue proliferation test, with BaF3/CRF2-4/IL-22RA cell screening IL-22

Antagonistic activity

A. use Alamar Blue proliferation test, with BaF3/CRF2-4/IL-22RA cell screening IL-22 activity

According to following description, use the existence of IL-22-CEE (embodiment 4) the test proliferation activity of purifying.According to following description, in this test, use the propagation of IL-22RA2-Fc4 (embodiment 1) the antagonism IL-22 of purifying to reply.

Centrifugation BaF3/CRF2-4/IL-22RA cell has added 10% heat-inactivated fetal bovine serum, 2ng/ml mouse IL-3 (mIL-3) (R with perfect medium; D, Minneapolis, MN), 2mM L-glutaMax-1 (Gibco BRL), 1mM Sodium.alpha.-ketopropionate (Gibco BRL) and PSN microbiotic (GIBCO BRL)) (RPMI substratum (JRH Bioscience Inc., Lenexa KS), does not still have the substratum rinsing (being called as " substratum of no mIL-3 " after this) of mIL-3.Cell centrifugation and rinsing 3 times are to guarantee to remove mIL-3.Use the blood counting instrument counting cells then.Cell is placed 96 orifice plates, 5000 every holes of cell, volume is 100 microlitres/hole, uses the substratum of no mIL-3.

Substratum with no mIL-3 is diluted to 50,10,2,1,0.5,0.25,0.13 with IL-22-CEE albumen, and the concentration of 0.06ng/ml is used to assess the propagation of BaF3/CRF2-4/IL-22RA cell.In the BaF3/CRF2-4/IL-22RA cell, add the described diluted protein of 100 microlitres.Total detection volume is 200 microlitres.Detect culture plate at 37 ℃, 5%CO ₂Hatched under the condition 3 days, at this moment add Alamar Blue (Accumed, Chicago, IL), every hole 20 microlitres.Then at 37 ℃, 5%CO ₂Culture plate 24 hours again under the condition.Alamar Blue provides the fluorescence metering reading according to the number of viable cell, and therefore comparing with negative control is exactly directly measuring of on cell proliferation.Culture plate is placed 37 ℃ again, 5%CO ₂The middle cultivation 24 hours.At Fmax ^TM(Molecular Devices Sunnyvale CA) goes up dull and stereotyped reading, uses SoftMax to read the plate instrument ^TMProgram, wavelength are 544nm (exciting) and 590 nanometers (emission).The result has confirmed that the BaF3/CRF2-4/IL-22RA cell has the response of dose-dependently propagation to IL-22-CEE.So the response of measuring is about 15 times of background 50ng/ml high-end, induces 2 times at the low side of 0.06ng/ml.BaF3 wild-type cell and BaF3/CRF2-4 cell do not produce propagation in response to IL-22-CEE, show that IL-22 is special for the CRF2-4/IL-22RA receptor heterodimer.For the activity of determining that IL-22RA2 whether can antagonism IL-22, use the solubility IL-22RA2/Fc4 of purifying to repeat above-described detection.When IL-22 and 10 μ g/ml IL-22RA2 combination, the response to IL-22 under all concentration all is lowered to background level.The proliferative effect that IL-22 has been eliminated in the existence of solubility IL-22RA has shown that IL-22RA is the strong antagonist of IL-22 aglucon.Can use the active antagonist of this detection test other IL-22 described herein, for example anti-IL-22RA antibody.

Embodiment 4

Purifying IL-22-CEE from the BHK570 cell

Unless otherwise indicated, all operations are all carried out at 4 ℃.Following step is used for IL-22 polypeptide (the SEQ ID NO:15 that purifying contains C end GluGlu (EE) label; Perhaps SEQ IDNO:16).With Amicon S10Y3 spiral cylinder concentrated conditioned medium on ProFlux A30 from the bhk cell of expressing IL-22-CEE.In spissated conditioned medium, add proteinase inhibitor solution, final concentration is 2.5mM ethylenediamine tetraacetic acid (EDTA) (EDTA, SigmaChemical Co.St.Louis, MO), 0.003mM leupeptin (Boehringer-Mannheim, Indianapolis, IN), 0.001mM pepstatin (Boehringer-Mannheim) and 0.4mM Pefabloc (Boehringer-Mannheim).Sampling analysis, with large volume-80 ℃ of storages, until the beginning purifying.By SDS-PAGE and Western trace, the proteic concentration of general objective that concentrates in the conditioned medium is analyzed, wherein use the anti-EE antibody of having puted together HRP.

The post (preparing according to following description) of about 100 milliliters of anti-EE G-Sepharose is injected Waters AP-5, in 5cm * 10cm glass chromatography column.(PerSeptive BioSystems, Framingham MA) go up dress post and use phosphate buffered saline buffer (PBS) pH 7.4 balances at BioCad Sprint.Spissated conditioned medium is melted, with 0.2 micron filter sterile filtration, be adjusted to pH7.4, go up sample then and spend the night to post, flow velocity is about 1 ml/min.Chromatography column is with phosphate buffered saline buffer (PBS) pH 7.4 washing of 10 column volumes (CVs), then with 200ml contain 0.5mg/ml EE peptide (Anaspec, San Jose, PBS CA) (pH 6.0) wash-out, flow velocity is 5 ml/min.The EE peptide that uses has sequence EYMPME (SEQ ID NO:15).PBS with 10CV washes post, uses the 0.2M glycine pH3.0 wash-out of 5CV then.Chromatography column pH to 7.0 with behind the 5XPBS adjusting glycine wash-out of 2CV uses PBS (pH7.4) balance then.In whole elution chromatography process, collect 5 milliliters fraction, and monitor 280 and the light absorption value of 215nm, keep effluent and purging compound and also analyze.Use SDS-PAGE silver to dye and Western trace (with the anti-EE antibody of having puted together HRP) evaluating objects albumen to EE-polypeptide elution peak fraction.Merge the target polypeptides elutriated fraction, the film that uses 10,000 Dalton molecular weights to hold back circles round, and (Millipore, Bedford MA), according to manufacturer's specification sheets, are concentrated into 5.0 milliliters with mixture from 60 milliliters to centrifugal thickener.

For the protein separation of IL-22-CEE and other copurifications is opened, with concentrate the polypeptide wash-out merge the POROS HQ-50 post that fraction is splined on pH8.0 (reinforcing yin essence ion exchange resin, from PerSeptive BioSystems, Framingham, MA).In 1.0 * 6.0cm chromatography column, injecting chromatography media and dress post on the BioCad Sprint.The chromatography column load counter ion, use 20mM Tris (pH8.0) balance (Tris, Tutofusin tris) then.With sample dilution in 1: 13 (to reduce the ionic strength of PBS), be splined on then on the PorosHQ chromatography column, flow velocity is the 5ml/ branch.Chromatography column washs with the 20mM Tris pH 8.0 of 10CV, uses the 20mM Tris/1M sodium-chlor gradient elution of 40CV then, and flow velocity is 10ml/ minute.In whole elution chromatography process, collect 1.5 milliliters fraction, and monitor 280 and the absorbancy at 215nm place.Use the SDS-PAGE argentation to analyze to the elution peak fraction.Merge the target fraction, (Millipore, Bedford MA), according to manufacturer's specification sheets, will merge thing and be concentrated into the 1.5-2 milliliter centrifugal thickener to use 10,000 Dalton molecular weight mwco membranes to circle round.

For the copurification albumen of IL-22-CEE polypeptide with free EE peptide and any pollution is separated, the concentrated fraction that is combined is at 1.5 * 90cm Sephadex S200 (Pharmacia, Piscataway, NJ) carry out size exclusion chromatography with flow velocity 1.0ml/min, use BioCad Sprint on the chromatography column, this post is balance and last sample in PBS.In whole chromatography process, collect 1.5 milliliters fraction, and monitor 280 and the absorbancy at 215nm place.The elution peak fraction uses the SDS-PAGE argentation to identify, only merges the purest fraction.This material has been represented the IL-22-CEE polypeptide of purifying.

At last the material of purifying is splined on 4 milliliters ActiClean Etox (Sterogene) chromatography column, to remove any residual intracellular toxin.Make sample four times by PBS equilibrated gravitational stratification post, chromatography column is with a PBS washing of 3 milliliters then, and washings mixes with " cleaning " sample.Use 0.2 micron filter sterile filtration then, and be stored under-80 ℃, until packing.

The Western trace, on coomassie brilliant blue staining and the silver-colored painted SDS-PAGE gel, the IL-22-CEE polypeptide presents a master tape.With the BCA analytical method (Pierce, Rockford IL) measure the protein concentration of purifying substance, with the protein packing, then according to the step of standard-80 ℃ of storages.

In order to prepare anti-EE-Sepharose, Protein G-Sepharose (Pharmacia with the 100 milliliters of bed volumes of 100ml PBS washing that contain 0.02% sodiumazide, Piscataway NJ) 3 times, wherein uses 0.45 micron filtration unit of Nalgene of 500 milliliters.Gel with the 200mM trolamine pH8.2 of 6.0 volumes (TEA, Sigma, St.Louis, MO) washing adds isopyknic antibody-solutions that comprises 900 milligrams of EE antibody then.After 4 ℃ of overnight incubation, according to top description washing resin, remove unconjugated antibody with the 200mM TEA of 5 volumes.With the resuspended resin of the TEA of 2 volumes, transfer in the suitable container, (Pierce, Rockford IL) join in Protein G-Sepharose gel, and final concentration is 36mg/ml with being dissolved in dimethylpimilimidate-2HCl among the TEA.Gel is jolted 45 minutes in room temperature, use above-described filtration unit to remove liquid.20mM thanomin by at room temperature using 5 volumes (in/200mM TEA) was hatched 10 minutes the non-specific site on the sealing glue jointly with gel.Then with the 5 volume PBS detergent gel that contain 0.02% sodiumazide, 4 ℃ of storages in this solution.

Embodiment 5

Effect in the body of IL-22 polypeptide

Mouse (female, C57BL/6N, 8 weeks are big; Charles River Labs, Kingston NY) is divided into three groups.Used standard method to prepare the adenovirus of expression IL-22 polypeptide (SEQ IDNO:6) in the past.At the 0th day, give parent's adenovirus or IL-22 adenovirus to first group (n=8) and second group of (n=8) mouse respectively by the tail vein, the dosage that every mouse obtains is in about 0.1 milliliter about 1 * 10 ¹¹Individual particle.The 3rd group (n=8) do not handle.The 12nd day, mouse is weighed and gets blood.Sample is carried out complete blood count (CBC) and serum chemistry analysis.Group with respect to handling with parent's adenovirus detects neutrophilic granulocyte and platelet count in statistically evident rising in the blood sample of IL-22 adenovirus administration group.And, with respect to the group of handling with parent's adenovirus, in the blood sample of IL-22 adenovirus administration group, detect lymphocyte and red blood cell count(RBC) and on statistics, significantly descend.In addition, the mouse weight loss of handling through the IL-22 adenovirus, and the mouse weight increase of handling through parent's adenovirus.And the 3rd day the time in the serum IL-22 level raise and glucose level reduces.In a word, IL-22 adenovirus mouse shows acute phase reaction, and this reaction also can be by other for example TNF-α, IL-1 β and the initiation of gp130 cytokine of pro-inflammatory cytokine.Acute phase reaction is that a series of what caused by the pattern recognition molecule is the reaction of inflammation disease.Acute phase protein provides the enhanced protection for the opposing microorganism, and by the influence of pair cell transportation and amboceptor release inflammatory reaction is modified.For example, SAA has powerful leukocyte activation function, comprises inducing chemotaxis, improving adhesion and the phagocytotic increase of white corpuscle to endotheliocyte.Understanding and cause and change acute phase reaction degree and the factor of time length, is that exploitation is to the anti-infective and important step inflammatory disease novel therapies.

Results suggest IL-22 influences hemoposieis, and promptly intravital hemocyte forms.Like this, IL-22 may have the biological activity of the different blood stem cells of influence, causes the increase or the minimizing of some differentiation hemocyte in the specific cells pedigree.For example, IL-22 seems to reduce lymphocyte, and this may be because to due to the inhibition of the directed progenitor cell that produces lymphoidocyte.IL-22 also reduces red corpuscle, and this has supported the viewpoint that IL-22 may work by the hemocyte that influences these processes of participation in anaemia, infection, inflammation and/or Immunological diseases.The antagonist of IL-22, for example antibody or its soluble receptors IL-22RA2 can be used as medicine in these diseases.

And these use the overexpression of the experiment prompting IL-22 of IL-22 adenovirus to increase neutrophilic granulocyte and hematoblastic level in the body in mouse.Can imagine in whole animal system, to also have other the factor (for example cytokine and modifying factor) to participate in replying to IL-22.But these data illustrate that strongly IL-22 participates in hemoposieis.Therefore, IL-22 and acceptor thereof are diagnosis and the suitable medicament/target for the treatment of multiple disorder, for example inflammation, immunologic derangement, infection, anaemia, hemopoietic system and other cancers or the like.

Embodiment 6

Express the transgenic mice of IL-22

A. express the preparation of the transgenic mice of mouse IL-22

Method preparation by standard is from containing 5 of lymph sample specificity E μ LCK promotor ' and 3 ' flanking sequence, mouse IL-22 (SEQ ID NO:10; Polypeptide shows in SEQ ID NO:11), the dna fragmentation of the transgene carrier of rat insulin II intron, IL-22 cDNA and human growth hormone poly A sequence, and the microinjection experimental technique of the standard of use, be used for the B6C3f1 (Taconic of microinjection to fertilization, Germantown is NY) in the mouse ovum.Referring to Hogan, B.et al., Manipulating the Mouse Embryo.A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1994.

In 154 young babies, find that 25 belong to transgenosis for mouse I L-22 and lymph sample specificity E μ LCK promotor.11 transgenic mice young babies death in the several hrs of birth back is carried out necrotomy to 9 transgenosis young babies that have bright looking at birth, and two long to growing up.Expression level is low in an adult mice.Preparation postmortem young baby's tissue, and carry out histological examination according to describing below.

Newborn young baby's bright appearance seems relevant with the sclerosis of skin, has soon done as skin, causes the decline of normal nurture.It is stiff that their action generally becomes.

B. the genotype of transgenic mice and expression analysis

In the IL-22 transgenic mice strain that is subjected to E μ Lck promoters driven from the above description, observing newborn young baby promptly had unusually first day (the birth same day), put to death and got tissue.Sign for all young babies with going up unique ear tag, note those have bright skin phenotype when putting to death individuality.In 12 young babies, observe for 6 and have bright skin phenotype, wherein two are called as and have " seriously " phenotype.The definition of serious phenotype is movable hardly, its skin light and very dry little young baby especially.Get skin from each young baby's left-external side, freezing in the Tissue-Tek embedding medium.

Gene type assay confirms that bright skin is a good transgenosis Status Flag, although do not collect the data of expressing.The refrigerated skin chunk is cut into 7 microns section on cryostat, the existence of CD3, CD4, CD8, mouse macrophage, B cell, CD80 and MHC II quasi-molecule is observed in dyeing.The dyeing scheme comprises with commercial antibody and tissue bond, uses two anti-detections through the superoxide enzyme labelling, observes dyeing through the DAB chromogen.

Find that transgenic animal contain higher levels of MHC II quasi-molecule and CD80, their dye respectively antigen presenting cell and dendritic cell.In serious and non-serious transgenic animal, the scavenger cell mark also detects than more cell is arranged in the wild-type animal, although the distribution of these cells is confined in the corium of top very much.The animal that is divided into serious phenotype all has the strongest dyeing for all three marks, shows to compare with wild-type, and cell density and quantity have significant rising.This variation may be because the expression level of IL-22 in these transgenosiss person of foundation young baby is different.MHC II class positive cell is positioned at bottom corium, be loose opening bunch shape and arrange, and the CD80 positive cell mainly is positioned at below the corium, and it is positioned at or has just exceeded muscle/lipid layer.These two groups of cells seem not overlap.It is every other that to be marked in all animals dyeing identical.Toluidine blue staining to mastocyte shows between wild-type and the transgenic animal to have only small difference or as broad as long.

C. the tissue to transgenic mice carries out microscopical analysis: the IL-22TG with EuLck promotor has newborn lethal histology

On the same day of birth, contain the genetically modified a brood of young baby of IL-22 and given euthanasia painlessly, be immersed in the 10% buffered formalin solution whole corpse fixing.Get 6 transgenosiss and 2 non-transgenic young babies do further inspection.Finding when carrying out euthanasia has 4 skins with light in 6 transgenic mices.To cut into 5 (head rip cuttings through the fixed tissue; Upper and lower thoracic cavity and upward abdomen crosscut).Organization embedding in paraffin, is handled routinely, and (Wetzlar Germany), uses H﹠amp for Jung 2065 Supercut slicing machines, Leica Microsystems in 5 microns sections; E dyeing.(Melville checks under NY) painted tissue for Nikon Eclipse E600, Nikon Inc. at opticmicroscope by the veterinary pathologist of (ACVP) council certificatory.

When carrying out microscopy, find that two transgenosis young babies' epidermis is thicker than other six young babies' (comprising contrast) epidermis.On the skin of any mouse and its hetero-organization, do not find the unusual of other.To under the 40X object lens, taking a picture from the representative skin region of thoracic cavity and belly respective regions, use be connected CoolSnap digital camera on the microscope (RoperScientific, Inc., San Diego, CA).Using-system morphology software (ScionImage for Windows (NIH Image), Scion Corp., Frederick, MD v.B4.0.2) measures the thickness of epidermis, shows in result's table 5 below.

Table 5

Genotype/phenotype	Average skin of chest thickness (μ m)	Average skin of abdomen thickness (μ m)
			Non-transgenic/normal	5.2	5.4
Transgenosis/non-light	5.0	6.7
			Transgenosis/light	8.2	7.4
Transgenosis/all	7.1	7.1

Because the mouse number is not enough, so can not determine significance,statistical; But transgenic mice particularly has the mouse of bright skin, compares with the contrast of non-transgenic mouse with the transgenic mice of non-bright skin and tends to have thicker epidermis.The light transgenic mice may have higher IL-22 expression level than non-bright transgenic mice; But do not measure its expression level for these mouse.These results suggest IL-22 role in psoriasis, arthritic psoriasis or other inflammatory skin illnesss or in other inflammatory diseases.

Embodiment 7

Effect in the body of IL-22 polypeptide

A. the mouse that infects the IL-22 adenovirus shows inducing of SAA

With mouse (female, C57BL/6N, 8 weeks are big; Charles River Labs, Kingston NY) is divided into three groups.Use standard method to prepare the adenovirus of expression IL-22 polypeptide (SEQ ID NO:6) in advance.At the 0th day, give parent's adenovirus or IL-22 adenovirus to first group (n=8) and second group of (n=8) mouse respectively by the tail vein, the dosage that every mouse obtains is in about 0.1 milliliter about 1 * 10 ¹¹Individual particle.The 3rd group (n=8) do not handle.The 12nd day, mouse is weighed and gets blood.At the 20th day of research, put to death mouse, the record body weight, and collect blood and tissue is used for analyzing.

The whole blood sample is carried out complete blood count (CBC) and serum chemistry analysis.With respect to the group of handling with the parent adenovirus, IL-22 adenovirus administration group detects neutrophilic granulocyte and platelet count significantly rising on statistics in the blood sample of the 12nd day and the 20th day.And with respect to the group of handling with the parent adenovirus, the lymphocyte cell in the 12nd day blood sample of IL-22 adenovirus administration group significantly descends statistically, but reverse effect occurs at the 20th day.In addition, the IL-22 adenovirus is handled the weight loss of mouse, and parent's adenovirus is handled the weight increase of mouse.And during two time points in the serum sample of IL-22 adenovirus treatment group G/W on average significantly reduce than the glucose level of parent's adenovirus treatment group.Serum albumin also significantly reduces during two time points.Blood urinary nitrogen level significantly reduced in the time of the 20th day.The serum globulin level of IL-22 adenovirus administration group significantly raises than parent's adenovirus treatment group during two time points.At microscopically, observe because the techtology change that IL-22 causes is the regeneration of kidney mesonephric tubule.Although unrare, the incidence and the seriousness of rising are arranged but in this treated animal.The feature of ephrosis is many focuses of basophilic cell zone of cortex uriniferous tubules epidermic cell.

Carry out the another one experiment identical with above-described experimental design, purpose is confirmed results and collects extra sample.In this research, write down a body weight in per three days, inject in adenovirus and got blood from mouse in back 3 days, put to death mouse and collect blood and tissue at the 10th day (every group of n=4) and the 20th day (every group of n=4).With respect to the group of handling with parent's adenovirus, in the blood sample of IL-22 adenovirus administration group, detect the neutrophilic granulocyte and the platelet count of rising once more.For neutrophilic granulocyte, this result is promptly obvious at the 3rd day, but for thrombocyte, significant difference occurs until the 10th talent.And at the 3rd day and the 10th day, IL-22 adenovirus administration group medium size lymphocyte counting is with respect to parent's adenovirus treatment group remarkable decline being arranged, and was still different with the research of front, do not raise at the 20th day.Equally, in whole research process, accept the mouse weight loss of IL-22 adenovirus, and the weight increase of contrast virus treated mouse and untreated mice.The result of serum chemistry parameter is consistent with the research of front.In this research, also proved conclusively with the IL-22 adenovirus and handled relevant kidney mesonephric tubule regenerated histology discovery.This also is consistent with the albuminuretic extra discovery of relevant moderate in the mouse that gives the IL-22 adenovirus (the 20th day).

Results suggest IL-22 influences hemoposieis, and promptly intravital hemocyte forms.Like this, IL-22 may have the biological activity of the different blood stem cells of influence, causes some the differentiation hemocyte in a certain specific cells pedigree to increase or reduce.For example, IL-22 seems to reduce lymphocyte, this may be owing to produce due to the directed progenitor cell of lymphoidocyte is suppressed, and this has supported IL-22 may be in anaemia, infection, inflammation and/or Immunological diseases to participate in the viewpoint that the hemocyte of these processes work by influence.The antagonist of IL-22, for example antibody or its soluble receptors IL-22RA2 can be used as medicine in these diseases.

And these use the overexpression of the experiment prompting IL-22 of IL-22 adenovirus to increase neutrophilic granulocyte and hematoblastic level in the body in mouse.Can imagine in whole animal system, to also have other the factor (for example cytokine and modifying factor) to participate in replying to IL-22.But these data support IL-22 to participate in hemoposieis strongly.Therefore IL-22, anti-IL-22 antibody, IL-22RA soluble receptors (for example SEQ ID NO:3) and anti-IL-22RA antibody are diagnosis and the suitable medicament/target for the treatment of multiple disease, and described disease for example is inflammation, dysimmunity, infection, anaemia, hemopoietic system and other cancers or the like.

The appearance of the expression of IL-22 and weight loss, acute phase protein SAA and to reduce the metabolism disorder that is reflected by serum glucose, albumin and urinary nitrogen relevant, prompting IL-22 is the cytokine that works in early days in some inflammatory reaction.The mouse that gives the IL-22 adenovirus may show a kind of state of chronic inflammatory diseases, as observed in IBD, ulcerative colitis, sacroiliitis, psoriasis, arthritic psoriasis, the asthma etc.Some deleterious inflammatory process can be by using IL-22 antagonist such as acceptor such as IL-22RA soluble receptors (for example SEQ ID NO:3) and anti-IL-22RA antibody or the like of anti-IL-22 antibody, IL-22 be suppressed.

B.IL-22 is a kind of pro-inflammatory cytokine: the Serum SA A level in the adenovirus IL-22 mouse

Carry out ELISA, measure the SAA level in the IL-22 adenovirus mouse, wherein use mouse SAA immunity detection reagent and method (Biosource International, California, USA).Enzyme plate in advance with anti-mouse SAA antibody sandwich, will be diluted good standard substance and unknown sample and the anti-mouse SAA of HRP-then and together join in the plate.Plate was hatched 1 hour at 37 ℃, washed according to the test kit specification sheets then.Use TMB plate to be developed the color 15 minutes, stop with 2M sulfuric acid in room temperature.(Molecular Devices, California USA) read the absorbancy at 450nm place to use Spectromax 190.With Softmax Pro (Molecular Devices, California, USA) and Excel (Microsoft Corp., Washington USA) analyze the data obtain.

Its mSAA level of mouse that infects the IL-22 adenovirus raises greatly, is more than 10 times of parent's adenovirus mouse contrast.

The flow cytometry analysis of C.IL-22 adenovirus infection mouse

Express the effect of IL-22 in vivo in order to analyze adenovirus, we have separated peripheral blood, spleen and marrow with the 20th day at metainfective the 10th day from the C57BL/6N mouse of IL-22 adenovirus infection.In the test tube of heparinization, collect about 100 μ l blood, remove clean erythrocyte (about 5 seconds of cell cracking in 4.5 milliliters distilled water adds 1.5 milliliter of 3.6% sodium-chlor afterwards) by the low-tension cracking then.Pulverize spleen with two obscure glass slide glasses, the cell that makes release precipitates then by Nytex film (cell filter screen).Femur is pulverized in mortar, cell by cell filter screen (Falcon), is obtained marrow.Cell is resuspended in (lavation buffer solution=HBSS/1%BSA/10mM HEPES) in the FACS lavation buffer solution.Count with trypan blue.Every type cell gets 1 * 10 ⁶Individual viable cell branch installs in 5 milliliters the polystyrene test tube.Rinsing and sedimentation cell, on ice with fluorescent mark (FITC, PE, and the CyChrome) monoclonal antibody (PharMingen that can discern different cell surface markers (being used to identify certain immunocyte subclass), San Diego, CA) mixture was hatched 20 minutes jointly.That these marks comprise is following (being listed in the 3rd group that we detect): dye for blood: CD3, Gr1, and B220; Dye for spleen: CD62L, CD44, and CD3; CD21, CD23, and B220; IgD, IgM, and B220; CD11b, Gr1, and CD8; For bone marrow stain: CD11b, Gr1, CD3; IgD, IgM, and B220.Cell also precipitates with 1.5 milliliters WB rinsing, is resuspended in then among 0.4 milliliter of WB, and (BectonDickinson, Mountain View CA) analyzes with CellQuest software on FACScan.

We find that the neutrophilic granulocyte fraction in the blood of IL-22 adenovirus processing mouse at the 10th day rising 4-13 has doubly raise 2-3 times at the 20th day.At the 10th day, this difference caused blood medium size lymphocyte and monocyte fraction to descend thereupon.In marrow, we find that the B total cellular score had reduced about 1.5 times in the 10th day, and the sum of the per-cent of sophisticated recirculation B cell and immature B cells has only decline slightly.At the 20th day, have in these differences many no longer obvious, although we find that really the fraction of ripe recirculation B cell has slight rising.In spleen, the sum of B cell all had reduction slightly (1.5-2 doubly) at the 10th day and the 20th day, and at the 20th day, edge district B cell fraction (CD21+CD23-B220+) increased by 2 times, and the number of folliculus B cell (CD21+CD23+B220+) has reduced by 2 times.B cell in edge district is considered to resist the first line of defence of cause of disease, because compare with more general folliculus B cell, they are more responsive to B cell mitogen (for example LPS), and when they run into their isogeneic, they will be divided into antibody secreting cell rapidly.IL-22 might improve follicular cells to the transformation of edge district B cell or optionally remove comparatively jejune follicular cells.The change of the B cell number of finding in marrow can reflect the increase of preceding/pro B lymphocyte or immature B cells differentiation, and perhaps recirculation B cell increases from the interior stream of blood/spleen, and may follow immature B cells to increase to the outward transport of peripheral blood.The actual number of ripe BMB cell does not increase, so IL-22 may not strengthen their propagation.Perhaps, IL-22 may block, reduces or suppress the differentiation of immature B cells, thereby improves the relative proportion of mature B cell.

D.IL-22RA2-Fc4 in vivo in and the activity of IL-22: SAA ELISA shows to be expressed by IL-22 inductive SAA and is suppressed by the injection of IL-22RA2-Fc4:

Whether can suppress IL-22 and induce SAA in order to measure IL-22RA2, with mouse (female, C3H/HEJ, 8 weeks are big; Jackson Labs, Bar Harbor ME) is divided into 5 groups, 3 every group, shows that according to table 6 injecting protein by the intraperitoneal approach handles:

Table 6

Group #	IL-22	IL-22RA2
			Group 1:	-	-
Group 2:	-	100μg
			Group 3:	3μg	-
Group 4:	3μg	20μg
			Group 5:	3μg	100μg

IL-22RA2 is injected at the IL-22 injection to carry out before in 15 minutes.The intraperitoneal approach is all passed through in two kinds of proteic injections.Before processing from each mouse blood sampling, 2 and 6 hours blood samplings after processing then.From each specimen preparation serum, be used to measure SAA and IL-22.

According to front description carrying out ELISA, to measure with the SAA level in the mouse of IL-22 and solubility IL-22 acceptor described herein and IL-22RA2-Fc4 processing.Use to show in the common mouse of handling of IL-22RA2-Fc4 of 3 microgram IL-22 and 20-100 microgram concentration and be reduced to background level, show that IL-22RA2 has suppressed the SAA induced activity of IL-22 in vivo by the independent inductive SAA of IL-22 level.

Embodiment 8

In the inflammatory bowel mouse model, express IL-22

Inflammatory bowel (IBD) is a kind of multi-factor disease, is divided into two types, i.e. ulcerative colitis (UC) and Crohn disease (CD).The etiology of these diseases is not also known now, and clinical manifestation is variant.UC is limited to colon, and its symptom comprises bloody diarrhea, weight loss and stomachache.Feature comprises the colon of spot ulcer and shortening under the anatomical lens of UC.On the contrary, Crohn disease also can influence other parts of intestines.Symptom comprises diarrhoea (compare with UC, it is comparatively rare to have blood in stool), low fever and pain.Feature comprises fibrosis and stenosal intestines under the anatomical lens, has narrow, deep ulcer, crackle and fistula.

There are many animal models to simulate these human diseasess.Three kinds of colitis model commonly used that are used for the newtype drug screening are 2,4,6-trinitro-benzene-sulfonic acid (TNBS) inductive rat model, mouse T cell metastasis model and dextran sulfate sodium (DSS) inductive mouse model.The DSS model is from the model that Dr.S.Murthy sets up, wherein use disease activity index points-scoring system (S.N.S.Murthy, Treatment of Dextran Sulfate Sodium-Induced Murine Colitis by Intracolonic Cyclosporin, Digestive Diseasesand Sciences, Vol.38, No.9 (September 1993), pp.1722-1734).

In this research, in its drinking-water, feed DSS after 6 days when mouse, produced acute colitis.Animal shows body weight and reduces and bloody diarrhea, and this has simulated patient's UC situation.The Mechanism Study of DSS damage gets not clear, but thinks that DSS has induced non-specific struvite immunne response, and has simulated the environmental influence to intestines.Might produce hydrogen sulfide, its pair cell is deleterious.In addition, the change of bacteria flora in the chamber has appearred.Activated monocyte, scavenger cell and mastocyte in colon, have been proved.The amboceptor of all three kinds of animal models comprises struvite prostaglandin(PG), leukotriene metabolite and cytokine.

A. method

In Swiss Webster female mice (from Charles River Laboratories), induce colitis by the DSS absorption.Mouse is that 10 and 11 weeks are big when the research beginning.Mouse adds 4%DSS in drinking-water, drink 6 days (handling mouse), perhaps drinks (control mice) with normal tap water to mouse.Use disease activity index clinical score (DAI), it comprises the combination of some measured values, comprises quality, occult blood and the weight loss of ight soil.The DAI of each mouse is measured in beginning in first day after the DSS processing every day.After 6 days, from the tap water of handling mouse, remove DSS.All mouse are monitored by the DAI clinical score, until putting to death at the 2nd, 7 or 10 day after the research beginning.At the 2nd and the 7th day, each was put to death four DSS and handles mouse and a control mice.At the 10th day, put to death four DSS and handle mouse and two control mice.All measure the length of colon behind all sacrifice of animal.The colon section is fixed for histologic analysis in the formalin of 10% neutral buffered, the perhaps freezing mRNA that is used for extracts.

B. histological score and disease activity index (DAI) scoring

Obtain the scoring of histology index according to the method in the reference 1.Generally speaking, the colon section is determined folliculus scoring, hyperplastic epithelium, folliculus distortion and inflammation scoring by the pathologist under unwitting situation.

Every day according to mouse lose weight, the situation of dry and hard stool solidity and enteric hemorrhage carries out the clinical score classification to mouse.Lose weight, the increase of diarrhoea and amount of bleeding, then mark increases.The mark of every mouse every day is the mean value of three result/observed values.

C. result

At the 7th and the 10th day, the colon length of the mouse of handling through DSS slightly was shorter than the untreated control mouse, but result not necessarily significantly (not carrying out statistical test).Clinical DAI mark has reflected the increase of disease symptoms in the mouse that DSS handles, and this is similar to observed result in this Study of model of former use.The most serious about the 4th day and the 5th day occult blood, and lax ight soil is more remarkable at the 6th and the 7th day.Histopathology result is presented at the date of all execution, and disease divides number average different with the mark of control group, particularly the 7th day (peak) and the 10th day.Histopathology screening scoring is: contrast=0.5, second day DSS and handle day DSS processing mouse DSS processing in 21, the 10 days mouse=18, mouse=8.8, the 7.The clinical demonstration with the histopathology mark compared with untreated contrast, and the mouse of handling through DSS has significant colon diseases.The back is used the refrigerated tissue sample to carry out mRNA and is measured, and describes as following.

D uses RT-PCR to check the tissue expression of IL-22RNA in mouse IBD colon sample:

In order to be determined at the relative expression of mouse IL-22RNA in the inflammatory bowel disease model (SEQ ID NO:10 and SEQ ID NO:11), collect the far-end colon of the mouse of handling and freezing in liquid nitrogen rapidly through DSS.In this experiment, mouse uses DSS to handle, the sampling in the 2nd, 7 and 10 day after processing.Also collect from the sample of normal undressed mouse.Use the RNeasy Midiprep of standard then ^TM(Qiagen, Valencia is CA) according to specification sheets isolation of RNA from sample of manufacturer for Kit.

' (Life Technologies, Gaithersburg MD) carry out the RT-PCR reaction to Superscript One-Step RT-PCR System with PlatinumTaq. ' in use.Contain in each 25 microlitres reaction: 12.5 microlitre 2X reaction buffers, (0.5ul 20pmol/ μ l) ZC39,289 (SEQ ID NO:17), 0.5 μ l (20pmol/ μ l) ZC39,290 (SEQ ID NO:18), 0.4 μ l RT/Taq polysaccharase mixture, 10 μ l do not have the water of RNA enzyme, the total RNA of 1.0 μ l (100ng/ μ l).Amplified reaction is following to carry out: 50 ℃ of 1 circulations in 30 minutes; Be 94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ afterwards and carried out 35 circulations in 60 seconds; Be 72 ℃ of last extensions of 7 minutes at last.The PCR product of getting the 8-10 microlitre carries out the agarose gel electrophoresis of standard, and the concentration of sepharose is 2%.Correct cDNA clip size is observed as follows: the band a little less than in the of is all arranged in two samples of the 2nd day.Have two to produce strong band in three samples of the 7th day, the 3rd the 7th day sample produces very strong band.Three samples of the 10th day produce strong band.At last, two " normally " control samples do not produce any band.In the inflammatory reaction of these results suggest IL-22 some type in colon, comprise in those and IBD, the UC inflammatory reaction relevant having rise with CD.Data are summarised in the following table 7, wherein the relative expression are marked: 0=does not have band, the weak band of 1=, the strong band of 2=, the band that 3=is very strong.

Table 7

Tissue	Relative expression (0-3)
		Normal colon	0
Normal colon	0
		Handled back second day	1
Handled back second day	1
		Handled the back the 7th day	3
Handled the back the 7th day	2

Handled the back the 7th day	2
		Handled the back the tenth day	2
Handled the back the tenth day	2
		Handled the back the tenth day	2

Embodiment 9

IL-22RA2 has reduced IL-6 in the collagen-induced sacroiliitis of mouse (CIA) model

Level with SAA

A. collagen-induced sacroiliitis (CIA) model of mouse

Ten weeks, big male DBA/1J mouse (Jackson Labs) was divided into three groups, every group of 13 mouse.At the 21st day, to be formulated in the 1mg/ml chicken II class collagen in complete Freund's adjuvant (by Chondrex, Redmond, WA preparation) of animal by subcutaneous injection 50-100 microlitre.After three weeks, at the 0th day, to the LPS of injected in mice 100 microlitres (25 microgram) from intestinal bacteria 0111:B4, (Sigma, St.Louis were prepared into 250 mcg/ml in MO) to LPS from freeze dried aliquot.By the intraperitoneal injection administration, a Wednesday is inferior with IL-22RA2, and continuous 4 weeks were from the 0th day to the 25th day.Preceding two groups injected dose is the IL-22RA2 of every animal per agent 100 or 10 micrograms, the 3rd winding be subjected to vehicle Control PBS (LifeTechnologies, Rockville, MD).Behind lps injection, animal begins to show arthritic symptom, and most of animals occur inflammation at 2-3 in week.The disease degree of every pawl by using the kind of calliper pawl thickness and give clinical scores (0-3) to each pawl and estimate: 0=is normal, 0.5=the toe inflammation, pawl inflammation that 1=is slight, the inflammation of 2=moderate pawl, with the inflammation of 3=severe pawl, as described below.

Monitoring of diseases:

Soon, animal may begin to show the sign of pawl inflammation behind second time collagen injection, some animals even just occurred the sign of toe inflammation in the second time before the collagen injection.Sacroiliitis appears in all most of animals of 2-3 after booster shots, but some animals may need the more longer time.The sickness rate of disease 95-100% normally in this model in a research of using 40 animals, can see 0-2 responseless animal (observe in 6 weeks after definite) usually.Attention the of short duration unsettled rudimentary pawl or the inflammation of toe may occur along with the beginning of inflammation.For this reason, before occurring, pawl enlargement remarkable, that continue do not think that animal is ill.

Observe all animals every day, estimate the morbid state of its pawl, its way is that each pawl is provided quantitative clinical scores.According to the clinical disease situation, mark to 4 pawls of every animal every day.In order to determine clinical scores, can think that pawl has three district's bands: toe, pawl self (pin or pin) and ankle or ankle joint.To observing with respect to the inflammation degree and the seriousness of these district's bands, comprise whether observe all toes has arthroncus, disruptive toenail or rubescent, note whether oedema or rubescent sign are arranged in any pawl, and observe the disappearance whether tendon or bone have any fine dissection boundary, whether have any oedema or rubescent, and notice whether inflammation is close to leg if estimating ankle or ankle.The scoring 1,2,3 of pawl is at first based on the overall impression to seriousness, and next depends on has involved for how many district's bands.The grade standard that is used for clinical score is as follows:

Clinical score:

0=is normal

0.5=relate to one or multiway toe more, but have only the toe inflammation

1=relates to the mild inflammation (1 district band) of pawl, may comprise one or multiway toe more

The moderate inflammation of 2=on pawl may comprise some toes and/or ankle/ankle (two district's bands)

The hyperphlogosis (3 district band) of 3=on pawl, ankle/ankle and some or all toes

The quantitative scoring that the disease of establishing is decided to be the pawl inflammation is 2 or higher, and (continuous two days) continue to spend the night.In case the disease of occur establishing, record date is then suffered from " establishment disease " first day with it as this animal.

In whole experiment, get blood, the level of anticol original antibody in the monitoring serum.At the 21st day animal is carried out euthanasia, get blood and be used to obtain serum and white blood cell count(WBC).For each animal, collect an ill pawl, be fixed in and be used for histology among the 10%NBF, pawl is freezing and be stored in and be used for mRNA in-80 ℃ and analyze in liquid nitrogen.In addition, 1/2 spleen, 1/2 thymus gland, 1/2 mesenteric lymph nodes, a leaf liver and left kidney are stored in are used for RNA among the RNAlater and analyze; Be used for histology among 1/2 spleen, 1/2 thymus gland, 1/2 mesenteric lymph nodes, remaining liver and the right kidney stuck-at-0%NBF.Collect serum and-80 ℃ freezing, be used for the detection of immunoglobulin (Ig) and cytokine.

When the mark of pawl and take off data are analyzed, between each group, do not find the significant difference of statistics, although there is a prompting to show that a treatment group of accepting IL-22RA2 may postpone to some extent in the beginning of pawl inflammation and development.Body weight between the group, CBC parameter or anticol original antibody level do not have significant difference.These early stage results show that IL-22RA2 produces body weight, red or white corpuscle or antibody and do not have negative impact, but may be able to reduce inflammation.Further research well afoot (for example embodiment 10) to dosage, mechanism of action and effectiveness.

B. former ELISA data of the anticol in mouse CIA model

At attack (the 0th day) with respect to LPS the 0th, 7,14,21 and 28 day, from collagen-induced sacroiliitis mouse model, collect serum sample (above embodiment 9A).Titre with anticol original antibody in the ELISA screening serum sample.Compare with the PBS contrast, IL-22RA2 handles in 100 micrograms or the 10 microgram treatment group does not have remarkable influence on the statistics for anticol original antibody level.It below is the description of antagonism collagen ELISA material and method.

The reagent that is used for the former ELISA of anticol be Maxisorp 96 hole microtiter plates (NUNC, Rochester, NY), chicken II class collagen (Chondrex, Redmond, WA), SuperBlock (Pierce, Rockford, IL), puted together horseradish peroxidase (HRP) goat anti-mouse IgG+A+M (H+L) (Zymed, South San Francisco, CA) and o-phenylenediamine dihydrochloride substrate (Pierce, Rockford, IL).The damping fluid that uses in all detect is ELISA B dilution buffer liquid (PBS+0.1%BSA+0.05%Tween (Sigma, St.Louis, MO)), ELISA C lavation buffer solution (PBS+0.05%Tween) and NovoD colour developing damping fluid (0.063M Trisodium Citrate, 0.037M H citric acid), ₂O ₂(Sigma) and 1N H ₂SO ₄(VWR, Tukwilla, WA).

About 100 microlitre peripheral bloods of getting the blood collection behind the eye socket are placed in the serum separator tube (Becton Dickinson).By centrifugal (2-3min, 16,000xg, 4-6 ℃) separation of serum ,-20 ℃ of storages until analysis.For measuring the level of the former Ig antibody of anticol, wrap by the NUNC plate 4 ℃ of overnight incubation with 10 μ g/mL chicken II class collagens (Chondrex, Redmond WA).Wash plate with ELISA C, (IL) sealing (5 minutes, room temperature) is then with ELISA C washing for Pierce, Rockford with Super Block.The serum sample (5 times of serial dilutions in ELISA B, from 1: 5000 to 1: 625,000) of dilution is joined in the elisa plate, triplicate, with plate 4 ℃ of overnight incubation.After hatching, wash plate, add the goat anti-mouse Ig Fc (Zymed, 1: 2000 extent of dilution among the ELISA B) of peroxidase labelling with ELISA C.Hatch plate (room temperature 90 minutes), wash, use o-phenylenediamine dihydrochloride substrate colour developing (10mL NovoD+1 sheet OPD+10 μ L H with ELISA C once more ₂O ₂, Pierce).Reaction stops with 1N sulfuric acid.The relative optical density observed value of 1: 25000 dilution serum sample uses Spectra MAX 190 to record at 490nm, and (Molecular Devices Corporation, Palo Alto CA) analyzes data with SoftMax Pro software.

C. in mouse CIA model, IL-6 and SAA are analyzed

The 0th day, behind the LPS of intraperitoneal injection 25 micrograms 4 hours, collect serum sample (above embodiment 9A) from the CIA mouse.(, measure the concentration of IL-6 and serum amyloid sample substance A (SAA) in the test sample available from BiosourceInternational (Camarillo, CA)) with commercial ELISA test kit according to manufacturer's specification sheets.

Injecting 100 μ g IL-22RA2 respectively, in the mouse group of 10 μ g IL-22RA2 and PBS contrast, the level of IL-6 be respectively 9651+/-1563pg/ml, 10,865+/-1478pg/ml and 15,006+/-2,099pg/ml.The IL-6 concentration of injecting in the CIA mouse group of 100 microgram IL-22RA2 dosage is compared obvious reduction, p=0.0351 with the PBS control group mice.Use Fisher ' s PLSD counting statistics significance, significance level be 5% (ABACUS Concepts, INC, Berkeley, CA).

In addition, injecting 100 μ g IL-22RA2 respectively, in the mouse group of 10 μ g IL-22RA2 and PBS contrast, SAA concentration is respectively 381+/-40 μ g/ml, 348+/-37 μ g/ml and 490+/-50 μ g/ml.The SAA concentration of injecting in the CIA mouse group of 10 μ g IL-22RA2 dosage is compared obvious reduction, p=0.0257 with the PBS control group mice.Use Fisher ' s PLSD counting statistics significance, significance level be 5% (ABACUS Concepts, INC, Berkeley, CA).

Embodiment 10

Anti-IL-22RA monoclonal antibody or anti-IL-22 monoclonal antibody suppress disease in mouse CIA model

Seriousness

Collagen-induced sacroiliitis (CIA) model is the mouse model of rheumatoid arthritis, it reflected to a great extent this disease of in the mankind, seeing (Moore, Methods Mol.Biol. 225: 175-179,2003:Waksman, Scand.J.Immunol., 56: 12-34,2002).Use two doses in CFA emulsive collagen by tail base portion immune mouse.This causes the enlargement of pawl, and this enlargement increases as time passes, and can visual score and use kind of calliper.And serum anticol original antibody and severity of disease have good dependency.According to showing that IL-20 and IL-22 induce the data of inflammation, anti-IL-22RA and anti-IL-22 monoclonal antibody are given to organize through the mouse of collagen immunization, investigating it influences for the disease fractional.After anti-IL-22RA monoclonal antibody or the administration of anti-IL-22 monoclonal antibody, the mark of pawl and the thickness of pawl descend, prompting can promote ongoing immunne response at IL-20 and IL-22 in the autoimmunity model, and blocking-up, inhibition, reduction, antagonism or in and the function of IL-20 or IL-22 can suppress autoimmune disease.It may be useful to autoimmune disease that blocking-up IL-22RA is also pointed out in the inhibition of serum TNF a and anticol original antibody.

Therefore, whether influential in order to determine anti-IL-22RA monoclonal antibody or anti-IL-22 monoclonal antibody for autoimmunity power, at the rheumatoid arthritis model of mouse--in collagen-induced sacroiliitis (CIA) model they are tested.Especially, to DBA1J injected in mice collagen to induce similar rheumatism sample sacroiliitis.Inoculation at the 0th day is subcutaneous vaccination, inoculation be homogenizing mixture (50-100 μ l contains 2mg/ml collagen) by complete Freund's adjuvant (CFA) and II class collagen.Be injected near the tail base portion and carry out.At the 21st day, carry out the inoculation second time, unique difference is that homogenizing mixture is to use incomplete Freund's adjuvant (IFA) rather than complete Freund's adjuvant preparation.Measure the mark and the thickness of pawl every day.Since the collagen injection second time, mouse on the same group do not accept the monoclonal antibody contrast of PBS, 20-200 microgram homotype coupling respectively or the 20-200 microgram anti--IL-22RA monoclonal antibody or anti--IL-22 monoclonal antibody, 1-4 week is injected in intraperitoneal injection, 2-3 time weekly.Monitor mouse every day until the 30th day.Put to death mouse at the 30th day, get serum and carry out analysis of anticol original antibody and serum cytokines analysis (TNF α).

By resist-IL-22RA or anti--IL-22 monoclonal antibody be suppressed pawl mark, pawl thickness, serum TNF α and serum anticol original antibody, this prompting barrier IL-22RA in the autoimmunity model can in conjunction with, blocking-up, suppress, reduce antagonism or in and IL-22, and suppress ongoing immunne response, may suppress the autoimmunity disorder.

Embodiment 11

The expression of IL-22 acceptor IL-22RA in the DSS mouse model

Carry out quantitative RT-PCR, to measure the expression level of mouse IL-22RA in the mouse that suffers from DSS inductive IBD (embodiment 8) colon.The mouse of from normal mouse colon and through DSS, handling after after the processing 2,7 and 10 days from its far-end colon isolation of RNA.Use Applied Biosystems 7700 TaqMan instruments and method to carry out RT-PCR.In brief, instruct according to Applied Biosystems, use the primer (ZC39776 (SEQ ID NO:19) and ZC39777 (SEQ ID NO:20)) of " Primer Express " software design at mouse IL-22RA sequence, and the TaqMan probe of FAM/TAMRA mark (ZC38752 (SEQ ID NO:21)).In each reaction, add 25ng RNA, and PE/Applied Biosystems TaqMan EZ RT-PCR Core Reagents, primer above-mentioned and probe.Each RT-PCR reaction is all duplicate, under the following conditions reaction: 50 ℃ 2 minutes, 60 ℃ 30 minutes, 95 ℃ 5 minutes, and 94 ℃ of 20 seconds and 60 ℃ of totally 40 circulations in 1 minute.Compare with the typical curve that the synthetic mouse IL-22RA rna transcription product molecule of dose known amounts is made expressing numerical value, expression values is expressed as the absolute number of mouse IL-22RA molecule in each reaction.The preliminary Notes of Key Data is compared with the expression level in the normal mouse colon, and in the far-end colon of the mouse the 7th with DSS inductive IBD and the 10th day, the expression of mouse IL-22RA may be reduced slightly.

Embodiment 12

IL-22 in slight endotoxemia model and short scorching indicator: LPS inductive intracellular toxin

The mass formed by blood stasis mouse model

A.LPS inductive endotoxemia mouse model: in LPS inductive endotoxemia mouse model, pro-inflammatory cytokine and body temperature are measured

Experiment in the design body is to detect the effect of IL-22RA2 (IL-22RA2) in slight endotoxemia mouse LPS model.For this model of preliminary assessment, we have measured pro-inflammatory cytokine and body temperature, collect reference data at this model.

In brief, 25 μ g LPS (Sigma) peritoneal injections that will be dissolved among the aseptic PBS are given six months Balb/c (CRL) female mice.The 0th, 1,4,8,16,24, get serum sample from every group of 8 mouse in the time of 48 and 72 hours.Pro-inflammatory cytokine level in the serum analysis sample.The ELISA test kit of commodity in useization (is measured IL-1b, IL-6, TNFa, the level of IL-10 and serum amyloid sample substance A albumen (SAA) available from measuring BiosourceInternational (Camarillo, CA)).

Lps injection after 1 hour the TNFa level reach 4000pg/ml and the IL-10 level is 341pg/ml.Behind the lps injection 4 hours, IL-6, IL-1b and IL-10 were respectively 6,100pg/ml, 299pg/ml and 229pg/ml.Behind the lps injection 4 hours, the level of SAA was 0.405mg/ml in the serum.Behind the lps injection 24 hours, the level of SAA continues to increase to 3.9mg/ml in the serum, but be to use existing ELISA test kit to be difficult to measure the SAA level that is higher than 1-2mg/ml in the serum, reason is the interaction of SAA and other serum components accurate or repeatablely.These results show that except that IL-22 (embodiment 11B), pro-inflammatory cytokine is induced really in this model.Set up the biomarker of following standard as slight endotoxemia LPS model like this: lps injection is the TNFa serum level after 1 hour, and lps injection is IL-6 serum level and SAA serum level lps injection after 4 hours and 8 hours after 4 hours.

In other animal groups, the remote sensing survey device take temperature in 72 hours experiment that uses operation to implant.Behind the lps injection, the body temperature maximum of mouse has reduced by 2 ℃, from 37.07 ℃ of 30 minutes 34.98 ℃ behind the lps injection.

The IL-22RA2-Fc fusion rotein of 30 minutes injection 100 micrograms before lps injection can significantly be reduced in 4 hours and the SAA of 8 hours time points induces approximately 50%, and the IL-22RA2-Fc of 10 micrograms does not significantly act on.TNF-α and IL-6 level significantly do not change.Neutrophilic granulocyte counting when the IL-22RA2-Fc injection has reduced by 1 hour in the circulation.Show give IL-22RA2-Fc can in and the IL-22 activity of inducing SAA.

B. use the BaF3/CRF2-4/IL-22RA cell in Alamar Blue proliferation test, to detect the intraserous IL-22 activity of mouse in the LPS inductive endotoxemia mouse model

With BaF3/CRF2-4/IL-22RA cell centrifugation precipitation described herein and with PBS rinsing 2 times, be completely removed to guarantee mIL-3.Centrifugal then the 3rd time, be resuspended in the perfect medium (RPMI 1640,10%FBS, 1%GlutaMAX, 1% Sodium.alpha.-ketopropionate) (being called as " substratum of no mIL-3 " after this) that does not have mLL-3.Then with blood counting instrument pair cell counting.Cell is placed 96 orifice plates, every hole 5000 cells, volume is 100 microlitres/hole, uses the substratum of no mIL-3.

To in the uppermost delegation of 96 orifice plates, be diluted to 2% with the substratum of no mIL-3 from the serum of the LPS inductive endotoxemia mouse of describing among the top embodiment 11A, 1: 2 serial dilution in following 7 row then, the volume in every hole all is 100 microlitres.Then these dilute serums are joined in the 100 microlitre cells, the final concentration that makes serum is 1%, 0.5%, 0.25%, 0.125%, 0.063%, 0.031%, 0.016% and 0.018%, and total detection volume is 200 microlitres.To detect flat board and place 37 ℃, 5%CO ₂In hatched 4 days, add then Alamar Blue (Accumed, Chicago, IL), every hole 20 microlitres.And then with flat board at 37 ℃, 5%CO ₂In hatched 16 hours.Therefore Alamar Blue provides the fluorescence metering reading according to the number of viable cell, is directly measuring of the cell proliferation compared with negative control.(Wallac, Turku Finland) go up dull and stereotyped reading, and wavelength is 544 nanometers (exciting) and 590 nanometers (emission) at Wallac Victor 21420 Multilabel Counter.

The result shows, at 0 hour, 1 hour, 8 hours and 16 hours time points did not have to surpass significantly the propagation of background level.The serum sample of 4 hours time points shows 4 times of propagation that surpass background level that arrive greater than 10 times, shows in those samples to have IL-22.

C.LPS inductive endotoxemia mouse model: measure the experiment of IL-22RA2 effect

Test I L-22RA2 handles induced the capability of influence of the short scorching indicator of generation by single 25 microgram LPS dosage IP injection mouse.To all sample analysis SAA, IL-22 and circulation neutrophilic granulocyte counting.The part of every group of mouse is used to analyze specific cytokine levels (to 1 hour sample analysis TNF α, 4 hours sample analysis IL-6).Put to death animal at the time point shown in the table 8, get whole blood, collect serum, be divided into aliquot and be used for analyzing.

Inject to the IL-22RA2 that 72 C57BL/6N female mices (CRL) give single dose by the IP approach, shown in following table 8.Control mice is C57BL/6N (CRL).

After 30 minutes, they are accepted an other IP and inject the LPS (Sigma) that 100 microlitres contain 25 micrograms, with initial endotoxemia cascade process.The corresponding time point that mouse in every group is displayed in Table 8 is put to death, and gets 50 milliliters whole blood, measures circulation neutrophilic granulocyte sum, to the centrifugal serum that obtains of remaining blood, is divided into aliquot and is used for various different detection the described herein.

Table 8

Group	Numbering	Handle	LPS	Put to death	Sample
						A	8	100μg IL-22RA2 IP	25μg IP 30min post tx	1 hour	Serum is divided into aliquot; Blood is carried out complete blood count
B	8	10μg IL-22RA2 IP	25μg IP 30min post tx	1 hour	Serum is divided into aliquot; Blood is carried out complete blood count
						C	8	200μl PBS IP	25μg IP 30min post tx	1 hour	Serum is divided into aliquot; Blood is carried out complete blood count
D	8	100μg IL-22RA2 IP	25μg IP 30min post tx	4 hours	Serum is divided into aliquot; Blood is carried out complete blood count
						E	8	10μg IL-22RA2 IP	25μg IP 30min post tx	4 hours	Serum is divided into aliquot; Blood is carried out complete blood count
F	8	200μl PBS IP	25μg IP 30min post tx	4 hours	Serum is divided into aliquot; Blood is carried out complete blood count
						G	8	100μg IL-22RA2 IP	25μg IP 30min post tx	8 hours	Serum is divided into aliquot; Blood is carried out complete blood count
H	8	10μg IL-22RA2 IP	25μg IP 30min post tx	8 hours	Serum is divided into aliquot; Blood is carried out complete blood count
						J	8	200μl PBS IP	25μg IP 30min post tx	8 hours	Serum is divided into aliquot; Blood is carried out complete blood count
K	5	Contrast	Do not have	Before the LPS	Serum is divided into aliquot;

Blood is carried out complete blood count

SAA induces among the D.IL-22RA2-Fc4 and in the body: SAA ELISA is presented in the LPS inductive endotoxemia mouse model LPS inductive SAA and expresses and suppressed by the injection of IL-22RA2-Fc4

Induce in order to measure the SAA whether IL-22RA2 can suppress in the LPS inductive endotoxemia mouse model, gave injected in mice IL-22RA2 in preceding 30 minutes at lps injection, shown in the table 8 of top embodiment 11C.

According to manufacturer's specification sheets use Mouse SAA Immunoassay Kit (BioSource International California) carries out ELISA, measured 4 hours and 8 hours samples in the level of SAA.At 4 hours time point places, compare with the mouse of injection PBS, descend with significant SAA level on the dependent statistics of mouse show dose of 100 micrograms or 10 microgram IL-22RA2 processing.At 8 hours time point places, to compare with the mouse of injection PBS, the mouse of handling with 100 microgram IL-22RA2 continues to show that significant SAA level descends on the statistics.This shows that the existence of IL-22RA2 can suppress LPS inducing SAA in vivo.

Embodiment 13

Act in the body of IL-22 polypeptide to skin

A.IL-22 inductive acanthosis

With mouse (female, C3H/HEJ, 8 weeks are big; Jackson Labs, Bar Harbor ME) is divided into 3 groups (every group of 6 animals), and another organizes 4 animals.The IL-22 that people's bhk cell is produced makes the part of IL-22 and the IL-22 concentration in stable state serum-concentration and the pump proportional by the administration of miniature infiltration press pump continous pouring.In the miniature infiltration press pump (model 2002 of Alzet; Alza corporation Palo Alto, CA) (A601F 0.22mL) fills aseptic condition, and this protein dilutes in phosphate buffered saline buffer (pH 7.0) with IL-22 albumen down in, make the albumen in the pump that different concentration be arranged: to first group of mouse, concentration is 2mg/mL; To second group of mouse, concentration is 0.2mg/mL; To the 3rd group of mouse, concentration is 0.02mg/mL; To the 4th group of mouse, concentration is 0mg/mL (having only diluent).On skin of back, make 1 centimetre otch, pump is implanted in the mouse body through subcutaneous route, with aseptic wound suture wound by this otch.Pump is designed to carry its content with the speed of 0.5 microlitre per hour, carried 14 days.According to this apparent infusion rates, the dosage level that calculates the 1-4 group was respectively 24 μ g/ days, and 2.4 μ g/ days, 0.24 μ g/ days and 0 μ g/ days.

When finishing in the 14th day, with mouse euthanasia, from every about 1 square centimeter skin samples of mouse collecting pump peripheral region skin.With the fixing skin of 10% neutral buffered formalin.To handle by usual method through the skin samples paraffin embedding of formalin fixed, do 5 microns section, with phenodin and eosin dyeing.The veterinary pathologist that is awarded a license by the ACVP council under unwitting situation carries out microscope inspection to tissue.Tissues observed changes, and use following points-scoring system to carry out subjective scoring to acanthotic seriousness (being that epidermis thickens): 0-is normal, the acanthosis that 1-is the slightest, the slight acanthosis of 2-, 3-moderate acanthosis and 4-severe acanthosis.In addition, use CoolSnap digital camera (Roper Scientific, Inc., SanDiego CA) takes a picture to selected group skin, using-system morphology software (ScionImage for Windows, v.4.02, Scion Corp., Frederick MD) measures the thickness of epidermis.

With 2.4 and 24 micrograms/cause thickening of epidermis when the sky gives IL-22, show as of the scoring of average acanthosis scoring (see s) consistence ground greater than control group skin.And its epidermis of animal of handling through IL-22 also has monocyte infiltration.These instillations are not observed in the contrast with vehicle treated.

Each organizes demonstration in the measurement table 9 below of the acanthosis scoring of entocuticle thickness and skin thickness (is unit with the conventional unit pixel):

Table 9:

Group #	n＝	Pump	Average acanthosis	The thickness of measuring
					1	6	24 μ g IL-22/ days	3.0	ND
2	6	2.4 μ g IL-22/ days	2.4	67.5
					3	6	0.24 μ g IL-22/ days	2.2	ND
4	4	The PBS perfusion	1.8	45.6

B.IL-22RA2 is to the acanthotic influence of IL-22 inductive

With mouse (female, C3H/HEJ, 8 weeks are big; Jackson Labs, Bar Harbor ME) is divided into 8 groups, every group of 8 animals.With IL-22 by the administration of miniature infiltration press pump continous pouring, as the description among the embodiment 12A.In the miniature infiltration press pump (model 2001 of Alzet; Alzacorporation Palo Alto, CA) (A601F 0.22mL) fills aseptic condition, and this albumen dilutes with phosphate buffered saline buffer (pH 7.0) with IL-22 albumen down in, make the albumen in the pump that different concentration be arranged: to first and second group mouse, concentration is 0.22mg/mL; To third and fourth group mouse, concentration is 0.45mg/mL; To the 5th, six group of mouse, concentration is 0.9mg/mL; To the 7th, eight group of mouse, concentration is 0mg/mL (having only diluent).Pump is designed to carry its content with the speed of 0.5 microlitre per hour, carried 14 days.According to this apparent infusion rate, the dosage level that calculates each group be respectively one, two group 10 μ g/ days, three, four group 5 μ g/ days, five, six group of 2.5 μ g/ days and seven, eight group 0 μ g/ days.For the mouse that per two windings are subjected to same dose IL-22, one group is passed through intraperitoneal approach injection three (the 1st, 3 and 5 day) 0.1 milligram of human il-22 RA2 Fc albumen (described herein), injects carrier (PBS) in an identical manner for other one group.

At the 8th day that studies, with mouse euthanasia, from every about 1 square centimeter skin samples of mouse collecting pump peripheral region skin.With the fixing skin of 10% neutral buffered formalin.To handle by usual method through the skin samples paraffin embedding of formalin fixed, do 5 centimetres section, with phenodin and eosin dyeing.The veterinary pathologist that is awarded a license by the ACVP council under unwitting situation carries out microscope inspection to tissue.In the mode different this research is marked with the embodiment of front.The epidermis number of plies of mensuration from the stratum basale to the granular layer.To section scoring: 0-normal (layer 2-3), 1-slightly thickens (3-4 layer) according to the result, and the 2-moderate thickens (4-6 layer) and 3-severe thickens (more than 6 layers).

Dosage with 2.5,5 and 10 microgram/skies gives thicken (referring to table 10) that IL-22 has caused epidermis.And, in the epidermis of the animal that IL-22 handles, also have monocyte infiltration.These instillations are not being observed in the contrast of vehicle treated.Give 100 microgram IL-22RA2 (three injections) simultaneously and reduced the epidermis amount of swelling in the mouse through handling in 5 microgram IL-22/ days.

Each is organized in the acanthosis scoring table 10 below of epidermal thickness and shows:

Table 10:

Group #	n＝	Pump	Injection	Average acanthosis
					1	8	2.5 μ g IL-22/ days	100 μ L carriers (3 injections)	1.1
2	8	2.5 μ g IL-22/ days	100 μ L IL-22RA2 (3 injections)	0.8
					3	8	5 μ g IL-22/ days	100 μ L carriers (3 injections)	2.0
4	8	5 μ g IL-22/ days	100 μ g IL-22RA2 (3 injections)	0.6
					5	8	10 μ g IL-22/ days	100 μ L carriers (3 injections)	2.0
6	8	10 μ g IL-22/ days	100 μ g IL-22RA2 (3 injections)	1.9
					7	8	Carrier	100 μ L carriers (3 injections)	0.0
8	8	Carrier	100 μ g IL-22RA2 (3 injections)	0.0

Also having observed epidermis in people's psoriasis skin thickens and the immunity infiltration.The hypodermic skin phenotype of IL-22 also shows the latent effect of IL-22 in psoriasis is caused a disease.IL-22RA2-Fc can in and the prompting of the fact of IL-22 inductive skin phenotype, other IL-22 antagonists for example and anti-IL-22 neutralizing antibody or soluble receptors can be used for the treatment of psoriasis and other IL-22 inductive inflammatory diseases.

C.IL-22RA soluble receptors and anti-IL-22RA antibody are for IL-22 inductive or the acanthotic influence of IL-20 inductive

The acanthosis histology terminal point that use is caused by h inf IL-22 or IL-20 albumen is estimated according to similar mode the antibody inhibition IL-22 of IL-22RA soluble receptors or IL-22RA or the ability of IL-20 activity in vivo.In the embodiment of this model, according to the description among top embodiment 12 (A) and 12 (B), to the subcutaneous implantable miniature infiltration of C3H/HEJ mouse press pump.With process that IL-22 or IL-20 contact in, handle mouse by the IL-22 monoclonal antibody of injection purifying, perhaps inject carrier similarly in contrast.At the terminal point in IL-22 infusion stage, get skin samples from pump zone on every side and carry out histologic analysis.Similar to IL-22RA2 soluble receptors IL-22 antagonist, IL-22 of the present invention or IL-20 antagonist IL-22RA soluble receptors or anti--IL-22RA antibody are estimated can be so that soak into reduction because the epidermis that IL-22 or IL-20 cause thickens with immunocyte, therefore can be used as IL-22 or IL-20 antagonist, therapeutic is used in psoriasis and other IL-22 or IL-20 inductive inflammatory disease.

Embodiment 14

IL-22 obtains raising in people's psoriasis skin samples

The RNA sample:

Obtain normal skin sample and psoriasis human skin sample.The latter comprises ill skin and the contiguous not ill skin that obtains from stable patch type psoriasis.Use ordinary method isolation of RNA from the human skin sample.Use Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn Germany) that the integrity and the quality of RNA sample are analyzed.

The primer of quantitative RT-PCR is known probe

Before described use ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Inc., Foster City, CA) carry out real-time quantitative RT-PCR (referring to Heid, C.A.et al., Genome Research 6: 986-994,1996; Gibson, U.E.M.et al., Genome Research 6: 995-1001,1996; Sundaresan, S.et al., Endocrinology 139: 4756-4764,1998).This method has comprised and has contained the two the use of gene specific probe of report and cancellation fluorescence dye.When probe is excellent, because near quencher dyes, so the emission of reporting dyes is eliminated.In the PCR extension process of using extra gene specific forward and reverse primer to carry out, probe is by 5 of r Tth archaeal dna polymerase ' to the active cutting of 3 ' nucleic acid hydrolysis, this makes reporting dyes be able to be released from probe, causes the increase of fluorescent emission.

Being used for the primer of the real-time quantitative RT-PCR analysis that IL-22 expresses and probe is to use primer-design software Primer Express TM (PE Applied Biosystems, Foster City CA) designs.The human il-22 primer is designed to cross over the junction of intron and exon, makes genomic dna can not be amplified out.Use forward primer ZC42459 (SEQ ID NO ∷ 22) and reverse primer ZC42458 (SEQ ID NO ∷ 23) in the PCR reaction below, concentration is 800nM, the product of synthetic 72bp.Synthetic corresponding IL-22 probe ZC42460 (SEQID NO:24), and in ZymoGenetics, carry out mark.5 of this IL-22 probe ' end is with reporting (FAM) (PE Applied Biosystems) mark of fluorescence dye (6-Fluoresceincarboxylic acid), 3 ' end (TAMRA) (PEApplied Biosystems) mark of cancellation fluorescence dye (6-carboxyl-tetramethyl-rhodamine).

C. real-time quantitative RT-PCR

Use TaqMan EZ RT-PCR Core Reagents Kit (PE AppliedBiosystems) analyzing total RNA sample, determine the level relatively of IL-22mRNA.Prepare IL-22 transcription product out of control, produce typical curve and be used for quantitatively.This curve is formed (about 1e8 is to total copy number of the whole mRNA of 1e3 IL-22) by 10 times of serial dilution degree, and each typical curve point is analyzed in triplicate.Also to the level of the triplicate analyst IL-22 of the total RNA sample transcription product of skin, the level of analyzing hGUS is as internal contrast.Each RNA sample is all carried out TaqMan EZ RT-PCR reaction (PE Applied Biosystems), and cumulative volume is 25 microlitres, contains: about 25 nanograms be dissolved in total RNA in DEPC treated water (no DNA enzyme and RNA enzyme); Suitable primer (approximately 800nM ZC 42459 (SEQID NO:22) and ZC 42458 (SEQ ID NO:23)); Suitable probe (approximately 100nMZC 42460 (SEQ ID NO:24)); 1XTaqMan EZ damping fluid; The 3mM magnesium acetate; Each 300 μ M of d-CTP, d-ATP and d-GTP; D-UTP 600 μ M; R Tth archaeal dna polymerase (0.1U/ μ l); And AmpErase UNG (0.01U/ μ l).The condition of PCR thermal cycling is as follows: initial UNG treatment step, i.e. 50 ℃ of 1 circulations in 2 minutes; Then be reverse transcription (RT) step, i.e. 60 ℃ of 1 circulations in 30 minutes; Then be to make the UNG deactivation step, i.e. 95 ℃ of 1 circulations in 5 minutes; Be amplification then, promptly 94 ℃ of 20 seconds and 60 ℃ carried out 40 circulations in 1 minute.

Use the method for typical curve, description (UserBulletin #2:ABI Prism 7700 Sequence Detection System according to manufacturer PE Biosystems, RelativeQuantitation of Gene Expression, 1997-12-11), measure the level relatively of IL-22 RNA.Use the observed value of hGUS that the IL-22 level is carried out stdn.Show in the data table 11 below.

Table 11

Skin samples	IL-22
		Normally	0
Involved	0
		Ill	1149

Normal patient or involved the zone skin samples in do not detect IL-22mRNA.On the contrary, IL-22mRNA has rapid rise in psoriasis patient ill (being involved) skin.These data have proved IL-22 and people's psoriasis has the intensive disease association.

Show the overexpression of IL-22 at people's psoriasis breakage, have IL-22 to participate in prompter's psoriasis.And, as described herein, the overexpression of IL-22 in transgenic mice shown that epidermis thickens and the participation of immunocyte, this is the indication of psoriasis phenotype, and the injection of IL-22 in normal mouse shown that epidermis thickens and immunocyte participates in, this is the indication of psoriasis phenotype, and these can be eliminated by soluble receptors antagonist IL-22RA2.Data further point out the IL-22 that short inflammatory is arranged in psoriasis to participate in such body.Similarly, the active antagonist of IL-22, anti-human il-22 monoclonal antibody for example of the present invention and soluble receptors and antibody all are useful in the therapeutic treatment of diseases associated with inflammation, particularly can be used as the antagonist of IL-22 in psoriasis treatment.And, the active antagonist of IL-22, anti-human il-22 monoclonal antibody for example of the present invention and soluble receptors and antibody, also be useful in the therapeutic treatment of other diseases associated with inflammation, atopic dermatitis for example, IBD, colitis, endotoxemia, sacroiliitis, rheumatoid arthritis and arthritic psoriasis, adult respiratory disease (ARD), septic shock, multiple organ failure, struvite pulmonary lesion is asthma or bronchitis for example, bacterial pneumonia, psoriasis, eczema, atopic dermatitis and contact dermatitis and inflammatory bowel be ulcerative colitis and Crohn disease for example.

Embodiment 15

IL-22 is raised in people's atopic dermatitis skin samples

Obtain normal skin sample (n=4) and from the skin samples of atopic dermatitis patient (n=4).Use ordinary method isolation of RNA from the human skin sample.Use Agilent 2100Bioanalyzer (Agilent Technologies, Waldbronn Gemany) that the integrity and the quality of RNA sample are analyzed.

The primer and the probe that are used for quantitative RT-PCR

Originally described use ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Inc., Foster City, CA) carry out real-time quantitative RT-PCR (referring to Heid, C.A.et al., Genome Research 6: 986-994,1996; Gibson, U.E.M.et al., Genome Research 6: 995-1001,1996; Sundaresan, S.et al., Endocrinology 139: 4756-4764,1998).Comprise in this method and use the gene-specific probe that contains report and cancellation fluorescence dye.When probe is excellent, because very near quencher dyes, so the emission of reporting dyes is eliminated.In the PCR extension process of using extra gene specific forward and reverse primer to carry out, probe is by 5 of r Tth archaeal dna polymerase ' to the active cutting of 3 ' nucleic acid hydrolysis, this makes reporting dyes be able to be released from probe, causes the increase of fluorescent emission.

The primer and the probe that are used for the real-time quantitative RT-PCR analysis of IL-22 expression are to use primer-design software Primer Express ^TM(PE Applied Biosystems, Foster City CA) designs.The primer of human il-22 is designed to cross over the junction of intron and exon, makes genomic dna can not be amplified out.Use forward primer ZC42459 (SEQ ID NO ∷ 22) and reverse primer ZC42458 (SEQ ID NO ∷ 23) in the PCR reaction below, concentration is 800nM, the product of a synthetic 72bp.Synthesize corresponding IL-22 probe ZC42460 (SEQ ID NO:24), and carry out mark in ZymoGenetics inside.This IL-22 probe 5 ' end is by (FAM) (PE AppliedBiosystems) institute mark of report fluorescence dye (6-Fluoresceincarboxylic acid), at 3 ' end by (TAMRA) (PE Applied Biosystems) institute's mark of cancellation fluorescence dye (6-carboxyl-tetramethyl-rhodamine).

C. real-time quantitative RT-PCR

Use TaqMan EZ RT-PCR Core Reagents Kit (PE AppliedBiosystems) analyzing total RNA sample, measure the level relatively of IL-22mRNA.Prepare IL-22 transcription product out of control, produce typical curve and be used for quantitatively.This curve is made up of 10 times of serial dilution degree, and the total copy number from 1e8 to the whole mRNA of 1e3 IL-22 is analyzed in triplicate to each typical curve point.Also to the triplicate analyst IL-22 of the total RNA sample transcription product level of skin, and the level of analyzing hGUS is as internal contrast.Each RNA sample is all carried out TaqMan EZ RT-PCR reaction (PE Applied Biosystems), and cumulative volume is 25 microlitres, contains: about 25 nanograms be dissolved in total RNA in DEPC treated water (no DNA enzyme and RNA enzyme); Suitable primer (approximately 800nM ZC 42459 (SEQ IDNO:22) and ZC 42458 (SEQ ID NO:23)); Suitable probe (approximately 100nM ZC42460 (SEQ ID NO:24)); 1XTaqMan EZ damping fluid; The 3mM magnesium acetate; Each 300 μ M of d-CTP, d-ATP and d-GTP; D-UTP 600 μ M; R Tth archaeal dna polymerase (0.1U/ μ l); And AmpErase UNG (0.01U/ μ l).The condition of PCR thermal cycling is as follows: initial UNG treatment step, i.e. 50 ℃ of 1 circulations in 2 minutes; Then be reverse transcription (RT) step, i.e. 60 ℃ of 1 circulations in 30 minutes; Then be the UNG deactivation step, i.e. 95 ℃ of 1 circulations in 5 minutes; Be amplification step then, promptly 94 ℃ of 20 seconds and 60 ℃ carried out 40 circulations in 1 minute.

Use the method for typical curve, description (UserBulletin #2:ABI Prism 7700 Sequence Detection System according to manufacturer PE Biosystems, RelativeQuantitation of Gene Expression, 1997-12-11), measure the level relatively of IL-22 RNA.Use the observed value of hGUS that the IL-22 level is carried out stdn.

In the skin samples of normal patient, detect less than IL-22mRNA.On the contrary, in the patient of 4 atopic dermatitiss, there are three IL-22mRNA to occur and significantly raise (approximately 400-2300 copy).These data have supported between IL-22 and the people's atopic dermatitis intensive disease-related is arranged.

The overexpression that has shown IL-22 in human atopic dermatitis skin, prompting IL-22 relates to the morbidity of human atopic dermatitis.And as described herein-in, the overexpression of IL-22 in transgenic mice shown that epidermis thickens and the involving of immunocyte, and this is the indication of atopic dermatitis phenotype; In addition, IL-22 injected in normal mouse shown that epidermis thickens and the involving of immunocyte, this is the indication of atopic dermatitis phenotype; This can eliminate by soluble receptors antagonist IL-22RA2.Data further point out short scorching IL-22 to participate in the atopic dermatitis morbidity in these bodies.Like this, the active antagonist of IL-22, anti-human il-22 RA monoclonal antibody for example of the present invention, and soluble receptors and antibody thereof, in the therapeutic treatment of diseases associated with inflammation, be useful, particularly can be in the treatment of atopic dermatitis as the antagonist of IL-22.And, the active antagonist of IL-22, anti-human il-22 monoclonal antibody for example of the present invention, and soluble receptors and antibody thereof, therapeutic at other diseases associated with inflammation also is useful in treatment, for example in the disease below treatment as the antagonist of IL-22: atopic dermatitis, IBD, colitis, endotoxemia, sacroiliitis, rheumatoid arthritis, arthritic psoriasis, adult respiratory disease (ARD), septic shock, multiple organ failure, struvite injury of lung is asthma or bronchitis for example, bacterial pneumonia, atopic dermatitis, oedema, atopic dermatitis and contact dermatitis and inflammatory bowel such as ulcerative colitis and Crohn disease.

Embodiment 16

The human il-22 polyclonal antibody

Use prepares anti-IL-22 polyclonal antibody from two female New Zealand white rabbits of ripe recombinant human il-2 2 polypeptide (amino-acid residue 22 (Ala) is to 167 (Ile) the SEQ ID NO:6) immunity of the purifying of bhk cell (IL-22-BHK) preparation.Every rabbit is by the initial injection of intraperitoneal (IP) approach (200 microgram purifying proteins in Freund's complete adjuvant), next every carrying out booster immunization three weeks one time, promptly is injected at 100 microgram peptides in the Freund's incomplete adjuvant by the IP approach.Strengthen for the second time and injected back (three injections altogether) 7 to 10 days, to animal blood sampling and collection serum.Per then three all booster shots are also blood sampling once.

Every gram CNBr-SEPHAROSE uses the recombinant protein human il-22-BHK of 10 milligrams of specific antigen purifying to prepare CNBr-SEPHAROSE 4B protein chromatographic post (Pharmacia LKB), utilize this post from the rabbit anteserum of immunity, the special polyclonal antibody affinity purification of human il-22 to be come out 20 * dialysed overnight in PBS.The human il-22 specific antibody is carried out ELISA research, and the recombinant protein human il-22-BHK that uses the 500ng/ml purifying is as antibody target.On specificity purification of Recombinant antigen human il-22-BHK, the anti-human il-22 affinity purification of rabbit detection of antibodies lower limit (LLD) is 280pg/ml.

Further the recombinant human il-2 2-BHK of the special polyclonal antibody blocking-up of research human il-22 purifying is to the ability (" neutralization test ") of the cell-proliferation activity of BaF3/CRF2-4/IL-22RA cell (embodiment 2 and embodiment 3).50 times of excessive special polyclonal antibodies of human il-22 of volumetric molar concentration are enough to suppress cell proliferation.

Embodiment 17

Anti-human il-22 monoclonal antibody

Use is from (IL-22-BHK) four female Sprague-Dawley rat (Charles River Laboratories of immunity of ripe recombinant human il-2 2 polypeptide (amino-acid residue 22 (Ala) is to 167 (Ile) the SEQ ID NO:6) of the purifying of bhk cell preparation, Wilmington, MA), preparation monoclonal antibody.To every rat by the initial Freund's complete adjuvant (Pierce that is injected at of intraperitoneal approach, Rockford, IL) 100 microgram purifying people recombinant il-2s, 2 albumen in, next per two weeks are carried out booster immunization one time, promptly are injected at 50 microgram purification of recombinant proteins in the Freund's incomplete adjuvant by the IP approach.Strengthened the injection back for the third time 7 to 10 days, to animal blood sampling and collection serum.

Human il-22 specificity rat blood serum sample is carried out ELISA research, with the biotinylated human il-22-BHK of 500ng/ml and biotinylated mouse IL-22 of 500ng/ml and biotinylated mouse IL-22-intestinal bacteria (R+D Systems, Minneapolis is MN) as antibody target.Three rat blood serum samples are 1 to the titre of specific antibody target-biotinylation human il-22-BHK: the 1E5 extent of dilution is 1 to specific antibody target-colibacillary titre of biotinylation mouse IL-22-: the 1E4 extent of dilution.

Results splenocyte and lymph-node cell from the rat of two high titres, in two fusion steps of carrying out respectively, merge with PEG1500 and SP2/0 (mouse) myeloma cell that (splenocyte and myeloma cell are 4: 1 integration percentages, Antibodies A LaboratoryManual, E.Harlow and D.Lane, Cold Spring Harbor Press).Merge back growth 10 days,, use biotinylated recombinant protein human il-22-BHK and biotinylated recombinant protein mouse IL-22-intestinal bacteria, identify the hybridoma mixture that produces specific antibody respectively as antibody target by ELISA.The hybridoma mixture that all is positive in two ELISA experiments is further analyzed their blocking-up or reduced the ability (" neutralization test ") of the reorganization mouse IL-22-intestinal bacteria of purifying to the cell-proliferation activity of BaF3/CRF2-4/IL-22RA cell (embodiment 2 and embodiment 3).

Use limiting dilution assay to clone at least again twice to the hybridoma mixture that only in ELISA, produces positive findings or at the hybridoma mixture that ELISA and " neutralization test " all produce positive findings in the two.

The research monoclonal antibody that purifying comes out from tissue culture medium (TCM) is used for the purposes of quantitative assay mouse and human serum sample's reorganization and natural human IL-22 content in ELISA.In detection by quantitative, the detection of antibodies lower limit of two selections is about 1ng/ml recombinant human il-2 2-intestinal bacteria in 100% human serum.

The recombinant human il-2 2-intestinal bacteria of blocking-up of the research monoclonal antibody that purifying comes out from tissue culture medium (TCM) or reduction purifying or mouse IL-22-intestinal bacteria are to the ability (" neutralization test ") of the cell-proliferation activity of BaF3/CRF2-4/IL-22RA cell (embodiment 2 and embodiment 3).Identify six kinds of " neutrality " monoclonal antibodies in this way.According to budapest treaty expression described above is preserved in U.S. typical case at the hybridoma of the neutralizing monoclonal antibody of human il-22 as original preservation thing and cultivates preservation center (ATCC; Manassas VA) patent collection place, and obtain following ATCC login numbering: clone 266.16.1.4.4.1 (ATCC patent preserving number PTA-5035); Clone 266.5.1.2.2.3 (ATCC patent preserving number PTA-5033); Clone 267.17.1.1.4.1 (ATCC patent preserving number PTA-5038); Clone 267.4.1.1.4.1 (ATCC patent preserving number PTA-5037); Clone 266.12.6.1.3.2.1 (ATCC patent preserving number PTA-5034); Clone 266.19.1.10.5.2 (ATCC patent preserving number PTA-5036); And clone 267.9.1.1.4.1 (ATCC patent preserving number PTA-5353).

Embodiment 18

Anti-IL-22RA monoclonal antibody

(Rockland Immunochemicals, Gilbertsville PA), prepare monoclonal antibody through four Lewis rats of the recombination fusion protein mouse IL-22-Fc (SEQ ID NO:4) of cutting and purifying immunity in use.Every rat is by the initial Freund's complete adjuvant (Pierce that is injected at of intraperitoneal approach, Rockford, IL) in) the recombination fusion protein of 100 microgram purifying, once strengthen the IP approach next per two weeks and be injected at 50 microgram purification of recombinant proteins in the Freund's incomplete adjuvant, around the immunity.After immunity finishes around first, 50 micrograms that will be in Freund's incomplete adjuvant are through recombinant protein and carrier proteins keyhole  hemocyanin (KLH, Pierce, the Rockford of cutting and purifying, IL) phase link coupled product carries out booster immunization one time by per two weeks of IP approach, around the immunity.Strengthened the injection back for the 4th time 7 to 10 days, to animal blood sampling and collection serum.

Mouse IL-22RA specificity rat blood serum sample is carried out ELISA research, wherein use the purified recombination fusion protein mouse IL-22RA-Fc of 500ng/ml as the specific antibody target, a kind of irrelevant fusion rotein is as the non-specific antibody target.

From the rat of a high titre, gather in the crops splenocyte, use the fusion experiment scheme (Rockland Immunochemicals) of the PEG mediation of optimizing to merge with SP2/0 (mouse) myeloma cell.Merge back growth 12 days, the hybridoma mixture that produces specific antibody is by ELISA, and the recombination fusion protein mouse IL-22RA-Fc-Bv that uses purifying is as the specific antibody target, and a kind of irrelevant fusion rotein is identified as the non-specific antibody target.The hybridoma mixture that further analysis only is positive to the specific antibody target is blocked or is reduced the ability (" neutralization test ") of the reorganization mouse IL-22-intestinal bacteria of purifying to the cell-proliferation activity of BaF3/CRF2-4/IL-22RA cell (embodiment 2 and embodiment 3), and measures the ability of its combination as the BaF3/CRF2-4/IL-22RA cell (embodiment 2 and embodiment 3) of antibody target by facs analysis.

The hybridoma mixture use limiting dilution assay that produces specific positive findings and produce positive findings in ELISA in FACS or " neutralization test " is cloned twice at least again.

Their block or are reduced in the recombinant protein mouse IL-22-intestinal bacteria of purifying or the ability of BaF3/CRF2-4/IL-22RA cell (embodiment 2 and the embodiment 3) propagation that there is growth down in human il-22-BHK to the monoclonal antibody research in the tissue culture medium (TCM).Identify 14 " neutrality " monoclonal antibodies, and cloned 9 monoclonal antibodies.

According to budapest treaty, the hybridoma of expressing at the neutralizing monoclonal antibody of mouse IL-22RA described above is preserved in American type culture collection (ATCC as original preservation thing; Manassas VA) patent collection place, and obtain following ATCC login numbering: clone R2.1.1G11.1 (ATCC patent preserving number [####); Clone R2.1.5F4.1 (ATCC patent preserving number [####]); Clone R2.1.5H8.1 (ATCC patent preserving number [####]); Clone R2.1.12G7.1 (ATCC patent preserving number [####]); Clone R2.1.13C8.1 (ATCC patent preserving number PTA-5037); Clone R2.1.15E2.1 (ATCC patent preserving number [####]); Clone R2.1.16C11.1 (ATCC patent preserving number [####]); Clone R2.1.18C8.1 (ATCC patent preserving number [####]); And clone R2.1.21G8.2 (ATCC patent preserving number [####]).

Embodiment 19

The binding affinity of two kinds of rat anti-mouse IL-22RA monoclonal antibodies

Goat Chinese People's Anti-Japanese Military and Political College mouse IgG-Fc γ specific antibody (Jackson) is fixed on the CM5 Biacore chip.To detect optimization, and make each monoclonal antibody catch combination on the surface, stride across the IL-22RA that monoclonal antibody is injected a series of concentration then, investigate in conjunction with (Ka) and dissociate (Kd) Chinese People's Anti-Japanese Military and Political College mouse.After tentative experiment, observe non-specific combination between the surface catching of fusion rotein and chip.Obtain a pipe and cut the IL-22RA of Fc4 label by zymoplasm, detect then, showing does not have background influence.After operation each time, use double injection 20mM hydrochloric acid, make surface regeneration become Chinese People's Anti-Japanese Military and Political College's murine antibody.Obtain data for each monoclonal antibody, operational analysis software (BIAevaluation software version 3.2, Pharmacia BIAcore, Uppsala, Sweden) kinetics of anti-IL-22RA of mensuration and IL-22RA protein bound, as shown in following table 12:

Table 12

Clone R2.1.5F4.1**		Clone R2.1.15E2.1**
				ka(M-1s-1)	1.49E+06	ka(M-1s-1)	1.76E+06
kd(s-1)	1.70E-04	kd(s-1)	2.55E-04
				KA(M-1)	8.76E+09	KA(M-1)	6.66E+09
KD(M)	1.14E-10	KD(M)	1.504E-10
				Chi2	2.08	Chi2	1.5

* has shown the binding equilibrium (Ka) of each anti-IL-22RA monoclonal antibody and the balance of dissociating (Kd) rate constant, and numerical value drops in the limits of machine.Chi2 refers to binding curve and estimates the sum of squares of difference between the matched curve.Near 0, the degree of confidence of data is high more more.

By shown in the table 12, two kinds of anti-IL-22RA monoclonal antibodies all combine with IL-22RA albumen is strong, and are shown as combining of the monoclonal antibody and the IL-22RA (cutting away the Fc4 label through zymoplasm) of picomole concentration.By low Chi ²The numerical value visual data has good degree of confidence, and data presentation monoclonal antibody clone R2.1.5F4.1 has slightly strong affinity to the IL-22RA acceptor.

Embodiment 20

The immunohistochemical analysis of IL-22 albumen expression in vivo in tissue sample

A. general introduction

Use anti-human il-22 (anti-hIL-22) monoclonal antibody (Mab266.19.1.10.5.2), carry out the immunohistochemical methods (IHC) of IL-22 protein expression in the tissue sample below and analyze and the location: people multi-Normal Grid and Tumor Grid; Human pancreas's inflammation, lung and kidney disease sample; People's psoriasis skin samples; The pancreas of INS IL-22 TG (reaching) and wild-type mice by the rat insulin promoter sublist; MuIL-22-EuLCKTG and wild-type mice skin samples; And DSS (wild-type and IL-22KO) mouse colon sample.And, compared anti-hIL-22 monoclonal antibody MAB 266.19.1.10.5.2 (embodiment 17) and polyclonal antibody (rabbit is anti--hIL-22) the dyeing pattern of (embodiment 16).

Rat anti human il-22 monoclonal antibody MAb 266.16.1.4.4.1 and MAb266.19.1.10.5.2 (embodiment 17) are tested, show that they can make most of BHK/ human il-22s (＞50%) dyeing, but some BHK/ mouse IL-22 cells (1-5%) are colored also.Use this tissue distribution and the expression of two kinds of antibody research IL-22 in patient and animal model sample, and be used for comparing, so that the result is proved conclusively with the dyeing pattern of multi-clone rabbit antibody.

B. material and method

The section that to be cut into 5 microns through the cell and the tissue of formalin fixed, paraffin-embedded people source and mouse model.The bhk cell of expressing human or mouse IL-22 and wild-type cell are respectively as positive control and negative control.People's tissue comprises: many tissue contrast slide glass (Normal Grid ^TMBiomeda, Foster City, CA), the section (for example brain, hypophysis, suprarenal gland, breast, kidney, heart, stomach, small intestine, large intestine, fetal liver, liver, skin, pancreas, lung, tonsil, ovary, testis, prostate gland, uterus, placenta, Tiroidina and spleen) that wherein has 50 various health adult tissues; Many tissue contrast slide glass (TumorGrid ^TMBiomeda, Foster City CA), wherein has section (for example adenocarcinoma of lung, liver gland cancer, renal adenocarcinoma, adenocarcinoma of colon, mammary cancer, thyroid carcinoma, adenocarcinoma of stomach, prostate cancer, carcinoma of the pancreas, the adenocarcinoma ovaries of 50 various people's tumours, lymphoma, myelomatosis, Ewing's sarcoma, epithelial tumor, malignant fibrous histiocytoma, the Rhabdo knurl, innocent tumour, undifferentiated cancer, mesothelioma, teretoma and spermocytoma); From CHTN (Cooperation HumanTissue Network, Cleveland, lung cancer Ohio); Normal Pancreas, have chronic pancreatitis pancreas, have the lung of chronic surrounding blood vessel inflammation, from NDRI (NationalDisease Research Interchange, Philadelphia, has the kidney of ball film hyperplasia glomerulonephritis or has the kidney of sclerotic kidney bead interstitial fibrosis at the kidney with many focuses property glomerulosclerosis PA); And people's psoriasis skin samples.The tissue of mouse comprises: the colon of inflammatory bowel animal model (DSS model disclosed herein, Swiss Webster female mice); IL-20 wild-type and the colitis animal model (DSS mouse, the female mice that wild-type and IL-20 knock out) that knocks out add carrier in tap water or 4%DSS handled 7 days; And the skin samples of transgenic animal model, comprise mIL-22-EuLCK TG and mIL-22-INS contrast and transgenic animal.A section phenodin and eosin (H﹠amp on each slide glass; E) histological examination is carried out in dyeing, and remaining section is checked IL-22 protein expression and location with immunohistochemical staining.

For immunohistochemical methods, cell and tissue slice are placed ChemMate ^TMOn the Capillary GapPlus slide (BioTek, Winooski, Vermont), 60 ℃ baking oven inner dryings 60 minutes, use standard program to dewax: in the dimethylbenzene 5 minutes three times, in 100% ethanol 4 minutes, in 100% ethanol 3 minutes, in 95% ethanol 2 minutes.Then to tissue slice at 37 ℃ of enzyme induction epi-position repair processes that carry out 20 minutes with stomach en-(NeoMarkers Fremont CA), then according to the (Zymed of production firm, South San Francisco, specification sheets CA) carry out the sealing step of avidin/biotin.Use TechMate500 ^TMAutomated Immunostainer and Immunoperoxidase (IP) immunohistochemical experiment scheme, (VentanaBiotek Systems, Tucson AZ) dyes with avidin-vitamin H complex body detection system.TechMate ^TM500 AutomatedImmunostainer have used capillary principle, and the IP experimental program has adopted the immunostaining of " sandwich " technology of a kind of being called as.Cut into slices with the normal goats serum (Vector of 5% (in PBS), Burlingame CA) seals 10 minutes in advance, use 1 * damping fluid, 1 (Signet then, Dedham MA) rinsing, anti-(monoclonal antibody 266.19.1.10.5.2) (embodiment 17) PAS purifying with anti-IL-22 is 2.04mg/ml again, dilution in 1: 800) incubated at room is 30 minutes, uses 1 rinsing of 5 * damping fluid then.One is anti-with TechMate 500 ^TMAntibody dilution buffer (Ventana) dilution.The biotinylated goat mouse IgG of the Chinese People's Anti-Japanese Military and Political College (Vector) dilutes with PBS at 1: 200, and the normal goats serum of adding 5% and 2.5% skim-milk as two anti-at room temperature hatching 25 minutes, are used 1 * damping fluid 1 and damping fluid 2﹠amp respectively afterwards; 3 (Signet) rinsing.Tissue slice (Ventana) sealed 3X7 minute with 3% hydrogen peroxide (HP), used 3 * damping fluid 2﹠amp then; 3 rinsings.Use superoxide DAB test kit (Ventana) to carry out the immunoperoxidase mark, hatched 30 minutes, so use 5 * damping fluid 2﹠amp with avidin-vitamin H complex body (ABC); 3 rinsings were handled 4 * 4 minutes with diaminobenzidine (DAB), used 2 * damping fluid 2﹠amp afterwards; 3 rinsings, 1 * washing (Signet, catalog number 2340).Using methyl green (Dako, catalog number S1962) that tissue was carried out negative staining 10 minutes then, next is 2 * damping fluid 2﹠amp; 3 rinsings and 3 * washing.Contrast comprises the elementary serum of non-immunity, uses rat one anti-homotype contrast (Zymed) to replace one to resist.

Use 0lympus BH-2 microscopic examination immunostaining, (Roper Scientific, Tucson AZ) take photograph with CoolSNAP HQ digital camera.

C. result

Positive and negative control cell is: monoclonal antibody 266.19.1.10.5.2, a kind of anti-human il-22 monoclonal antibody, at the bhk cell of expressing human IL-22 (+++) with express in the bhk cell (+) of mouse IL-22 and shown positive staining, not dyeing (-) in the wild-type bhk cell.Use rat homotype negative control replacement one to resist all positives and negative bhk cell system are dyeed, all do not show dyeing (-), this shows that described antibody is special to the IL-22 aglucon.This antibody has cross-immunoreactivity to the IL-22 of people and mouse.

People's tissue: to people multi-Normal Grid and Tumor Grid; Pancreas, lung and kidney disease sample; And people's psoriasis skin samples is checked.These people's tissue comprises 1) at many tissue contrast slide glass (NormalGrid ^TM) on brain, hypophysis, suprarenal gland, breast, kidney, heart, stomach, small intestine, large intestine, fetal liver, liver, skin, pancreas, lung, tonsil, ovary, uterus, testis, placenta, Tiroidina and spleen/health adult tissue; 2) adenocarcinoma of lung, liver gland cancer, renal adenocarcinoma, thyroid carcinoma, adenocarcinoma of stomach, prostate cancer, carcinoma of the pancreas, the adenocarcinoma ovaries on many tissue contrast slide glasses (TumorGrid), lymphoma, melanoma, Ewing's sarcoma, epithelial tumor, malignant fibrous histiocytoma, Rhabdo knurl, innocent tumour, undifferentiated cancer, mesothelioma, teretoma and spermocytoma/people's abnormal structure/tumour; 3) Normal Pancreas, the pancreas with chronic pancreatitis, the lung with chronic surrounding blood vessel inflammation, lung cancer, the kidney with many focuses property glomerulosclerosis, the kidney with ball film hyperplasia glomerulonephritis and the kidney with sclerotic kidney bead interstitial fibrosis are from CHTN and/or NDRI; 4).

Mouse tissue: the pancreas of having checked INS IL-22 transgenosis and wild-type mice.The cell that scatters in the pancreas islet of INS IL-22 transgenosis pancreas shows and uses monoclonal antibody 266.19.1.10.5.2 that strong positive staining (+++) is arranged, and the not dyeing (-) of wild-type pancreas.

The comparison of polyclone and monoclonal antibody: shown that anti-IL-22 polyclonal antibody (embodiment 16) is a sensitive, and monoclonal antibody MAB 266.19.1.10.5.2 is special.Polyclonal antibody (+++) on the bhk cell of expressing human IL-22, express on the bhk cell of mouse IL-22 on (+), various people and the mouse tissue sample (+) and in the pancreas islet of INS mIL-22 transgenic mice (+++) shown positive colour developing.Compare with wild-type, the pancreas islet in the transgenic mice has and contains positive staining more at high proportion.The dyeing of transgenosis pancreas islet is distributed in the pancreas islet (+++) widely, and the dyeing in the wild-type pancreas islet generally is confined to the periphery (+) of pancreas islet.But this antibody has shown unspecific staining (+) to wild-type BHK negative control cell.

Monoclonal antibody 266.19.1.10.5.2 (+++) on the bhk cell of expressing human IL-22, express on the bhk cell of mouse IL-22 (+) and in the pancreas islet of INS mIL-22 transgenic mice (+++) shown positive colour developing.The dyeing of transgenosis pancreas islet is distributed in the pancreas islet (+++) and not dyeing (-) in the wild-type pancreas islet widely.

Embodiment 21

IL-20 raises in people's psoriasis skin samples

The A.RNA sample

Obtain normal skin samples and psoriasis patient's the influenced sample of skin.The latter comprises and relates to psoriasic skin and be close to unaffected skin.Use ordinary method isolation of RNA from the human skin sample.Use Agilent 2100 Bioanalyzer (AgilentTechnologies, Waldbronn Germany) that the integrity and the quality of RNA sample are analyzed.

B. the primer of quantitative RT-PCR and probe

Use ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Inc., Foster City had originally been described, CA) carry out real-time quantitative RT-PCR (referring to Heid, C.A.et al., Genome Research 6:986-994,1996; Gibson, U.E.M.et al., Genome Research 6:995-1001,1996; Sundaresan, S.et al., Endocrinology 139:4756-4764,1998).This method has comprised using and has contained the two gene-specific probe of report and cancellation fluorescence dye.When probe is excellent, since close closely with quencher dyes, so the emission of reporting dyes is eliminated.In the PCR extension process of using extra gene specific forward and reverse primer to carry out, probe is by 5 of r Tth archaeal dna polymerase ' to the active cutting of 3 ' nucleic acid hydrolysis, this makes reporting dyes be able to be released from probe, causes the increase of fluorescent emission.

Being used for the primer of the real-time quantitative RT-PCR analysis that IL-22 expresses and probe is to use primer-design software Primer ExpressTM (PE Applied Biosystems, Foster City CA) designs.Use forward primer ZC40541 (SEQ IDNO ∷ 25) and reverse primer ZC40542 (SEQ ID NO ∷ 26) in the PCR reaction below, its concentration is 800nM, the product of a synthetic 71bp.Synthesize corresponding IL-20 TaqMan  probe ZC 40544 (SEQ ID NO:27), and carry out mark by PE Applied Biosystems.On 5 of IL-20 probe ' end mark, report fluorescence dye (6-Fluoresceincarboxylic acid) (FAM) (PE AppliedBiosystems), 3 ' the end mark on cancellation fluorescence dye (6-carboxyl-tetramethyl-rhodamine) (TAMRA) (PE Applied Biosystems).

C. real-time quantitative RT-PCR

Use TaqMan EZ RT-PCR Core Reagents Kit (PE AppliedBiosystems) analyzing total RNA sample, determine the level relatively of IL-20mRNA.Prepare (runoff) IL-20 transcription product out of control, produce a typical curve and be used for quantitatively.Curve is made up of to total copy number of the whole mRNA of 1e3 IL-22 from about 1e8 10 times of serial dilutions, and each typical curve point is analyzed in triplicate.Also the level of human IL-2's 0 transcription product in total RNA sample of skin is analyzed in triplicate, the level of analyzing hGUS is as internal contrast.Each RNA sample all carries out TaqMan EZ RT-PCR reaction (PE AppliedBiosystems), and cumulative volume is 25 microlitres, contains: about 25 nanograms be dissolved in total RNA through DEPC treated water (no DNA enzyme and RNA enzyme); Suitable primer (approximately 800nMZC40541 (SEQ ID NO:25) and ZC40542 (SEQ ID NO:26)); Suitable probe (approximately 100nM ZC 40544 (SEQ ID NO:27)); 1XTaqMan EZ damping fluid; The 3mM manganese acetate; Each 300 μ M of d-CTP, d-ATP and d-GTP; D-UTP 600 μ M; RTth archaeal dna polymerase (0.1U/ μ l); And AmpErase UNG (0.01U/ μ l).The condition of PCR thermal cycling is as follows: 50 ℃ of 1 circulations in 2 minutes of initial UNG treatment step; Then be reverse transcription (RT) step, 60 ℃ of 1 circulations in 30 minutes; Then be to make the UNG deactivation step, 95 ℃ of 1 circulations in 5 minutes; Be that 40 circulations are carried out in the amplification in 1 minute of 94 ℃ of 20 seconds and 60 ℃ then.

Use the method for typical curve, description (UserBulletin #2:ABI Prism 7700 Sequence Detection System according to manufacturer PE Biosystems, RelativeQuantitation of Gene Expression, 1997-12-11), measure the level relatively of IL-20 RNA.Use the measurement of hGUS that the IL-20 level is carried out stdn.Show in the data table 13 below.

Table 13

Skin samples	IL-20
		Normally	2903
Involved	7233
		Ill	27,695

Although in normal patient or involved in the skin samples in zone and can detect IL-22mRNA, the mRNA of IL-20 raises in psoriasis patient's the ill skin.The receptor subunit of IL-20 comprises that IL-20RA, IL-22RA (IL-22RA) and IL-20RB have expression in the normal and ill skin of people.These data have supported IL-20 and people's psoriasis to have the intensive disease association.

Show the overexpression of IL-20 at people's psoriasis breakage, have IL-20 to participate in prompter's psoriasis.And as described herein, the overexpression of IL-20 in transgenic mice shown that epidermis thickens and immunocyte is participated, and this is the indication of psoriasis phenotype.Data are further pointed out in such body has IL-20 to participate in psoriasis.Like this, the active antagonist of IL-20, anti-human il-22 RA monoclonal antibody for example of the present invention and soluble receptors and antibody, and anti-IL-20 neutrality and monoclonal antibody, for example can be used as the antagonist of IL-20 in psoriasis and other treatment of conditions treatments disclosed herein at diseases associated with inflammation.

Embodiment 22

IL-20 raises in people's atopic dermatitis skin samples

The A.RNA sample

Obtain normal skin sample and atopic dermatitis patient's skin samples.Use ordinary method isolation of RNA from the human skin sample.Use Agilent 2100 Bioanalyzer (AgilentTechnologies, Waldbronn Germany) that the integrity and the quality of RNA sample are analyzed.

B. the primer of quantitative RT-PCR and probe

Originally described use ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems, Inc., Foster City, CA) carry out real-time quantitative RT-PCR (referring to Heid, C.A.et al., Genome Research 6: 986-994,1996; Gibson, U.E.M.et al., Genome Research 6: 995-1001,1996; Sundaresan, S.et al., Endocrinology 139: 4756-4764,1998).This method comprises using and contains the two gene-specific probe of report and cancellation fluorescence dye.When probe is excellent, since closely close with quencher dyes, so the emission of reporting dyes is eliminated.In the PCR extension process of using extra gene specific forward and reverse primer to carry out, probe is by 5 of rTth archaeal dna polymerase ' to the active cutting of 3 ' nucleic acid hydrolysis, this makes reporting dyes be able to discharge from probe, causes the increase of fluorescent emission.

Being used for the primer of the real-time quantitative RT-PCR analysis that IL-20 expresses and probe is to use primer-design software Primer ExpressTM (PE Applied Biosystems, Foster City CA) designs.Use forward primer ZC40541 (SEQ IDNO:25) and reverse primer ZC40542 (SEQ ID NO:26) in the PCR reaction below, concentration is 800nM, the product of a synthetic 71bp.Synthetic corresponding IL-20TaqMan ^Probe ZC40544 (SEQ IDNO:27), and carry out mark by PE Applied Biosystems.This IL-20 probe 5 ' report fluorescence dye (6-Fluoresceincarboxylic acid) (FAM) (PE AppliedBiosystems) on the end mark, cancellation fluorescence dye (6-carboxyl-tetramethyl-rhodamine) (TAMRA) (PE Applied Biosystems) on 3 ' end mark.

C. real-time quantitative RT-PCR

Use TaqMan EZ RT-PCR Core Reagents Kit (PE AppliedBiosystems) analyzing total RNA sample, determine the level relatively of IL-20mRNA.Prepare IL-20 transcription product out of control, produce a typical curve and be used for quantitatively.Curve is made up of to total copy number of the whole mRNA of 1e3 IL-20 from 1e8 10 times of serial dilutions, and each typical curve point is analyzed in triplicate.Also the level of human IL-2's 0 transcription product in total RNA sample of skin is analyzed in triplicate, and the level of analysis hGUS is as internal contrast.Each RNA sample is all carried out TaqMan EZ RT-PCR reaction (PE AppliedBiosystems), and cumulative volume is 25 microlitres, wherein contains: about 25 nanograms be dissolved in total RNA in the water (no DNA enzyme and RNA enzyme) that DEPC handles; Suitable primer (approximately 800nM ZC40541 (SEQ ID NO:25) and ZC40542 (SEQ ID NO:26)); Suitable probe (approximately 100nM ZC40544 (SEQ ID NO:27)); 1XTaqMan EZ damping fluid; The 3mM manganese acetate; Each 300 μ M of d-CTP, d-ATP and d-GTP; D-UTP 600 μ M; RTth archaeal dna polymerase (0.1U/ μ l); And AmpErase UNG (0.01U/ μ l).The condition of PCR thermal cycling is as follows: 50 ℃ of 1 circulations in 2 minutes of initial UNG treatment step; Then be reverse transcription (RT) step, 60 ℃ of 1 circulations in 30 minutes; Then be to make the UNG deactivation step, 95 ℃ of 1 circulations in 5 minutes; Be that 40 circulations are carried out in the amplification in 1 minute of 94 ℃ of 20 seconds and 60 ℃ then.

Use the method for typical curve, description (UserBulletin #2:ABI Prism 7700 Sequence Detection System according to manufacturer PE Biosystems, RelativeQuantitation of Gene Expression, 1997-12-11), measure the level relatively of IL-20 RNA.Use the observed value of hGUS that the IL-20 level is carried out stdn.

In skin samples, can detect low-level IL-20mRNA (about 800 copies).On the contrary, IL-20mRNA has rise (about 8600 copies) in from the patient's of atopic dermatitis skin.In people's normal and ill skin, the IL-20 receptor subunit comprises that IL-20RA, IL-22RA and IL-20RB have expression.These data have supported IL-20 and people's atopic dermatitis that the intensive disease-related is arranged.Show the overexpression of IL-20 at people's atopic dermatitis skin place, have IL-20 to participate in prompter's atopic dermatitis.And as described herein, the overexpression of IL-20 in transgenic mice shown that epidermis thickens and immunocyte is participated, and this is the indication of atopic dermatitis phenotype.Data are further pointed out in such body has IL-20 to participate in atopic dermatitis.Like this, the active antagonist of IL-20, anti-human il-22 RA monoclonal antibody for example of the present invention and soluble receptors and antibody, and anti-IL-20 neutrality and monoclonal antibody, for example can be used as the antagonist of IL-20 in atopic dermatitis and other treatment of conditions treatments disclosed herein at diseases associated with inflammation.

Embodiment 23

The rise of the IL-8 that causes by IL-20

With newborn keratinocyte (NHEK) (from the Clonetics) bed board of the 2nd generation normal people epidermis, and in 12 hole tissue culturing plates, grow to intensive.KGM (Keratiocyte growth substratum) is available from Clonetics.When cell reached intensive, using the KGM substratum that does not conform to somatomedin was KBM (keratinocyte minimum medium) rinsing.In KBM, make cell carry out serum starvation 72 hours, add compound to be measured then.The trypsinase of 1 I.U./mL zymoplasm and 25nM is as positive control.Every hole adds 1 milliliter substratum.Only use KBM as negative control.

In the KBM substratum, prepare IL-20, and join in the experiment with different concentration: first the experiment in from 2.5 μ g/ml to 618ng/mL; In second experiment from 2.5 μ g/mL to 3ng/mL.

Cell was hatched 48 hours in 37 ℃ of 5% carbonic acid gas.Remove supernatant ,-80 ℃ of freezing many levels that detect IL-8 and GM-CSF in the future.According to manufacturer's specification sheets, end user IL-8 immunity detection reagent #D8050 (RandD Systems, Inc.) and human GM-CSF immunity detection reagent # HSGMO (RandD Systems Inc.), measures production of cytokines.

The result shows that the expression of IL-8 and GM-CSF is induced by IL-20.

Embodiment 24

IL-20 causes the rise of struvite cytokine

With human keratinized cell is that HaCaT cultivates a few days in 37 ℃ in the T-75 tissue culture flasks, to reach intensive after.At this moment remove normal growth medium (DMEM+10%FBS), change to the substratum of serum-free.Then 37 ℃ of incubated cells 2 days.Remove DMEM then, each is handled and uses 4 bottles of cells, use following conditions to handle respectively, hatched 4 hours: the IL-20 of IL-20, the 1 μ g/mL of rh IL-1 α+1 μ g/mL of rh IL-1 α, the 5ng/mL of the recombinant human of 5ng/mL (rh) IL-1 α, 20ng/mL or the rh IL-10 of 10ng/mL at 37 ℃.

Cytokine is removed substratum after handling, and uses the guanidine thiocyanate solution lysing cell.Spend the night by caesium chloride density gradient centrifugation, from cell lysate, extract total RNA.Second day resuspended and use ethanol sedimentation with TE/SDS solution with RNA precipitation.Use spectrophotometer that RNA is carried out quantitatively, then according to Clontech ' s Atlas ^TMThe V.B. part of cDNA Expression Arrays UserManual (version PT3140-1/PR9X390,1999-11-5 publishes) is carried out the DNA enzyme to RNA and is handled.Prove conclusively the quality of RNA sample according to the observation of purity that calculates by spectrophotometric reading and agarose gel electrophoresis.Use the β actin gene in the pcr analysis RNA sample, discharge the pollution of genomic dna thus.

Use the synthetic and Atlas of polyA+ enrichment, probe of Clontech ^TMThe method of hybridization array (referring to top, and Atlas ^TMPure Total RNA Labeling System UserManual, PT3231-1/PR96157,1999-6-22 publishes).In brief, (Clontech, Paolo Alto CA) isolate polyA+RNA with the magnetic-particle separator from total RNA of 50 milligrams by the magnetic bead of streptavidin bag quilt in use.Use α 32P-dATP that PolyA+RNA is carried out mark by RT-PCR then.Use the CDS primer of Clontech in reaction, these primers are to Atlas ^TMHuman cell factor/be subjected to 268 genes in the volume array (catalog number 7744-1) is special.The probe of mark separates with the method for column chromatography, uses liquid flashing counting then.

To Atlas ^TMThe salmon sperm DNA that array uses Clontech ExpressHyb to add the 100mg/mL thermally denature carries out prehybridization, and prehybridization is at least 30 minutes under constantly shaking under 68 ℃.With 1.9 * 10 ⁶CPM/mL (amounts to 1.14 * 10 ⁷CPM) film is spent the night 68 ℃ of hybridization, constantly shake.Second day, use 2X SSC, 68 ℃ of rinsing films of 1%SDS 4 times, each 30 minutes, use 68 ℃ of rinsing films of 0.1X SSC, 0.5%SDS 1 time 30 minutes then, at room temperature washed film one time 5 minutes at last with 2X SSC.The film of array placed in the Kodak plastics bag seal, and phosphorescence imaging screen (phosphor imager screen) at room temperature exposed spend the night.Used the AtlasImage of Clontech with phosphorescence imager scanning phosphorescent screen in second day ^TM1.0 software is analyzed.

The gene that is raised by IL-20

1. tumour necrosis factor (TNF) is raised 1.9-2.4 doubly by IL-20;

2. placenta growth factor 1﹠amp; 2 (PLGF) are raised 1.9-2.0 doubly by IL-20;

3. the prothrombin acceptor is raised 2.0-2.5 doubly by IL-20;

4. Calcitonin Receptor is raised 2.2-2.3 doubly by IL-20;

5.TNF induction type hyaluronic acid binding protein TSG-6 is raised 2.1-2.2 doubly by IL-20;

6. vascular endothelial growth factor (VEGF) acceptor-1 precursor, tyrosine protein kinase acceptor (FLT-1) (SFLT) are raised 2.1-2.7 doubly by IL-20;

7.MRP-8 (calcium binding protein in the scavenger cell, MIF is correlated with) raised 2.9-4.1 doubly by IL-20;

8.MRP-14 (calcium binding protein in the scavenger cell, MIF is correlated with) raised 3.0-3.8 doubly by IL-20;

9. Relaxin H2 is raised 3.14 times by IL-20;

10. transforming growth factor-beta (TGF β) receptor II I 300kDa is raised 2.4-3.6 doubly by IL-20;

Reveal synergitic gene with the IL-20+IL-1 processing list:

1. bone morphogenetic protein 2a raises 1.8 times with the IL-20 individual curing time, raises 2.5 times during with the IL-1 individual curing, raises 8.2 times when handling jointly with IL-20 and IL-1.

2.MRP-8 with the IL-20 individual curing time, raise 2.9 times, raise 10.7 times during with the IL-1 individual curing, raise 18.0 times when handling jointly with IL-20 and IL-1.

3. red corpuscle differential protein (EDF) raises 1.9 times with the IL-20 individual curing time, raises 9.7 times during with the IL-1 individual curing, raises 19.0 times when handling jointly with IL-20 and IL-1.

4.MRP-14 (calcium binding protein in the scavenger cell, MIF is correlated with) raises 3.0 times with the IL-20 individual curing time, raise 12.2 times during with the IL-1 individual curing, rise is 20.3 times when handling jointly with IL-20 and IL-1.

5. heparin associativity class EGF somatomedin raises 2.0 times with the IL-20 individual curing time, raises 14 times during with the IL-1 individual curing, raises 25.0 times when handling jointly with IL-20 and IL-1.

6. β-thromboglobulin sample albumen raises 1.5 times with the IL-20 individual curing time, raises 15 times during with the IL-1 individual curing, raises 27 times when handling jointly with IL-20 and IL-1.

7. brain derived neurotrophic factor (BDNF) raises 1.7 times with the IL-20 individual curing time, raises 25 times during with the IL-1 individual curing, raises 48 times when handling jointly with IL-20 and IL-1.

8. MCAF MCAF raises 1.3 times with the IL-20 individual curing time, raises 32 times during with the IL-1 individual curing, raises 56 times when handling jointly with IL-20 and IL-1.

Embodiment 25

IL-20 transgenosis phenotype

Use the IL-20 of various promotor overexpression people and mouse in transgenic mice.Bring into use the expression of the mouse albumin promoter instructor IL-20 of liver specificity, to attempt to obtain the albumen of cyclical level.Keratin sulfate 14 (K14) promotor is used in ensuing research, and it mainly is targeted to expression epidermis and other stratified squamous epithelium; Mouse metallothionein(MT) 1 promotor, it has expresses pattern widely; E μ LCK promotor, its kinesin is expressed in the cell of lymph pedigree.Obtained similar result in all four kinds of situations, this may be because these promotors have all caused the IL-20 of cyclical level to express.

In all situations, the genetically modified transgenosis young baby of expression IL-20 is little than not genetically modified brood young baby, has the appearance of light, tightens the skins of many wrinkles, and death in several leading day after birth.In young baby's the stomach milk is arranged, show that they can suck.These mouse have four limbs, tail, nostril and the mouth region of swelling, and move difficulty.In addition, mouse is very thin and weak, lacks the visible fatty tissue, and the development delay of ear and toe.Low-level expression in liver (less than 100 mRNA molecule/cells) just is enough to cause newborn young baby to cause death and skin unusual.The transgenic mice of no visible phenotypic or do not have express transgenic, perhaps expression level is detectable below horizontal, or mosaic.

The skin of IL-20 transgenic mice is carried out fabric analysis shows, with not genetically modified brood mouse relatively, the transgenic mice epidermis thickens, hyperkeratosis and fine and close stratum corneum.Sometimes observe plasmocyte scab (incrustation).Electron microscopic analysis to transgenic mice skin shows Intramitochondrial lipoid inclusion body, macular keratohyalin particle and quite few tonofilament (to observed similar in people's psoriasis skin and mouse skin disease model).In addition, many transgenic mices have the thymic lymphocytes of apoptosis.Histopathological analysis is not found the unusual of other.These histologies and Electronic Speculum support as a result and expanded observed cardinal principle skin change.

Embodiment 26

Be used to express the structure of the expression vector of soluble human IL-22RA-muFc

Human il-22 RA soluble receptors-muFc fusion rotein (called after IL-22RA-C (mG2a)) that merge mutually in the extracellular domain that preparation contains IL-22RA and mouse γ 2a heavy chain Fc district (mG2a).By homologous recombination, use two independently dna fragmentation and expression vector pZMP40, make up the expression plasmid that contains IL-22RA-C (mG2a).Primer below using, produce the polynucleotide sequence fragment of IL-22RA (SEQ ID NO:1) and mG2a (SEQ IDNO:39) by pcr amplification: (a) IL-22RA primer ZC45,593 (SEQ IDNO:28), and ZC45,592 (SEQ ID NO:29); And (b) mG2a primer ZC45,591 (SEQ ID NO:30), and ZC45,594 (SEQ ID NO:31).

First fragment contains IL-22RA extracellular domain coding region, and it is to use IL-22RA polynucleotide (for example SEQ ID NO:1) to prepare as template.This first fragment comprises and the pZMP40 carrier sequence of part eclipsed 5 ' end, IL-22RA fragment and contain joint sequence and 3 ' lap of part mG2a sequence to some extent.PCR condition: 1 circulation, 94 ℃, 5 minutes; 35 circulations, 94 ℃, 1 minute, 55 ℃, 2 minutes, 72 ℃, 3 minutes; 1 circulation, 72 ℃, 10 minutes.

Second fragment comprises and joint sequence and part IL-22RA sequence eclipsed 5 ' end, mG2a fragment and 3 ' lap of containing part pZMP40 carrier sequence to some extent.Mouse γ 2a heavy chain Fc district (mG2a) (SEQ ID NO:39) is produced by the clone of mouse Ig γ 2a heavy chain cDNA.MG2a contains hinge, the C of mouse immuning ball protein γ 2a CH _H2 and C _H3 structural domains.PCR condition: 1 circulation, 94 ℃, 5 minutes; 35 circulations, 94 ℃, 1 minute, 55 ℃, 2 minutes, 72 ℃, 3 minutes; 1 circulation, 72 ℃, 10 minutes.

The PCR reaction mixture is carried out 1% agarose gel electrophoresis, use QIAquick ^TMGelExtraction Kit (Qiagen) gel extraction is corresponding to the band of inset size.

To be used for inserting fragment with the pZMP40 plasmid after the BglII cutting and carry out the three-dimensional reorganization with two PCR.The pZMP40 plasmid is a kind of mammalian expression vector, wherein contains expression cassette, the multiple clone site that this expression cassette has the MPSV promotor and is used to insert encoding sequence; The intestinal bacteria replication orgin; The Mammals selectable marker gene is expressed unit (comprising SV40 promotor, enhanser, replication orgin, DHFR gene and SV40 terminator); And screening and duplicate required URA3 and CEN-ARS sequence in S.cerevisiae.The pZMP40 plasmid is by (being deposited in American type culture collection to pZMP21,10801 UniversityBoulevard, Manassas, VA 20110-2209 is numbered PTA-5266) multiple clone site in add several restriction enzyme sites and make up.

With competence yeast (S.cerevisiae) cell of 100 microlitres respectively with 10 microlitre inset DNA and the cutting of 100 nanograms after the pZMP40 carrier mixed, mixture transferred in 0.2 centimetre the electroporation cuvette.Yeast/DNA mixture is used power supply, and (BioRadCaboratories, Hercules CA) are provided with 0.75kV (5kV/cm), infinitely great ohm, and 25 μ F shock by electricity.The 1.2M sorbyl alcohol that adds 600 microlitres in cuvette is got on the coating of 100 microlitres and 300 microlitres electric shock product and the two URA-D flat boards, hatches for 30 ℃.After about 72 hours, will be from the Ura in the flat board ⁺Yeast transformant is resuspended with 1 ml water, slight centrifugation yeast cell.Cell precipitation is resuspended with 0.5 milliliter of lysis buffer (100mM NaCl, 10mM Tris, pH 8.0,1mM EDTA for 2%Triton X-100,1%SDS).The lysate of 500 microlitres joined contains in the Eppendorf pipe of 250 microlitres with the granulated glass sphere of pickling and 300 microlitre phenol-chloroforms, vortex vibration 3 minutes, in the Eppendorf whizzer with maximum velocity centrifugation 5 minutes.The water of 300 microlitres is transferred in the clean pipe, with 600 microlitre ethanol and 30 microlitre 3M sodium-acetate deposit D NA, then with maximum velocity centrifugation 30 minutes.The supernatant that inclines is with 1 milliliter of 70% washing with alcohol precipitation.The supernatant that inclines is with the resuspended DNA precipitation of 30 microlitre TE.

Use the cerevisiae dna extract of 5 microlitres and the cell transformation electroreception attitude e. coli host cell (DH12S) of 50 microlitres.At 2.0kV, 25 μ F and 400ohms be the electric shock cell down.Behind the electroporation, add 1 milliliter SOC (2%Bacto ^TMTryptones (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10mM NaCl, 2.5mM KCl, 10mM MgCl ₂, 10mM MgSO ₄, 20mM glucose), get 50 microlitres and 200 microlitre cells then and coat (LB substratum (Lennox), 1.8%Bacto on two LB penbritin flat boards ^TMAgar (Difco), the 100mg/L penbritin).

Inset to three clones being used for construct carries out sequential analysis.Select a clone who contains correct sequence for each construct.(Valencia CA), carries out plasmid in large scale DNA according to manufacturer's specification sheets and separates the test kit of commodity in useization for QIAGENPlasmid Mega Kit, Qiagen.

Embodiment 27

Human soluble IL-22RA-muTc polypeptide expression and purifying

Use 200 PvuI of unit to cut 3 hours at 37 ℃ of enzymes respectively IL-22RA-C (mG2a) construct (embodiment 22) of three group of 200 microgram, use the IPA precipitation then, centrifugation is in 1.5 milliliters of miniature centrifuge tubes.The supernatant that inclines, precipitation be with 1 milliliter of 70% washing with alcohol, then incubated at room 5 minutes.With centrifugal 10 minutes of centrifuge tube 14000rpm in Eppendorf centrifuge, supernatant inclined.In gnotobasis, will precipitate with the PF-CHO substratum of 750 microlitres resuspendedly, hatch 30 minutes for 60 ℃.Three by all means in, every pipe precipitation 5E6 APFDXB11 cell uses the DNA culture medium solution resuspended.The DNA/ cell mixture is placed 0.4 centimetre of breach cuvette, use following parameter to shock by electricity: 950 μ F, high capacitance, 300 volts.Content in the cuvette is taken out, mixed and be diluted to 25 milliliters with the PF-CHO substratum, place 125 milliliters the bottle that shakes.Place on the vibrator in the incubator shaking bottle, 37 ℃, 6%CO ₂The 120rpm vibration is hatched.

Pair cell system carries out nutrition to be selected, and is progressively to increase afterwards, and wherein the concentration of methotrexate (MTX) is brought up to 500nM at last to 1 μ M from 100nM.Carry out the cd8 cell sorting afterwards.Use stable mixture, adopt the anti-CD8 antibody of mono-clonal FITC (BD PharMingen, cat# 30324X), under the concentration that the manufacturer recommends, about 5E6 cell is suitably dyeed, finish the cd8 cell sorting through 1 μ M MTX amplification.Painted cell is handled and sorting on FACS Vantage (BD) flow cytometer.5% cell above collecting is also cultivated it.Use the Western trace that expression is proved conclusively, clone is carried out amplification culture, and use standard method to carry out protein purification.

Embodiment 28

From immunization in the serum of mouse of huIL22RA-mG2a and huIL-22RA

A. utilize based on the inhibition of the neutralization test of cell test to IL-20 and/or IL-22.Use is a BaF3 (BAF/IL-22RA/IL-20RB cell through the pre B cell that depends on the factor of IL-22RA and IL-20RB (pDIRS1) cotransfection; Embodiment 38), by the IL-20 of antagonism on the IL-22RA/IL-20RB acceptor, estimate the neutralising capacity of anti-IL-22RA antibody.Similarly, use is through BaF3 (the BAF/IL-22RA/CRF2-4 cell of IL-22RA and IL-10RB (CRF2-4) cotransfection; Embodiment 2), by the IL-22 of antagonism on the IL-22RA/IL10RB acceptor, estimate anti-IL-22RA antibody in and potentiality.Use the Alamar Blue that describes among the embodiment 3 to detect, estimate when IL-20 or IL-22 exist respectively, express respectively IL-20 and IL-22 clone propagation and when antagonist antibodies exists to this inhibition of proliferation.In this detection, are the active indications of neutralization to the inhibition of these cell proliferations.

B. in neutralization test based on cell, in the anti-IL-22RA antiserum(antisera) and IL-20 and IL-22 the two.Use the detection of describing among the embodiment 28A, to from immunization the serum of the IL-22RA knock-out mice of huIL-22RA-muG2a (embodiment 30 (A) (1)) carry out serial dilution (1%, 0.5%, 0.25%, 0.13%, 0.06%, 0.03%, 0.02% and 0%) also adds.Detect culture plate at 37 ℃, 5%CO ₂In hatched 4 days, this moment add Alamar Blue (Accumed, Chicago, IL), every hole 20 microlitres.Then at 37 ℃, 5%CO ₂Culture plate 16 hours again under the condition.Alamar Blue provides the fluorescence metering reading according to the number of viable cell, and comparing with negative control like this is exactly directly measuring of on cell proliferation.(Wallac, Turku read plate on Finland), and wavelength is 530 nanometers (exciting) and 590 nanometers (emission) at WallacVictor 2 1420 Multilabel Counter.The result shows, from the serum of all seven immune animals can both in and huIL-22 and the huIL20 signal conduction by huIL-22RA.For example, be 1% o'clock in concentration, neutralized fully by huIL-22 inductive propagation from the serum of five animals (16517,16518,16519,16520 and 16527), under lower concentration, inhibition of proliferation shown with dose-dependent form and reduce.And, be 1% o'clock in concentration, suppressed the about 90% of the propagation that causes by huIL-22 from the serum of other two animals (16471 and 16701), under lower concentration, inhibition of proliferation shown with dose-dependent form and reduce.Similarly, be 1% and 0.5% o'clock in concentration, from five animals (16517,16518,16519,16520, with 16527) serum neutralized fully by huIL-20 inductive propagation, under lower concentration, inhibition of proliferation shown with dose-dependent form and reduces.And, be 1% o'clock in concentration, neutralized fully by huIL-20 inductive propagation from the serum of animal 16701, under lower concentration, inhibition of proliferation shown with dose-dependent form and descend.In concentration is 1% o'clock, from neutralized about 95% by huIL-20 inductive propagation of the serum of animal 16471, under lower concentration inhibition of proliferation shown with dose-dependent form and descends.Therefore, from the serum of all seven animals can both in and IL-22 or the IL20 propagation by the huIL-22RA receptor-inducible.These results have further shown really can be in the activity of short scorching aglucon IL-20 of antagonism under the lower concentration and IL-22 at the antibody of IL-22RA.

These results provide extra evidence to show, by combination, blocking-up, suppress, reduce, antagonism or in and the activity of IL-20 or IL-22 (separately or common) the IL-22RA activity is effectively blocked, for example via the neutralizing monoclonal antibody at IL-22RA of the present invention, aspect the effect in the body that alleviates IL-20 and IL-22 (independent or common) may be useful, and may alleviate IL-20 and/or IL-22 inductive inflammation, for example in IL-20 inductive skin sufferer and in the IL-22 inductive skin sufferer, for example in psoriasis, IBD, colitis or other comprise IBD by IL-20 and/or IL-22 inductive diseases associated with inflammation, sacroiliitis, asthma, arthritic psoriasis, colitis, inflammatory skin sufferer and atopic dermatitis.

Embodiment 29

The generation of P815/hIL-22RA cell and to the immunization of mouse

The generation of A.P815/hIL-22RA cell and injection mouse are to produce anti-hIL-22RA antibody

Scheme (Roche according to the manufacturer, Indianapolis, IN), use FugeneReagent, with the plamid vector transfection wild-type P815 cell (ATCC code T IB-64) that comprises hIL-22RA cDNA sequence (for example SEQ ID NO:1) and selectable tetracycline resistance marker.After the transfection, cell placed on the tetracycline select 48 hours.Clone the transfectant of anti-tetracycline by limiting dilution assay, use biotinylated human il-22 (huIL-22-vitamin H), by the hIL-22RA cell surface expression level of flow cytometry screening transfectant.In brief, make cell and 5 μ g/ml huIL-22-vitamin Hs hatch rinsing then jointly 30 minutes on ice.Use the streptavidin detection huIL-22-vitamin H of PE-mark and combining of cell of dilution in 1: 500.Analysis of cells on the Facscan flow cytometer, and use Cellquest software (Becton Dickinson, San Jose, CA).

The P815/IL-22RA cell growth that order is selected, results are used for injection then.Harvested cell, with PBS rinsing three times, counting, resuspended to density be every milliliter 1 * 10 ⁸Individual cell, with 10, the 000rads radiation.Then cell suspension is transferred in 1 milliliter the syringe, is expelled in the DBA/2 mouse body by the intraperitoneal approach.3 all backs are with the same manner booster immunization mouse, to its ability that combines with the hIL-22RA transfectional cell series of serum screening.In brief, with Facs damping fluid (HBSS, 2%BSA, 0.02%NaN ₃) with serum dilution 1: 100, hatch with 293 human kidney cells of the overexpression hIL-22RA of Fc sealing then.(AL) (dilution in 1: 200) anti-IL-22RA antibody of detection combines with cell for Southern Biotech, Birmingham to use the goat anti-mouse IgG of having puted together fluorescein then.Descriptive analysis cell according to the front.The booster immunization mouse is 3 times altogether once more, screens serum as mentioned above.Select two mouse to be used for the hybridoma fusion according to the level that its serum combines with the hIL-22RA transfectional cell, use the method for this area internal standard to be used to produce monoclonal antibody (embodiment 25).

Above method also is used for producing the assorted dimerization IL-22RA acceptor of expression, for example IL-22RA/CRF2-4 (P815/IL-22RA/CRF2-4 cell), IL-22RA/pDIRS1 (P815/IL-22RA/pDIRS1 cell), the perhaps P815 cell of IL-22RA/CRF2-4/pDIRS1 (P815/IL-22RA/CRF2-4/pDIRS1 cell), be used for for example immunized mice, produce monoclonal antibody and the anti-antibody that comprises the assorted dimerization acceptor of IL-22RA of anti-IL-22RA.

Embodiment 30

The generation of mouse-anti human il-22 RA (IL-22RA) monoclonal antibody

A. immunization produces anti-IL-22RA antibody

(1)Use solubility IL-22RA-muFc

To 6-12 week big IL-22RA knock-out mice (embodiment 26) by intraperitoneal injection immunization 25-50 microgram soluble human IL-22RA-muFc albumen (embodiment 23) and (v: v) mixture, per two all immunity once 1: 1 of Ribi adjuvant (Sigma).After the immunization 7-10 days for the third time, get the mode blood sampling of blood by the back eye socket, results serum and measure it with neutralization test (for example described herein) and suppress IL-22 or IL-20 and the two ability that combines with IL-22RA of IL-22, and with FACS dye assay method to transfection the ability of 293 cell dyeings of 293 cells of IL-22RA and untransfected.Continue immunized mice and blood sampling and mensuration according to top description, until in and titre reach a platform.At that time by the solubility IL-22RA-Fc protein (be dissolved in PBS) of approach in the blood vessel to injected in mice 25-50 microgram with senior middle school and titre.After three days, be used to produce hybridoma from these mouse acquisition spleens and lymphoglandula, for example use other suitable clones in mouse myeloma (P3-X63-Ag8.653.3.12.11) cell or this area, adopt standard method known in the art (for example referring to Kearney, J.F.et al. J Immunol. 123: 1548-50,1979; And Lane, R.D. J Immunol Methods 81: 223-8,1985).

(2) use the R815 transfectional cell of expressing the IL-22RA acceptor

By intraperitoneal injection 1 * 10 ⁵The transfection P815 cell of individual work, P815/IL-22RA cell for example, P815/IL-22RA/CRF2-4, (for example in 0.5 milliliter, cell density is 2 * 10 for P815/IL-22RA/pDIRS1 or P815/IL-22RA/CRF2-4/pDIRS1 cell (embodiment 24) ⁵Individual cells/ml), all big female DBA/2 mouse of 6-10 are carried out immunity.Before the injection, cell is maintained in the logarithmic phase.During injection, harvested cell with PBS rinsing three times, is resuspended among the PBS then, to density be 2 * 10 ⁵Individual cells/ml.In this model, mouse produces ascites tumour at 2-3 in week, if do not excite the immunne response to the target antigen of institute's transfection in advance, then mouse is in 4-6 death during week.The mouse that the time does not have obvious belly enlargement (indication of ascites tumour) three weeks according to above-mentioned interval with 2-3 week immunization again.After the immunization 7-10 days for the second time, by the blood sampling of taking a blood sample behind the eye, results serum, and measure it with neutralization test (for example described herein) and suppress IL-22 or IL-20 and the two ability that combines with IL-22RA of IL-22, and with FACS dye assay method measure its make transfection 293 cells of IL-22RA with respect to the ability of 293 cell dyeings of untransfected.Continue immunized mice according to top description, and blood sampling and mensuration, until in and titre reach a platform.At that time, by the intraperitoneal approach to injected in mice 1 * 10 with senior middle school and titre ⁵Individual work through the P815 of transfection cell.After four days, from these mouse results spleen and lymphoglandula, be used to produce hybridoma, for example use other suitable clones in mouse myeloma (P3-X63-Ag8.653.3.12.11) cell or this area, utilize standard method known in the art (for example referring to Kearney, J.F.et al. DittoAnd Lane, R.D. Ditto).

An alternative route of the immunization scheme of carrying out through transfection P815 cell of more than use living is every 2-3 week peritoneal injection 1-5 * 10 ⁶Individual radiating transfectional cell.In this mode, there is not animal to produce ascites and because ascites death.On the contrary, the blood sampling after the second time immunization is according to the neutrality immunne response of the method monitoring animal serum of summarizing above for IL-22RA.In case in and titre reach maximum horizontal, promptly merge intraperitoneal injection 5 * 10 in advance to having the mouse of high titre ⁶Individual through the radiating cell, be used to produce hybridoma from these mouse acquisition spleens and lymphoglandula after four days, for example use other suitable clones in mouse myeloma (P3-X63-Ag8.653.3.12.11) cell or this area, use standard method known in the art (for example referring to Kearney, J.F.et al. DittoAnd Lane, R.D. Ditto).

Whether B. screen the hybridoma fused cell produces and can and can suppress the antibody that IL-22 combines with IL-22RA in conjunction with IL-22RA

After fusion, the hybridoma supernatant carried out two different primary dcreening operations in 8-10 days.In first detects, detect the ability that the antibody in the supernatant combines with combination soluble human IL-22RA-muFc protein onboard by ELISA, wherein use goat anti-mouse κ and the anti-lambda light chain of having puted together HRP anti-, detect the bonded mouse antibodies as two.In order to prove the specificity of the IL-22RA part in the IL-22RA-muFc albumen, use the irrelevant albumen that merges mutually with identical mouse Fc district (mG2a) to check positive supernatant in first detection.Combining with IL-22RA-muFc not with those supernatants of fusion rotein bonded that contain irrelevant muFc in antibody be considered to the antibody special to IL-22RA.For second detection, check that with ELISA the antibody in all hybridoma supernatants suppresses the ability that biotinylated human il-22 combines with combination IL-22RA-muFc onboard.

Next detect all and contain with the supernatant of IL-22RA specificity bonded antibody and suppress combine with the Baf3 cell respectively ability of (and short cultivation effect simultaneously) of IL-20 or IL-22, no matter and whether these serum suppress combining of IL-22 and IL-22RA in ELISA detects through IL-22RA/IL-20RB and IL-22RA/CRF2-4 transfection.Make through the painted ability of IL-22RA cells transfected assessing its Baf3 cell with the male supernatant by facs analysis in being in suppressing to detect at IL-22 or in IL-20 and two kinds of inhibition detections of IL-22 subsequently with respect to untransfected.The purpose that designs this analysis be conclusive evidence the bonded of IL-22 and IL-22RA/CRF2-4 is suppressed or to IL-20 and the inhibition of IL-22RA/IL-20RB bonded really owing to due to the IL-22RA receptor-specific bonded antibody.In addition, because facs analysis is to use two anti-the carrying out of anti-IgG, special FACS positive findings shows that neutralizing antibody may be the IgG class.Identify a main aperture by these modes, it combines with IL-22RA in the mould assembly ELISA that hardens, in based on the inhibition test of ELISA, suppress combining of IL-22 and IL-22RA, blocking-up IL-20 or IL-22 respectively with interaction (embodiment 28) through the Baf3 cell of IL-22RA/IL-20RB and IL-22RA/CRF2-4 transfection, and in the dyeing of the Baf3 cell of IL-22RA/IL-20RB and IL-22RA/CRF2-4 transfection, presenting strong positive (use anti-mouse IgG anti-) as two.

D produces the clone of the hybridoma of anti-IL-22RA specific antibody

The low density of use standard dilution (every hole in less than a cell) method clone generates the hybridoma of anti-IL-22RA monoclonal antibody (combining of the BaF3 cell of this monoclonal antibody cross neutralization IL-20 and IL-22 and suitable transfection).Behind bed board about 5-7 days, use ELISA on the mould assembly human il-22 RA-muFc that hardens, the clone to be screened, use the fusion rotein that contain irrelevant muFc again to detect positive cell with ELISA according to top description then.To select, its supernatant and IL-22RA-muFc in conjunction with and the clone that do not combine with the fusion rotein that contains irrelevant muFc further proves conclusively wherein specific antibody activity by repeating neutralization test and facs analysis.All IL-22RA antibody positives of selecting clone all clones twice at least guaranteeing its clonality, and the stability that produces of assessment antibody.Carrying out further many wheel clones, and screen according to description, is male until institute's DCRP of at least 95% preferably for producing the anti-IL-22RA antibody of neutrality.

B. by the biochemical characteristic research of the molecule of anti-IL-22RA monoclonal antibody identification

The biochemistry conclusive evidence that the IL-22RA really of the target molecule of being discerned for the anti-IL-22RA monoclonal antibody of being inferred carries out be with standard the immunoprecipitation step, carry out SDS-PAGE analysis or Western trace step then and carry out, wherein all used the Baf3 cell of the IL-22RA transfection of hanging oneself and the dissolvable film prepared product of untransfected Baf3 cell.And the dissolvable film prepared product of use expressing the non-transfected cells system of IL-22RA shows that this monoclonal antibody can discern the natural receptor chain and the receptor chain of transfection.Perhaps, detect monoclonal antibody specific immunity precipitation or the proteic ability of Western trace solubility IL-22RA-muFc.

Embodiment 31

Right from the serum of having injected by the mouse of the P815 cell of huIL22RA institute transfection

The neutralization of huIL22RA

Use the neutralization test of describing among the embodiment 28 based on cell, to carry out serial dilution (1% from having injected the serum (embodiment 30.A.2) through the mouse of the P815 of huIL-22RA transfection cell of living, 0.5%, 0.25%, 0.13%, 0.06%, 0.03%, 0.02% and 0%) also adds.Detect culture plate at 37 ℃, 5%CO ₂In hatched 4 days, this moment add AlamarBlue (Accumed, Chicago, IL), every hole 20 microlitres.Then at 37 ℃, 5%CO ₂Culture plate 16 hours again under the condition.The result show from the serum of four animals can in and huIL-22 and the two signal conduction of huIL20 by huIL-22RA.

In concentration is 1% o'clock, has neutralized by the 50%-80% of huIL-22 inductive propagation from the serum of six animals (7125,7127,7128,7118,7124 and 7117), under lower concentration inhibition of proliferation is descended with dose-dependent form.And, be 1% o'clock in concentration, neutralized by the 40%-70% of huIL-20 inductive propagation from the serum of four animals (7125,7127,7118 and 7117), under lower concentration, inhibition of proliferation is descended with dose-dependent form.These results have further shown really can be in the activity of short scorching aglucon IL-20 of antagonism under the lower concentration and IL-22 at the antibody of IL-22RA.

These results provide extra evidence, shown by combination, blocking-up, suppress, reduce, antagonism or in and the activity of IL-20 or IL-22 (separately or common) and the IL-22RA activity is effectively blocked, for example via the neutralizing monoclonal antibody at IL-22RA of the present invention, aspect the effect in the body that alleviates IL-20 and IL-22 (independent or common) may be useful, and may alleviate IL-20 and/or IL-22 inductive inflammation, for example in IL-20 inductive skin sufferer and in the IL-22 inductive skin sufferer viewed those, for example in psoriasis, IBD, colitis or other comprise IBD by IL-20 and/or IL-22 inductive diseases associated with inflammation, sacroiliitis, asthma, arthritic psoriasis, colitis, inflammatory skin sufferer and atopic dermatitis.

Embodiment 32

The phenotype of IL-22RA knock-out mice

A. carry the generation of the mouse of hereditary change

1. express mouse IL-20, have the birth generation of the transgenic mice of bright appearance afterwards

A) construct by K14 promoter expression mouse IL-20

For biological function in the body of studying IL-20, preparation transgenic constructs, the wherein IL-20 of mouse (also referring to embodiment 21) under the control of people K14 promotor.Design oligonucleotides makes it to produce the PCR fragment that contains total Kozak sequence and mouse IL-20 coding region.These oligonucleotide are designed at its 5 ' end the FseI site is arranged, the AscI site are arranged, be cloned among the pRSK14 to help it at 3 ' end.PRSK14 is the transgene carrier of a standard, contains human keratinized cell and epidermic cell specificity promoter.

Use the oligonucleotide of 200ng mouse IL-20 template (SEQ ID NO:33) and design, amplify the IL-20 (SEQ ID NO:34) of total length by the PCR reaction.Use the method known in the art to determine the reaction conditions of PCR.Separate the PCR product by agarose gel electrophoresis, use QiaQuick ^TM(Qiagen) gel extraction kit purified product.Use FseI and AscI (Boerhinger-Mannheim) to carry out enzyme the dna fragmentation of isolating, correct size and cut, ethanol sedimentation and being connected in advance in the pRSK14 that FseI and AscI enzyme cut.The pRSK14 plasmid is designed to express target gene in keratinocyte in transgenic mice and the epidermic cell, and it contains the expression cassette that flank is about 3kb human keratinous specificity K14 promotor.

According to manufacturer's guidance, the connection product electroporation of about 1 microlitre is transformed into DH10BElectroMax ^TMCompetent cell (GIBCO BRL, Gaithersburg, MD) in, the cell after transforming is coated on the LB flat board that contains 100 μ g/ml penbritins overnight incubation.The picking clone also cultivates on the LB flat board that contains 100 μ g/ml penbritins.From clone's micropreparation DNA of picking, cut inset with agarose gel electrophoresis screening mouse IL-20 by using FseI and AscI associating enzyme.Transgenic constructs with correct cDNA inset is proved conclusively by sequential analysis.Correct pRSK14-mouse IL-20 is prepared in a large number.

B) generation of K14IL-20 transgenic mice and evaluation thereof

From containing 5 of Keratin sulfate specificity K14 promotor ' and 3 ' flanking sequence, mouse IL-20 (SEQ ID NO:33, polypeptide shows in SEQ ID NO:34), isolate the long NotI fragment of about 4kb in transgenosis (TG) carrier of Gormon intron, IL-20cDNA and human growth hormone polyA signal sequence.(NY) in the mouse ovum, the preparation of microinjection and transgenic mice is according to Hogan, B.et al. for Taconic, Germantown to the B6C3f1 of fertilization with its microinjection Manipulating the Mouse Embryo, 2 ^NdEd., ColdSpring Harbor Laboratory Press, NY, the description in 1994 is carried out.

Use TaqMan ^TMThe expression of RT-PCR reaction pair TG RNA is carried out quantitatively, wherein uses the special PCR primer of human growth hormone polyA signal section in the transgenosis.

All TG constructs of expressing IL-20 have shown high birth mortality ratio, and the TG young baby who is born shows " light " phenotype usually.Newborn young baby's bright appearance seems relevant with the sclerosis of skin, and skin causes the decline of normal nurture as soon having done.It is rigid that their action generally becomes.From the histopathology angle, the young baby of light has the epidermis that thickens, and the keratin layer densification.Great majority are dead in 5 days among the former source kind young baby of these lights, and generally express transgenic (analysis draws according to transcription product) or they are not mosaic (transgenosis are very low to offspring's transmission) for survival and young baby wean.

Established a strain by K14 promotor control expression mouse IL-20.Expression level in its skin and the thymus gland is low, and all newborn young babies have the phenotype of light when being born.Generally speaking, this strain has 20% TG offspring, shows that the transgenosis young baby of 50-60% is dead in uterus.(in a hemizygote mating, offspring's 50% should be TG.)

2. there is not the generation of the mouse of IL-22RA expression: the IL-22RA knock-out mice

A) mouse IL-22RA knocks out the preparation of (KO) construct

For biological function in the body of further studying IL-22RA, the preparation mouse knocks out (KO) strain to eliminate the expression of IL-22RA.At first, use mouse IL-22RA cDNA probe screening mouse 129/SvJ genome BAC library.Determine to contain the clone of IL-22RA genomic gene seat and it is characterized.Mouse IL-22RA polynucleotide sequence is seen SEQ ID NO:41, and peptide sequence is seen SEQ ID NO:42.

Use ET clone technology (Zhang et al.1998.A new logic for DNAengineering using recombination in E.coli.Nat.Genet.Vol.20:123-8) preparation knockout carrier, be used to eliminate the construct that knocks out of IL-22RA with generation.In brief, the KO carrier contains IL-22RA gene 3 ' arm (long-armed) of IL-22RA gene 5 ' arm (galianconism), IRES-LacZ/MClneo selective marker and the 10kb of 1.8kb.In the KO carrier, the exon 2 of IL-22RA genome sequence, 3 and 4 and intron 2 and 3 replaced by the IRES-LacZ/MClneo selective marker, so just in the ES cell, produced the disappearance of about 4.4kb by homologous recombination.

After using restriction enzyme PmeI to make the linearizing of KO carrier, its electroporation is transferred in the 129/SvJ ES cell.The selection of homologous recombination incident and reorganization ES clone's evaluation is according to Robertson, E.J.et al. Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, 2 ^NdEd., IRL Press Limited, Oxford, the description in 1987 is carried out.

B) generation and the analysis of the mouse that IL-22RA expresses have been eliminated

To the exon 2 of IL-22RA genome sequence, 3 and 4 and the positive ES clone that lacked of intron 2 and 3 increase.It is expelled in the blastocyst of C57B1/6j mouse.After the of short duration heavy amplification of blastocyst through injection, it is expelled in foster mother's body of false pregnancy, produce mosaic.According to Robertson, E.J.et al. Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, 2 ^NdEd., IRL PressLimited, Oxford, the description in 1987 suddenly change IL-22RA the blastocyst injection, allophenic mice is bred and ensuing kind system transmits.

Identify KO mutant mouse by pcr gene typing mirror strategy.In multi-PRC reaction, use three PCR primer ZC22901 (SEQ ID NO:35), ZC45039 (SEQ IDNO:36), ZC38573 (SEQ ID NO ∷ 37) detects wild-type allele and mutation allele.The dna fragmentation length that wild-type (WT) allelotrope produces is 229bp, and the dna fragmentation length that mutation allele produces is 371bp.

The pairing of hemizygote mouse produces homozygote (HOM), heterozygote (Het) and wild-type (WT) offspring and the normal sex ratio of normal ratio.By PhysioScreen (collecting body weight, tissue weight, full blood count, clinical chemistry, gross examination of skeletal muscle and histopathology) mouse is checked, found not obviously difference between HOM, Het and wild-type animal.

B.IL-22RA is essential for IL-22 inductive SAA: SAA ELISA demonstration is expressed in the IL-22RA knock-out mice by IL-22 inductive SAA and does not occur

In order to determine whether inducing of SAA needs IL-22RA in the mouse of having injected IL-22,, get blood after 6 hours with the IL-22 injection IL-22RA KO mouse of 5 micrograms.

Carry out ELISA, determining the SAA level in the serum sample, this ELISA uses mouse SAA Immunoassay Kit according to manufacturer's explanation (BioSoufceInternational California) carries out, and serum is diluted to 1: 1000.In five WT mouse, four demonstrate in IL-22 injection back SAA level and raise, and in five HOM IL-22RAKO mouse, four have shown basic SAA level.Two Het IL-22RA KO mouse being checked all have the SAA level of rising, but its SAA level is lower than the SAA level in the WT mouse that the SAA level raises.This shows that it is essential that IL-22RA induces generation SAA for IL-22.

These results provide evidence to show, block the IL-22RA activity effectively, for example by the IL-22RA gene knockout or similarly by IL-22RA neutralizing monoclonal antibody of the present invention, for example can be reduced in similarly in psoriasis, IBD, colitis, endotoxemia or other inflammatory disease of IL-22 inductive by IL-22 inductive inflammation.

C.IL-22RA thickens necessary for IL-22 inductive epidermis: the miniature infiltration press pump by subcutaneous implantation gives IL-22 pure protein, can not cause IL-22RA KO mouse that epidermis takes place and thicken.

Whether in order to determine that IL-22RA thickens for IL-22 inductive epidermis is essential, with IL-22 by miniature infiltration press pump through subcutaneous route to IL-22RA HOM and the administration of WT KO mouse.The speed that pump is delivered IL-22 is 18.4 microlitre/skies, successive administration 7 days.4 HOM and 6 WT IL-22RA KO mouse give IL-22 albumen, and 3 HOM and 1 WT give PBS.

In the Baf3 proliferation test, detect the serum sample of the mouse of handling, with existing of conclusive evidence IL-22 through IL-22.Baf3 cell through IL-22RA and CRF2-4 transfection needs the existence of IL-22 or mouse IL3 to breed.((JRH Bioscience Inc., Lenexa KS), replenish 10% heat-inactivated foetal calf serum, 2mM L-glutaMax-1 to the RPMI substratum with these cell centrifugations and with the perfect medium that does not contain mIL-3 ^TM(Gibco BRL), 1mM Sodium.alpha.-ketopropionate (Gibco BRL) and PSN microbiotic (GIBCO BRL)) (after this, be called " substratum of no mIL-3) rinsing.Sedimentation cell and rinsing three times are to guarantee removing of mIL-3.Use the blood counting instrument counting cells then, cell is placed 96 orifice plates, 5000 cells/well, final volume are 200 microlitres/hole, use the substratum of no mIL-3.The concentration of mice serum is 1%, 0.5% in the hole, 0.25% or 0.125%.To detect culture plate at 37 ℃, 5%CO ₂Hatched under the condition 3 days, at this moment add Alamar Blue (Accumed, Chicago, IL), every hole 20 microlitres.Then with plate at 37 ℃, 5%CO ₂Cultivated again under the condition 24 hours.Alamar Blue provides the fluorescence metering reading according to the number of viable cell, and comparing with negative control like this is exactly directly measuring of on cell proliferation.Culture plate is placed 37 ℃ again, 5%CO ₂Cultivated 24 hours.(Wallac, Turku read plate on Finland), and wavelength is 544 nanometers (exciting) and 590 nanometers (emission) at Wallac Victor 2 1420 Multilabel Counter.The result shows does not have an animal through the PBS injection to have the IL-22 activity, and in one, 4 the HOM animals in 1 Het animal 2 and 6 the WT animals 3 have detectable IL-22 activity.When 1ug/ml IL-22BP exists, blocked serum inductive propagation, proved that it is that IL-22 is specific.

The IL-22 that hangs oneself in the future handles and undressed IL-22RA HOM, Het knock out skin samples submerged fixed in 10% buffered formalin of (KO) and WT control mice.Tissue is pruned, be embedded in the paraffin, handle routinely, (Wetzlar Germany), uses H﹠amp for Jung 2065Supercut microtome, Leica Microsystems with 5 microns sections; E dyeing.(Melville NY) observes down painted tissue for Nikon Eclipse E600, Nikon Inc. at opticmicroscope by the veterinary pathologist of ACVP council certificatory.

Each skin samples is checked the severity of inflammation in its subcutaneous pump implantation position tissue on every side to the rank of 4 (seriously) according to 0 (not having), check that to the rank of 3 (disperses) epidermis thickens the degree of (acanthosis) according to 0 (having), at the number of the thick count table cortex of epidermis.Between HOM mouse that gives PBS and WT mouse, there is not difference.The result of these two groups is mixed in the result who becomes a PBS group together.Calculate the mean value and the standard deviation of each treatment group, list in the table 14 below.

Table 14

Handle	The PBS contrast	HOM KO：IL-22	WT：IL-22
				Number of mice	4	4	6
Epidermal thickness	3.5±1.0	3.2±0.5	5.9±2.3
				The acanthosis degree	0.5±1.0	0.2±0.5	1.9±1.3
Inflammation	1.5±1.0	1.2±1.0	2.0±1.0

The result shows, accepting the trend that WT mouse mesocuticle thickness that IL-22 handles and acanthosis have increase, and IL-22RA HOM mouse its epidermis when accepting IL-22 and handle thickens with acanthosis less.

These results provide evidence to show, block the IL-22RA activity effectively, for example by the IL-22RA gene knockout or similarly by IL-22RA neutralizing monoclonal antibody of the present invention, can be reduced in the skin sufferer in for example psoriasis, IBD, colitis or other inflammatory diseases of IL-22 inductive similarly.

D.IL-22RA is essential for the newborn young baby's light of IL-20 inductive skin: transgenic mice and the mating of IL-22RA KO mouse of expressing mouse IL-20 produce the transgenosis young baby who does not have bright skin

In order to determine whether IL-22RA is essential for the newborn transgenosis young baby's of IL-20 inductive bright skin, and the K14muIL-20 transgenosis is hybridized in the IL-22RA KO strain, observes whether bright phenotype is arranged among the newborn young baby.

Bear 69 young babies, they meet Mendelian gene type ratio.All TG are bright under Het KO background, but not do not have one to be bright among the TG under TG or the HOM KO background.

The Baf3 cell of use expressing IL-20RA and IL-20RB carries out alamar blue proliferation test, to determine existing of in mice serum IL-20.These cells can produce IL-20 or mouse IL3 and reply and breed.The step of test and the top C step in partly is identical.The result of test shows that all TG mouse all have similar IL-20 activity, and is identical with IL-20TG level under the C57BL/6N background.The bright phenotype that any newborn young baby do not occur shows that bright newborn young baby's phenotype depends on the existence of IL-22RA.Proliferation test shows that all TG mouse all have suitable IL-20 activity, and identical with IL-20TG level under the C57BL/6N background.The bright phenotype that any newborn young baby do not occur shows that bright newborn young baby's phenotype depends on the existence of IL-22RA.

The 3rd day postpartum, the mouse that comes to contain the nest of K14 muIL-20TG under the comfortable IL-22RA KO background is carried out painless euthanasia, whole corpse is immersed in 10% buffered formalin fixes, the fixed tissue is cut, the crosscut piece that is divided into thoracic cavity and abdominal cavity, paraffin embedding is handled, routinely with 5 microns sections (Jung 2065 Supercut microtome, LeicaMicrosystems, Wetzlar Germany), uses H﹠amp; E dyeing.(Melville NY) observes down painted tissue for NikonEclipse E600, Nikon Inc. at opticmicroscope in unwitting mode by the veterinary pathologist of ACVP council certificatory.The epidermis number of plies of record organization abnormal conditions and counting swept-back hair end thorax.

Tissue from non-TG (the non-TG/IL-22RA KO HOM) mouse under IL-20TG (the IL-20TG/IL-22RA KO HOM) mouse under three HOM IL-22RA KO backgrounds and three the HOM IL-22RA KO backgrounds is carried out microscopy, do not find not contain unusual.Also checked the tissue of the mouse of the IL-20 TG (IL-20TG/IL-22RA KO Het) under two Het IL-22RA KO backgrounds.Epidermal area number in all animal mesocuticle layers is similar.But, have two IL-20TG/IL-22RA KO Het mouse to compare, eosinophilia in its epidermis, and the forfeiture that shows graininess in the granular layer with other animals.In the skin of any mouse or its hetero-organization, do not observe other abnormal conditions.

These results provide evidence to show, block the IL-22RA activity effectively, for example by the IL-22RA gene knockout or similarly by IL-22RA neutralizing monoclonal antibody of the present invention, for example can be reduced in similarly psoriasis, IBD, colitis or by in IL-20 and/or other inflammatory diseases of IL-22 inductive (comprising IBD, sacroiliitis, asthma, arthritic psoriasis, colitis, inflammatory skin illness and atopic dermatitis) by IL-20 and IL-22 inductive skin disorder.

Embodiment 33

The tissue morphology imaging analysis of IL-22RA knock-out mice

Set up a K14 IL-20m transgenosis (TG) mouse species, the newborn young baby of its TG shows the phenotype of light.This transgenosis is by the K14 promoter expression, and it makes to express to occur in the skin and produces in the keratic cell.Also set up an IL-22RA and knocked out (KO) mouse species, in mouse not under attack, do not observed significant change.With two incrosses, obtain newborn young baby: (1) TG/-HOM: under the background that does not have IL-22RA to express, express k14 IL-20m transgenosis with following four kinds of different genotype; (2) TG/-Het: under the background of some IL-22RA of IL-22RA genetic expression of a copy, expressing the k14IL-20m transgenosis; (3) WT/HOM: under the background that does not have IL-22RA to express, do not express k14 IL-20m transgenosis; And (4) WT/Het: under the background of some IL-22RA of IL-22RA genetic expression of a copy, do not expressing k14 IL-20m transgenosis.At the 3rd day, euthanasia (table 15) was carried out to 34 newborn young babies with range gene type in about 48 hours of birth back:

Table 15

	TG/-HOM* (group 1)	TG/-Het* (group 2)	WT/HOM* (group 3)	WT/Het* (group 4)
					Amount to	n＝10	n＝10	n＝9	n＝5

The TG=transgenosis; The WT=wild-type; HOM=isozygotys; The Het=heterozygosis; N=young baby's number.

Every young baby is decaptitated, become three parts (head end chest, tail end chest and belly) along the health crosscut.In the formalin with tissue samples stuck-at-0% neutral buffered of 4.0-5.0 mm thick, be treated to paraffin mass, phenodin and eosin (H﹠amp; E) dyeing is used for conventional histological examination and tissue morphology imaging analysis.Select the epidermis of spinal cord dorsal area in each tissue sample to be used for the tissue morphology imaging analysis, use Olympus BH-2 microscope, pick up camera (Dage-MTI, Michigan City, IN) and BioQuant True Color windows 98 software (R﹠amp; M Biometrics, Inc.Nashville, TN 37209), use following setting: parameter: mag.10X, Z off set 0; Array: length (mm); Measure: artificial and add the epidermis of each skin samples of syntype and the thickness (micron) of stratum corneum or keratinization layer is measured respectively 10 times in each 10 * field of microscope, the interval is 0.1 millimeter between each the measurement, calculates by Excel and obtains mean value, SD and SEM.All sections are upset order at random, under prior ignorant situation, measure.The measurement back is separated blind to section, result and treatment group are complementary.Net result according to treatment group is classified as follows: 1. the mean epidermal thickness (micron) of head end chest, tail end chest and belly is further divided into the mean epidermal thickness of (a) head end chest; (b) mean epidermal thickness of tail end chest and (c) mean epidermal thickness of chest.2. the average cuticle thickness (micron) of head end chest, tail end chest and belly is further divided into the average cuticle thickness of (a) head end chest; (b) the average cuticle thickness of tail end chest and (c) the average cuticle thickness of chest.3. head end chest, tail end chest and abdominal skin add cuticular mean thickness.Use GraphPad InStat software (GraphPad Software, Inc., San Diego, CA 92121) the gained result is analyzed.Use one-way analysis of variance (ANOVA) and check the significance,statistical of the 1st group to the 4th group mean value difference.Use the check of Tukey-Kramer multiple comparisons, determine significant difference (* P＜0.05 of mean value between two groups; * P＜0.01; * * P＜0.001; * * * P＜0.0001).It is significant that the observations of P＜0.05 is considered to.

(1) tissue morphology result

(a) mean epidermal thickness (micron) of head end chest, tail end chest and belly

With the IL-20 transgenosis young baby (TG/-HOM that does not have IL-22RA to express, P=0.001***) and contrast brood young baby (WT/HOM, P=0.001*** and WT/Het, P=0.001***) compare respectively, the IL-20 transgenosis young baby mesocuticle that lacks a copy IL-22RA gene (TG/-Het) thickens significantly (table 16).TG/-Het young baby shows not that the angling epidermis thickens, and this is likely because the keratinocyte hypertrophy causes.This increase may relate to all three non-cutinized layers (stratum basale, spinous layer and granular layer), but the most frequent stratum spinosum epidermidis that influences.It is about 25% that TG/-Het young baby's epidermal thickness increases, and it is obvious that stratum spinosum epidermidis becomes.And TG/-HOM young baby's epidermal thickness is compared with contrast (WT/HOM and WT/Het) only to have slightly and is increased, and statistics shows do not have significant difference between group (P＞0.05).Epidermal thickness to head end chest, tail end chest and belly also compares.Abdominal skin very thin under the normal circumstances is thicker than tail end chest epidermis, and tail end chest epidermis is than head end chest epidermis thick (table 16).

Table 16

	TG/-HOM (N＝28)	TG/-Het (N＝30)	WT/HOM (N＝27)	WT/Het (N＝15)
					Mean value	32.58±1.25	41.05±2.04	31.31±1.08	30.83±1.43

The result has represented mean value ± SEM.The slice numbers that N=is measured.

Squamous epithelium thickness in TG/-Het young baby's head end chest skin increases, and follows the hypertrophy of epithelial cell (keratinocyte); But relatively there is not significant difference (P=0.1565, table 17) with other each groups (TG/-HOM, WT/HOM and WT/Het).This may be because the histology illusion causes, and for example the otherness between section and the section, the natural framework of epidermis perhaps do not have very big effect in the thin layer skin of head end chest.Attention: the histology step of head end chest or the uncomfortable cooperation tissue morphology of tissue slice possibility are analyzed and are obtained significance,statistical.

Table 17

	TG/-HOM (N＝10)	TG/-Het (N＝10)	WT/HOM (N＝9)	WT/Het (N＝5)
					Mean value	29.18±2.24	33.20±2.24	27.28±0.62	29.38±1.77

The result has represented mean value ± SEM.The slice numbers that N=is measured.

(((P＜0.01**) compare respectively has only the mean value of IL-20 (TG/-) epidermal thickness of a copy IL-22RA gene (Het) to increase (table 18) for P＜0.001***) and WT/Het for P＜0.05*), WT/HOM with TG/-HOM.Statistics shows very significantly (P＜0.0001****) of group difference.Compare with the WT/Het epidermis, the TG/-Het epidermis increases about 29%.Do not have IL-20 (TG/-) young baby's of IL-22RA (HOM) phenotype partly to be similar to and lack a copy IL-22RA gene (Het) and have the young baby's who thickens epidermis phenotype, and different with the brood young baby's of contrast (WT/HOM and WT/Het) phenotype, but statistics shows with contrast there is not statistical discrepancy (P＞0.05).The TG/-HOM epidermis thickens about 14% than WT/HOM epidermis.Different with IL-20TG/-young baby, IL-22RAm acceptor defect young baby (WT/HOM and WT/Het) shows relative thinner epidermal thickness.Be significantly, the tissue morphology result of tail end chest epidermal thickness is consistent constant, and relevant with the belly mean epidermal thickness with head end chest, tail end chest, this shows that the histology step of tail end chest and tissue slice are very suitable for the tissue morphology imaging analysis.

Table 18

	TG/-HOM (N＝10)	TG/-Het (N＝10)	WT/HOM (N＝9)	WT/Het (N＝5)
					Mean value	35.91±1.37	43.79±2.35	30.83±1.86	30.94±2.83

The result has represented mean value ± SEM.The slice numbers that N=is measured.

Except TG/-HOM compare with the brood young baby of contrast do not have difference (WT/HOM and WT/Het, P＞0.05) in addition, belly mean epidermal thickness result (table 19) and tail end chest mean epidermal thickness result (table 18) are similar.Tissue slice has some variations, in the TG/-HOM group two slice loss is arranged, and does not promptly cover the epidermis section of dorsal area.

Table 19

	TG/-HOM (N＝8)	TG/-Het (N＝10)	WT/HOM (N＝9)	WT/Het (N＝5)
					Mean value	32.35±1.44	46.33±3.10	35.81±1.90	32.16±2.97

The result has represented mean value ± SEM.The slice numbers that N=is measured.

(b) the average cuticle thickness (micron) of head end chest, tail end chest and belly

Although do not increase for not expressing IL-22RA (HOM) or only expressing IL-20 transgenosis young baby (TG/-) mesocuticle thickness that copies this gene (Het) in background, but compare with the brood young baby of contrast (WT/HOM and WT/Het), in TG/-HOM and TG/-Het skin, observe stratum corneum or the keratinization layer thickness obviously reduces, statistics shows group difference very significantly (P＜0.0001****, table 20).(P＜0.01**), ((P＜0.001***) compare respectively, keratic amount has reduced by 36%, 50% and 49% respectively to WT/HOM on TG/-Het young baby's epidermal surface for P＜0.001***) and WT/Het with TG/-HOM.(WT/HOM, P＜0.05*) compare, TG/-HOM young baby's cuticle thickness have shown about 22% remarkable decline, and compare with WT/Het, have only descended 17%, do not have significant difference (P＞0.05) with its contrast.Cuticle thickness among contrast young baby WT/HOM and the WT/Het much at one, obviously, tail end stratum corneum in the heart is than the cutin bed thickness of belly, and the belly stratum corneum is than head end chest cutin bed thickness.

Table 20

	TG/-HOM (N＝8)	TG/-Het (N＝10)	WT/HOM (N＝9)	WT/Het (N＝5)
					Mean value	33.26±2.69	21.41±1.27	42.54±2.01	40.31±3.82

The result has represented mean value ± SEM.The slice numbers that N=is measured.

The cuticular in the heart mean thickness of head end (table 21) is similar to the cuticle thickness (table 20) of head end chest, tail end chest and belly, but cuticular remarkable minimizing only appear at TG/-Het and TG/-HOM (P＜0.05*) compare and with WT/HOM (P＜0.01**) compare.Standard deviation peace average value standard deviation height, this may be because chipping qualities is poor, skin samples is lost, the natural framework of epidermis or cause the effect of head end chest is not obvious.Attention: the histology step of head end chest or tissue slice may be not suitable for carrying out the tissue morphology analysis and obtain valuable results.

Table 21

	TG/-HOM (N＝28)	TG/-Het (N＝30)	WT/HOM (N＝26)	WT/Het (N＝14)
					Mean value	34.96±3.53	18.14±3.99	40.47±4.38	32.96±8.11

The result has represented mean value ± SEM.The slice numbers that N=is measured.

The cuticular in the heart mean thickness result of tail end (table 22) is similar to the cuticle thickness of head end chest, tail end chest and belly, has only following three exceptions: (1) TG/-HOM compares with TG/-Het and TG/-HOM compares with WT/HOM and shows there is not statistical discrepancy (P＞0.05); (2) TG/-HOM compares with WT/Het and shows significant difference (P＜0.01**); (3) stratum corneum among the WT/Het significantly thickens, and this may be because the illusion of organized processing causes, and for example keratoprotein is placing hypotonic solution or occurring swelling in water-bath storage period when long or expand.

Table 22

	TG/-HOM (N＝10)	TG/-Het (N＝10)	WT/HOM (N＝8)	WT/Het (N＝4)
					Mean value	35.64±3.4	24.22±1.54	44.35±3.51	53.77±7.21

The result has represented mean value ± SEM.The slice numbers that N=is measured.

Have only TG/-HOM to compare with WT/HOM and TG/-Het compares with WT/HOM and shows significant difference on the statistics, the P value is respectively P＜0.05* and P＜0.001*** (table 23).TG/-young baby and the brood mouse of its contrast (WT/HOM and WT/Het) show that relatively the belly cuticle thickness reduces.

Table 23

	TG/-HOM (N＝8)	TG/-Het (N＝10)	WT/HOM (N＝9)	WT/Het (N＝4)
					Mean value	28.84±4.36	21.86±1.30	42.45±3.15	33.25±3.96

The result has represented mean value ± SEM.The slice numbers that N=is measured.

(c) head end chest, tail end chest and abdominal skin add cuticular mean thickness (micron)

Compare with the brood mouse of contrast (WT/HOM and WT/Het), TG/-Het young baby shows the remarkable increase of epidermal thickness and the remarkable decline of cuticle thickness, and TG/-HOM young baby produces similar result, but influence degree low (table 24).

Table 24

	TG/-HOM (N＝10)	TG/-Het (N＝10)	WT/HOM (N＝9)	WT/Het (N＝4)
					Stratum corneum	32.58	41.05	31.31	30.83
Epidermis	33.26	21.41	42.54	40.31

The result has represented mean value ± SEM.The slice numbers that N=is measured.

(d) IL-20 is by the two signal conduction of IL-20RA and IL-22RA

Epidermis is an epithelial cell stratified, that bring in constant renewal in, and its balance that depends between cell proliferation, differentiation and the death reaches stable state.In normal epidermis, the active basal layer cell of mitotic division produces keratinocyte (keratinocyte becomes seedless scale from skin at last and comes off to external migration), the Keratin sulfate of final differentiation or is arranged in the cutinized layer of stratum corneum.Although known have many albumen to work in keeping the epidermis stable state, understand also considerably less to the synergy of the molecule between these incidents.IL-20 is a kind of novel receptor interacting protein, its IL20RA or IL-22RA acceptor (IL-22RA) conducted signal by expressing in the layer relevant with keratinocyte propagation in skin.The newborn young baby of IL-20 transgenosis shows unusual thickening and bright skin phenotype.The defective of IL-22RAm in the mouse (HOM) shows the not reaction of processing for IL-22, and the wild-type mice with IL-22RA gene shows significant epidermal thickness increase (P＜0.001*** after using IL-22 to handle, referring to the result of IL-22RAm KO/IL-22 tissue morphology imaging analysis, PID 59.2).For whether the disappearance of studying IL-22RA has influence to observed bright phenotype in the K14 IL-20m TG newborn mice, isozygoty defective (HOM) or IL-22RA heterozygosis defective (Het) mouse of the transgenic mice of ectopic expression IL-20 and IL-22RA carries out mating.Epidermal thickness is carried out the quantitative imaging analysis, several young babies were once carried out (i.e. 19 young babies in this tail end chest research in this research, section of every young baby, totally 19 sections), but there is not significance,statistical, reason is that the animal number of being studied is limited, and variant in the group.The purpose of this research is that the more skin samples in the head end chest of all young babies in the identical research, tail end chest and the belly are carried out quantitatively (i.e. 34 young babies of tissue morphology, 3 sections of every young baby, totally 102 sections), with the biology of probing into IL-20 and obtain reliable quantitative result.In order to obtain effective imaging analysis, we guarantee respectively to organize all individualities among the young baby for all, and the direction of the skin in paraffin mass is consistent in all skin samples, and the measurement of skin samples is carried out from same relative position.Carried out following two kinds of measurements: (1) to each skin samples, to the thickness measurement of epidermis 10 times, all is positioned at the spinal cord dorsal part, with the effect of research IL-20 in mediation keratinocyte propagation and differentiation in each 10 * microscopic field; (2) measure the thickness of stratum corneum or keratinization layer in the same manner, just the bright skin appearance among result and the newborn young baby of IL-20TG is connected.

The tissue morphology imaging analysis of epidermal thickness shows, the newborn young baby of TG/-Het (express the K14IL-20m transgenosis under the background of some IL-22RA of copy IL-22RA genetic expression) shows the epidermis that thickens, and the newborn young baby of TG/-HOM (expressing K14 IL-20m transgenosis under the background that does not have IL-22RA to express) does not have considerable change.The IL-20 transgenosis young baby (TG/-HOM) who all lacks with the IL-22RA gene of two copies and contrast brood mouse (WT/HOM and WT/Het) and compare, in the IL-20 transgenosis young baby (TG/-Het) who lacks a copy IL-22RA gene, epidermal thickness significantly increases, and the P value is respectively P=0.001***, P=0.001*** and P=0.001***.TG/-Het young baby's non-keratinocyte epidermis thickens, and mainly is because due to the keratinocyte hypertrophy in the spinous layer.TG/-Het young baby's epidermal thickness is compared with contrast (WT/HOM and WT/Het) has increased about 25%, and TG/-HOM young baby's epidermal thickness has only increase slightly, about 4-5%, statistics shows that TG/-HOM compares with its contrast WT/HOM and do not have significant difference (P＞0.05).

Cuticular tissue morphology result shows, although thickening appears in TG/-Het young baby's epidermis, but compare with the brood young baby of contrast (WT/HOM and WT/Het), observe Keratin sulfate in TG/-HOM and the TG/-Het skin or the keratinization layer thickness obviously reduces, statistics shows very significantly (P＜0.0001****) of group difference.(P＜0.01**), ((P＜0.001***) compare respectively, the Keratin sulfate amount of TG/-Het young baby's epidermal surface has reduced about 36%, 50% and 49% respectively to WT/HOM for P＜0.001***) and WT/Het with TG/-HOM.(have an appointment 22% remarkable decline of WT/HOM, P＜0.05*) compare, TG/-HOM young baby's cuticle thickness, and comparing with WT/Het, 17% (P＞0.05) has only descended with its contrast.Cuticle thickness among contrast young baby WT/HOM and the WT/Het much at one.As if the reduction of the newborn young baby's mesocuticle of TG/-HOM and TG/-Het mean thickness corresponding with the result of gross examination of skeletal muscle, promptly IL-20 (TG)/newborn young baby's of IL-22RA (Het) skin luminance brightness reduces (for example having less Keratin sulfate) on the cardinal principle level, be called gloss, and IL-20 (TG)/IL-22RA (HOM) not luminous (for example having more Keratin sulfate).On histology, the Keratin sulfate in TG/-young baby's stratum corneum is tightr than the Keratin sulfate among the WT young baby.Generally speaking, relevantly with loose keratinocyte among the newborn young baby of IL-20 transgenosis thickened epidermis and thin stratum corneum possible explanation why they have shown the phenotype of bright skin.

In the newborn young baby of the IL-20 transgenosis of the background (Het) that the IL-22RA gene with a copy is knocked out by target, observe the hypertrophy of keratinocyte and the final differentiation that is interfered.Cutaneous manifestations goes out the hypertrophy of keratinocyte, but can not break up fully, lacks Keratin sulfate or stratum corneum.The newborn young babies of the IL-20 transgenosis of two copy IL-22RA genes destroyed (HOM), the phenotype that shows is similar to TG/-Het skin, but only shows lower or MIN influence (Figure 12-15).The disappearance (HOM) that seems IL-22RA has influence partly for observed bright phenotype among the newborn young baby of K14IL-20mTG, and the disappearance of IL-22RA (Het) has only slight influence or almost not influence to bright phenotype.In other words, the signal conduction of IL-20 (a kind of novel acceptor interaction albumen by IL-20RA or IL-22RA acceptor conducted signal) is not probably because the defective of a copy IL-22RA gene is expressed (Het) to be hindered, but the equal defective expression of two copy IL-22RA genes can make its signal conduction be subjected to the obstruction of part.

These results provide evidence to show, block the IL-22RA activity effectively, for example by the IL-22RA gene knockout or similarly by IL-22RA neutralizing monoclonal antibody of the present invention, for example can be reduced in similarly psoriasis, IBD, colitis or by in IL-20 and/or other inflammatory diseases of IL-22 inductive by IL-20 and the influence of IL-22 inductive skin.

Embodiment 34

IL-22 is to the influence of IL-22RA knock-out mice

By the micropump or the micropump self of subcutaneous implantation belt conduit, give IL-22 or PBS (table 25) to 36 mouse that comprise 23 IL-22RA KO (HOM) mouse and 13 contrasts (WT) mouse:

Table 25

	HOM/PBS (group 1)	HOM/IL-22 (group 2)	WT/PBS (group 3)	WT/IL-22 (group 4)
					Male female	n＝3 n＝0	n＝10 n＝10	n＝3 n＝0	n＝8 n＝2
Amount to	n＝3	n＝20	n＝3	n＝10

From each animal, obtain the skin samples tissue of 1.5-2.5 cm long, 4.0-5.0 mm thick, be used for conventional histological examination and tissue morphology imaging analysis by the implantable pump position.In the formalin with all tissue samples stuck-at-0% neutral buffered, be processed into paraffin mass.Do 6 sections from each skin samples of every animal, 5 micron thickness are between the contiguous slices 10 microns at interval (all surface all has skin to cover), with phenodin and eosin (H﹠amp; E) dyeing.The tissue morphology imaging analysis of tissue sample be to use Olympus BH-2 microscope, pick up camera (Dage-MTI, Michigan City, IN) and BioQuant True Color windows98 software (R﹠amp; M Biometrics, Inc.Nashville, TN 37209) and use following setting to carry out: parameter: mag.10X, Z off set 0; Array: length (mm); Measure: manual and add syntype.In each 10 * field of microscope, measure respectively 5 times, catch 4 visuals field (10 * visual field=0.1 centimetre for example, four 10 * visual field=0.4 centimetre), meter skin thickness (μ m) thus from each skin biopsy center 0.4 centimeters.Every animal is measured six sections altogether, calculates by Excel and obtains mean value, SD and SEM.All sections upset order at random, measure under prior ignorant situation.The measurement back is separated blind to section, result and treatment group are complementary.Net result according to treatment group is classified as follows: the epidermal thickness of the male and female mice of 1.HOM and WT; 2.HOM and the epidermal thickness of WT male mice.Use GraphPad InStat software (GraphPad Software, Inc., San Diego, CA 92121) the gained result is analyzed.Use one-way analysis of variance (ANOVA) and check the significance,statistical of the 1st group to the 4th group mean value difference.Use check of Tukey-Kramer multiple comparisons and unpaired T-to check the significance of mean value difference between definite two groups.It is significant that the observations of P＜0.05 is considered to.

III. the epidermal thickness (micron) of the male and female mice of tissue morphology result (1) HOM and WT

Compare with the WT/PBS contrast, significantly increase (P=0.0001) at the WT mouse skin mesocuticle thickness that uses IL-22 (WT/IL-22) to handle.Compare with the HOM/PBS contrast, the IL-22RAm KO mouse skin indicator gauge skin thickness mean value that uses IL-22 (HOM/IL-22) to handle increases, but statistics shows there is not significant difference (P＞0.05) between two groups.Compare with the WT mouse, observe epidermal thickness in the IL-22RA KO mouse and descend significantly that (for example HOM/IL-22 and WT/IL-22 are relatively: P＜0.001) (table 26).

Table 26

	HOM/PBS (N＝3)	HOM/IL-22 (N＝19)	WT/PBS (N＝3)	WT/IL-22 (N＝10)
					Mean value	14.15±0.19	19.01±1.03	23.34±5.49	43.08±1.85

The result has represented mean value ± SEM.The N=number of animals.

(2) epidermal thickness (micron) of HOM and WT male mice

Compare with the male contrast of WT/PBS, increase about 2 times (P=0.0001) at the WT male mice skin mesocuticle thickness that uses IL-22 (WT/IL-22) to handle, but compare with the male contrast of HOM/PBS, the IL-22RAm KO male mice epidermal thickness that uses IL-22 (HOM/IL-22) to handle has only slightly and thickens (P＞0.05).It should be noted that with its contrast WT male mice and compare that the epidermal thickness of IL-22RAm KO mouse shows remarkable reduction, and (for example HOM/PBS and WT/PBS order compare: P＜0.05; HOM/IL-22 compares with WT/IL-22: P＜0.001) (table 27).

Table 27

	HOM/PBS (N＝3)	HOM/IL-22 (N＝9)	WT/PBS (N＝3)	WT/IL-22 (N＝8)
					Mean value	14.15±0.19	15.86±0.75	23.34±5.49	41.41±1.71

The result has represented mean value ± SEM.

(3) epidermal thickness of HOM and WT mouse (μ m), male and female comparison

Thick (for example HOM/IL-22/ is male compares with HOM/IL-22/ is female the epidermis of finding female mice: P＜0.01 than the epidermis of male mice; WT/IL-22/ is male to be compared with WT/IL-22/ is female: P＜0.05) (table 28).

Table 28

	HOM/IL-22 (male, N=9)	HOM/IL-22 (female, N=10)	WT/IL-22 (male, N=8)	WT/IL-22 (female, N=2)
					Mean value	15.86±0.75	21.85±1.3	41.41±1.71	49.75±4.82

The result has represented mean value ± SEM.

(4) epidermal thickness of HOM mouse (μ m), IL-22 passes through pump+catheter drug delivery by pump administration and IL-22

The epidermal thickness that uses pump and conduit to give in IL-22RAm KO (HOM) mouse of IL-22 will be apparently higher than the epidermal thickness in IL-22RAm KO (HOM) mouse of only using the pump administration (P＜0.0001 is not by matching the T-check) (table 29).

Table 29

	HOM w/IL-22 pump (M=8 ﹠ F=2, N=10)	HOM w/IL-22 pump+conduit (M=2 ﹠ F=8, N=10)
			Mean value	15.85+0.65	23.30+1.36

The result has represented mean value ± SEM.

M: male; F: female; N: total mice.

IV discusses

As a whole, the purpose of this research is that research IL-22RAm KO and WT mouse are accepted the influence of IL-22 processing back to epiderm skin, and these are found to connect with clinical symptom.Carry out the quantitative imaging analysis to determine H﹠amp; The thickness of the painted skin biopsy mesocuticle of E.Skin samples to every animal is measured (being that 6 segmentation sections of 20 times * every mouse of each section are measured for=120 times) 120 times by the techtology method, calculates by Excel and obtains mean epidermal thickness.Tissue morphology measurement studies have shown that IL-22 has caused the remarkable increase of epidermal thickness, particularly in having the WT mouse of IL-22RA acceptor, (calculate by ANOVA, P＜0.0001, be considered to very remarkable), and influence is lower or have only slight influence (P＞0.05) in having IL-22RAm KO (HOM) mouse of IL-22RA acceptor.Compare with the mouse of using PBS to handle, that the wild-type mice epidermal thickness of handling through IL-22 has increased is about 43% (WT/PBS for example, P＜0.001), comparing with contrast (HOM/PBS) and through IL-22RAm KO (HOM) the mouse skin thickness that IL-22 handles has only increased about 26% (P＞0.05).Compare with the WT mouse, IL-22RAm KO mouse shows thinner epidermis (P＜0.001).In a word, IL-22 points out this factor may participate in the regulation and control of epidermal growth and propagation to the biological impact of mouse skin.

Embodiment 35

The pharmacokinetics of anti-human IL-2's 0 monoclonal antibody (clone #262.7.1.3.2.4)

To test monoclonal antibody-anti-human IL-2's 0 monoclonal antibody (clone #262.7.1.3.2.4) is that the form of 1.08 mg/ml (being determined by the 280nm uv-absorbing) provides with 3 * 3 milliliters of equal portions, concentration, be stored in-80 ℃ standby.Carrier is 1 * PBS (50mM sodium phosphate, 109mM sodium-chlor) pH 7.3.Before using monoclonal antibody is at room temperature melted, first and second parts are used for respectively by intravenously and subcutaneous route administration (100 micrograms dose group).Half of the 3rd part is with 1 * PBS1: 2 dilutions are used for 50 microgram subcutaneous dosage groups, and half is with 1 * PBS1 in addition: 10 dilute and are used for 10 microgram subcutaneous dosage groups.Obtain female SCID mouse (n=96) from Charles River Labs.Check its health condition when animal is arrived at and divide assembling cage (three animals of every cage).Mouse is that 12 weeks are big during the research beginning, and mean body weight is 22 grams.

A. dosage

Female SCID mouse (n=24/ dosage group) is assigned randomly in 4 dosage groups (referring to table 30).The 1st group gives anti-huIL-20 monoclonal antibody by IV injection, by about 93 microlitres of tail vein injection; 2nd, 3,4 groups give monoclonal antibody by the SC approach, inject about 93 microlitres at nape.

B. sample collection

Before collecting blood, mouse is used fluothane and isoflurane holonarcosis.Collect blood sample at all time points by heart cathetrization, just get blood at 168 hours time points by eye, same animal was got blood once more by heart cathetrization in the time of 504 hours.Collect blood in the serum separator, made it to solidify 15 minutes.Then with sample centrifugal 3 minutes at 14000rpm.After centrifugal, the aliquot sample of 125-150 microlitre is packed in the good eppendorf tubule of mark, frozen at-80 ℃ immediately until analyzing (table 30).

Table 30

Group #	Dosage (route of administration)	Animal	The PK time point
				1	100μg(IV)	3 mouse/time point *	0.25,1,4,8,24,72,168,336 and 504 hours
2	100μg(SC)	3 mouse/time point *	0.25,1,4,8,24,72,168,336 and 504 hours
				3	50μg(SC)	3 mouse/time point *	0.25,1,4,8,24,72,168,336 and 504 hours
4	10μg(SC)	3 mouse/time point *	0.25,1,4,8,24,72,168,336 and 504 hours

* use identical animal 168 with 504 hours time points.

C. by ELISA the anti-huIL-20 monoclonal antibody in the serum is carried out quantitatively

Develop enzyme-linked immuno-sorbent assay (ELISA), make it can be used for the analysis of mice serum sample qualifiedly.Described serum is from resisting in the pharmacokinetic-mouse of IL-20 monoclonal antibody 267.7.1.3.2.4.It is commercial two anti-and use TMB to carry out colorimetric detection that this detection is designed to utilize.Change the extent of dilution be used for typical curve, with the definition of the linear portion that improves typical curve.The dilution range that is used for typical curve is the continuous 2 times dilutions of 100ng/mL to 0.231ng/mL, can be used for the quantitative of mice serum sample.The QC sample with 10% SCID mice serum be diluted to 1: 100,1: 1000 and 1: 10000, and return survey by typical curve.

D. pharmacokinetic analysis

Serum-concentration and time data download to WinNonlin Professional 4.0 (Pharsight, Inc.; Cary NC) is used for pharmacokinetic analysis in the software.Use the model analysis of non-chamber, determine pharmacokinetic parameter according to the average data of each time point.

E. result

The anti-human IL-2's 0 monoclonal antibody concentration of average serum is listed in table 31 after giving 100 microgram IV and 100,50 and 10 microgram SC:

Table 31

Time (hr)	100 μ g IV concentration (μ g/mL)	10 μ g SC concentration (μ g/mL)	50 μ g SC concentration (μ g/mL)	100 μ g SC concentration (μ g/mL)
					0.25	196(12)	LTR	0.101(0.065)	0.481(0.485)
1	154(18)	0.356(0.146)	1.61(0.52)	3.48(1.72)
					4	118(20)	2.42(0.53)	10.4(3.4)	19.7(4.7)
8	112(20)	3.41(0.30)	18.9(3.6)	40.2(6.4)
					24	103(13)	4.95(0.05)	26.3(0.7)	50.1(6.2)
72	101(16)	4.27(0.79)	21.0(3.4)	43.4(2.7)
					168	45.6(15.4)	2.92(0.53)	19.6(2.7)	37.6(3.4)
336	36.4(16.6)	3.60(0.31)	23.5(3.5)	34.4(5.8)
					504	28.8(3.8)	2.74(0.39)	20.5(3.6)	25.7(2.1)

LTR: be lower than and report numerical value

After the IV administration, the concentration of monoclonal antibody is two index decreased to the curve of time.After the SC administration, monoclonal antibody it seems absorption stage slowly, has been followed the restrictive removing of uptake rate.Serum pharmacokinetic parameter based on the average data of each time point is listed in table 32.

Table 32

Parameter	Unit	100μg IV	10μg SC	50μg SC	100μg SC
						C 0(IV)；C maλ(SC) T maλ t 1/2，λz AUC (0-t) AUC (0-mf)AUC (% extrapolation) V ss(IV)；V z/ F (SC) Cl (IV); Cl/F (SC) F (biological availability)	μg/mL hr hr hroμg/m L hroμg/m L ％ mL mL/hr ％	212 N/A 509 27059 48269 43.9 1.34 0.002 N/A	4.95 24 ND 1730 ND ND ND ND ND	26.3 24 ND 10845 ND ND ND ND ND	50.1 24 612 18110 41561 56.4 2.12 0.002 86.1

ND: can't measure owing to lack the interior data of final removing stage in concentration-time changing curve

After the IV administration, monoclonal antibody has shown low-down removing (CI=0.002mL/hr) and long removing half life (t _{1/2, λ z}About 21 days).Monoclonal antibody shows the volume of distribution (V of stable state _Ss=1.3 milliliters), less than the blood volume (about 1.7 milliliters) of mouse, the prompting monoclonal antibody almost is not distributed to beyond the lacuna vasorum.Return the peak concentration (C that calculates ₀) beguine will height according to the concentration of the volume expectation of injected dose and mouse blood.This point, and little V _Ss, point out together in the serum fraction that described monoclonal antibody may be confined to blood to a great extent.

After the SC administration, C _MaxNumerical value is linear with dosage to be increased.During 100 microgram SC dosage, the t of monoclonal antibody _{1/2, λ z}Be about 25 days, remove and afterwards similar of apparent volume of distribution and IV dosage.Biological availability is 86%.When two lower SC dosage, most of pharmacokinetic parameters are owing to have and can not do not estimated for the final removing stage of measuring, even when sample is got 504 hours.It seems that the absorption of monoclonal antibody reach a stable state after the SC administration, and removing in the whole process of research all has generation.

Embodiment 36

At CD4+CD45RB ^Hi(CD25-) IL20 and the IL-22 in colitis and the psoriasis model

Antagonist

A. general introduction

With CD4+CD45RB ^HiOr the CD4+CD25-T cell transfer can cause mouse colitis to occur in homology SCID mouse.Corotation moves modulating T cell (CD4+CD25+ or CD4+CD45RB ¹⁰) can suppress this colitis.With after the CD4+CD25-T cell transfer is in the mouse, if give injected in mice staphylococcal enterotoxin B (SEB) again, colitis not only appears in mouse, and psoriasis occurs.After cell transfer, give antibody or the solubility IL-22RA acceptor of anti-IL-22RA, IL-20, IL-22, IL-20R and/or IL-22RA in 0-21 days, monitor colitis and psoriasic symptom simultaneously.The decline of psoriasis scoring or the inhibition (histology) of colitis show that IL-21 can suppress these autoimmune diseases.

B. research and design

Separating spleen and inguinal lymph nodes from the B10.D2 mouse.Preparation single cell suspension and counting.Use Miltenyi Bead system, select to sub-elect the CD25+ cell by the positive.CD25-PE (BD Pharmingen) pair cell with dilution in 1: 100 dyes, and hatches 15 minutes.Rinse excessive antibody, with cell and the anti-PE pearl of 10 microlitres/10 ⁶Individual cell was hatched 20 minutes jointly.With PBS rinsing cell, and by LS post (Miltenyi Biotech).Reservation is used for further analysis by the cell (CD25-) of post.In these CD25-cells, add the mixture (1: 100) (Stem Cell technologies) of enrichment CD4, hatched 15 minutes.With PBS rinsing cell.In cell, add the antibiotin tetramer of dilution in 1: 10, hatched 15 minutes, add magnetic colloid (60 microlitres/10 afterwards ⁶Individual cell) hatches 15 minutes (all from Stem Cell Technologies).With cell by the negative chromatography column (0.5 ", Stem cell Technologies) of selecting.The cell that passes through is the CD4+CD25-cell.Use flow cytometer that purity is analyzed.In CB-17SCID, inject 0.4 * 10 by the IV approach ⁶Individual cell, cumulative volume are 200 microlitres.Second day (D1) passes through the IP approach to injected in mice 10 microgram SEB.The from the 2nd to the 5th week was followed the tracks of the symptom of psoriasis and colitis.According to following standard mouse is carried out the psoriasis scoring: 0-not damaged, 1-neck minor injury, 2-neck and back (trunk) major injury, 3-mouse neck, back and the very serious damage of belly.Measure ear simultaneously and thicken, as to one of measuring of disease seriousness.1-30 days, several groups of mouse were injected the anti-IL-22RA of PBS, 100 microgram control antibodies or 10-100 microgram, IL-20 by the IP approach, IL-22, IL-20R or IL-22R antibody, perhaps solubility IL-22RA uses different dosage regimen (on every Wendesdays time or twice weekly).

C. result and conclusion

Be suppressed through the mouse heifer tinea of antibody treatment and the symptom of colitis, showing that inhibition to IL-20 and/or IL-22 function can suppress the autoimmunity symptom in this psoriasis and the colitis model.

Embodiment 37

IL-20 and IL-22 antagonist in SCID-hu transplanting psoriasis model

People's psoriasis skin of transplanting on the SCID mouse can keep that it is clinical several weeks, light microscopic and immunohistochemical methods psoriasis character.This model provides a system with damaged organized renewing for the methods of treatment of normal phenotype for estimating.In case people's skin is successfully transplanted, anti-IL-22RA, IL-20, IL-22, the antibody of IL-20R and/or IL-22R or solubility IL-20 or IL-22 acceptor can administration several weeks, can the analytical table skin thickness, to estimate these antagonists to psoriasic influence.

B. research and design

Take out 6 millimeters punchings of full thickness biopsies from healthy adult volunteer and psoriasis damaged skin, its corium by whole epidermis and several millimeters is formed.Obtain 4-6 biopsy from each donor.A punching biopsy of each donor is transplanted to recipient SCID mouse (CB-17, skin of back surface Taconic).With animal rearing in the environment of no cause of disease.(transplanting back 2-3 week) after success is transplanted begins by following processing: a biopsy is used to use the soluble receptors of anti-human il-22 RA, anti-human IL-2 0, anti-human il-22 monoclonal antibody or IL-20 or IL-22 to handle (intraperitoneal injection as negative control (PBS or homotype monoclonal antibody), a biopsy as positive control (Ciclosporin A), a 2-3 biological tissue, on every Wendesdays time continuous 2-4 week, carry out) according to the M-W-F schedule.

C. quantitative analysis

In whole experiment, regularly carry out clinical observation and evaluation, and carry out record.Severity to the psoriasis breakage under the situation of not knowing sample is in advance estimated according to many scales property, sclerosis and erythema.Use 3 standards that these parameters are given a mark: 0=does not have skin to involve fully; 1=slightly involves; The 2=moderate is involved; 3=seriously involves.When the administration stage finishes all animals are implemented euthanasia, collection organization is used for histology and immunohistochemical methods.(1) fixation of tissue is partly dyeed in 10% formalin and with phenodin and eosin.Use the NIH imaging software, cuticle region is measured as the function of the epidermal thickness change of unit length.A plurality of areas to each graft carry out quantitatively, amass so that high n value and average epidermis side to be provided.(2) go up each high power field (struvite monocytic number in 0.103 * 0.135mm) in the corium; (3) Parakeratotic degree is with the standard scoring of relative unit according to 0-3, wherein 0 is no parakeratosis, part less than 1/3 in the 1 representative section has parakeratosis, greater than 1/3 but have parakeratosis less than 2/3 part, the part greater than 2/3 in the 3 representative sections has parakeratosis in the 2 representative sections.(4) remaining harpoon is dyeed (mark of proliferative keratinocyte) to Ki67, with the number of Ki67 circulation keratinocyte in every mm length in the evaluation section.The reduction of the psoriasis severity of measuring with epidermal thickness shows that the neutralization to IL-20 and IL-22 function may be effective in this psoriasis model.For the reduction of psoriasis severity is carried out quantitatively, we have measured epidermal thickness, have gone up the number of struvite cell in the corium, the number of Ki67 circulation keratinocyte and Parakeratotic grade.Compare with control group mice, all four parameters all significantly reduce, and show the potential therapeutic value of IL-20 and IL-22 antagonist.

Embodiment 38

Use Alamar Blue propagation to detect and use the BaF3/IL-22RA/IL-20RB cell screening

The IL-20 antagonistic activity

With IL-22RA and IL-22RB cotransfection factor dependency pre B cell is BaF3 (referring to the method among the embodiment 3), and uses the I1-20 of different concns to handle.According to the description among the embodiment 3, with alamar blue assay assessment propagation.Under the concentration of estimating to have in cytokine, IL-20 stimulates proliferation in dose-dependent mode, shows IL-20 combination and the assorted dimerization IL-22RA/IL-20RB acceptor of activation under the concentration that the cytokine expectation should have.The negative control that contains untransfected BaF3 cell is not bred.

For the activity of determining that anti-IL-22RA antibody whether can antagonism IL-20, use anti-IL-22RA antibody as the active antagonist of IL-20, carry out above-described detection.When IL-20 and this antagonist are combined, the response of IL-20 just is lowered to the level of background.It is exactly the antagonist of IL-20 aglucon for the existence proof of the antagonist of the proliferation function of elimination or reduction IL-20.This detection can also be used to detect active other antagonists of IL-20 described herein, for example comprises the antagonist polypeptide of solubility IL-22RA acceptor.

Embodiment 39

Anti-huIL22RA monoclonal antibody is to IL-20 and the active neutralization of IL-22

The neutralization test that use is described in embodiment 28 based on cell, the mouse anti huIL-22RA monoclonal antibody (embodiment 30 (D)) of purifying is added with the serial dilution degree, for example concentration is 10ug/ml, 5ug/ml, 2.5ug/ml, 1.25ug/ml, 625ng/ml, 313ng/ml, 156ng/ml and 78ng/ml.With assay plate at 37 ℃, 5%CO ₂In hatched 4 days, add afterwards Alamar Blue (Accumed, Chicago, IL), every hole 20 microlitres.Then at 37 ℃, 5%CO ₂In continued to hatch plate 16 hours.The result show purifying anti-huIL-22RA monoclonal antibody can in and the signal conduction that produces by huIL-22RA of huIL-22 and huIL-20.When concentration was 10ug/ml, antibody had neutralized fully by huIL-22 or huIL-20 inductive propagation, under lower concentration inhibition of proliferation is shown with dose-dependent form decline.Negative control mouse monoclonal antibody with the homotype coupling is tested under concentration described above, and the result shows that the proliferation function to two kinds of cytokines does not all have to suppress.These results further proof really can be in the activity of short scorching aglucon IL-20 of antagonism under the lower concentration and IL-22 at the monoclonal antibody of IL-22RA.

These results provide extra evidence to show, the IL-22RA activity is effectively blocked, for example via IL-22RA neutralizing monoclonal antibody of the present invention, can help blocking-up, suppress, reduce, antagonism or in and the activity in vivo of IL-20 and IL-22 (separately or common), and may alleviate IL-20 and/or IL-22 inductive inflammation, for example in IL-20 inductive skin sufferer and in the IL-22 inductive skin sufferer viewed those, psoriasis for example, IBD, colitis or IL-20 and/or other diseases associated with inflammation of IL-22 inductive comprise IBD, sacroiliitis, asthma, arthritic psoriasis, colitis, in inflammatory skin sufferer and the atopic dermatitis.

Embodiment 40

Use the anti-IL-22RA monoclonal antibody of neutrality to handle conceived IL-20 and IL-22 commentaries on classics

Dna murine

Active for neutralization in the body that detects rat anti-mouse IL-22RA monoclonal antibody (mAb), in the IL-20 of pregnancy transgenosis (Tg) and IL-22 transgenic mice, inject anti-mouse IL-22RA monoclonal antibody by the intraperitoneal approach.Check that whether " light " phenotype that shows these strain mouse features among the newborn young baby usually exists.

Especially, with the C57BL/6N mouse mating in male IL-20Tg (use Keratin sulfate 14 promotors preparation) or IL-22Tg (use insulin promoter) mouse and rutting sedson, judged by the inspection vaginal suppository whether female mice mating takes place in second day.The female mice that each is only conceived is isolated in the independent cage, monitoring every day.Treatment group comprises at least 4 every group pregnant female mouse, so that Tg and non-Tg young baby are analyzed in significance ground on the energy statistics.According to the former experience on these transgenic mices, each nest has about 6-8 young baby usually, and wherein 2-3 only is a transgenic positive.

Post-coitum 7-9 days (embryo age 7-9; E7-9), the 250-500 microgram rat anti-mouse IL-22RA monoclonal antibody (rat IgG2a homotype) among the 200-250 microlitre PBS is injected to female mice by the intraperitoneal approach.The syringe needle of use weak point, shallow injection angles are to avoid being injected directly into intrauterine.Female mice to pregnancy is injected three days (Monday, Wednesday and Friday) weekly with this mode, injects for two weeks (until producing), and purpose is successfully to contact the embryo who is growing.Control group (every group is no less than 4 female pregnant mouse) comprises following: homotype control rats IgG2a monoclonal antibody, anti-people/mouse IL-22 monoclonal antibody (rat IgG1 homotype) and homotype control rats IgG1 monoclonal antibody.As in and the contrast of mouse IL-20, give conceived female mouse injection can in conjunction with and in and solubility IL-20R-Fc4 albumen or the Fc4 reference protein of people and mouse IL-20.

Birth back 1-2 days, whether the close observation young baby bright skin phenotype occurs.Second day, the young baby is implemented euthanasia, get the part of tail and extract DNA, to determine every young baby's genotype (Tg or non-Tg).Collect skin samples and carry out histologic analysis to estimate the cuticular cellulose that thickens whether the young baby has these Tg mouse features of common performance.Get young baby's trunk blood (and eye blood of getting mother (dams) after being born one day), the anti-IL-22RA monoclonal antibody level in every mice serum is carried out quantitatively by ELISA.Because these monoclonal antibodies are effective I L-20 and/or IL-22 inhibitor in vivo, Tg young baby has normal skin (promptly not having epidermis to thicken or " light " appearance).

Embodiment 41

IL-20 in organ culture psoriasis model and IL-22 antagonist

People's psoriasis patch skin can maintain in the organ culture, and the abnormal structure of damaged skin feature can be maintained under the condition that does not have the appositional growth factor.Can give antibody or solubility IL-20 or the I1-22 acceptor of anti-IL-22RA, IL-20, IL-22, IL20R and/or IL-22R, the histologic characteristics of psoriasis damaged skin is improved.

B. research and design

Take out 2 millimeters punchings of full thickness biological tissues from healthy adult volunteer or psoriasis damaged skin, its corium by whole epidermis and several millimeters is formed.Immediately tissue is immersed into after the biopsy sampling that (Clonetics Inc, Walkersville is MD) in the tissue culture medium (TCM) of Zu Chenging by keratinocyte minimum medium (KBM).In substratum, add calcium chloride and make Ca ²⁺Final concentration reach 1.4mM (Varani et al, 1993,1994).Then biological tissue is placed the hole of 96 orifice plates, wherein contain 200 microlitres and replenish Ca ²⁺KBM, use anti-IL-20, IL-22, IL22RA antibody or solubility IL-20 or IL-22 acceptor to handle or do not handle.Culture was cultivated 8 days in 37 ℃ of atmosphere that contain 95% air and 5% carbonic acid gas.

C. quantitative analysis

When hatching end, will be organized in 10% buffered formalin and fix, and after with phenodin and eosin dyeing, carry out tissue examination.Psoriasis is organized in the outward appearance that the outward appearance that is exposed to behind antibody or the soluble receptors can similar healthy tissues, comprise following observed phenomenon: originally amorphous, unbodied substrate epidermic cell develops into the outward appearance of more apparent column, has the polarity of recovery; Epidermis net ridge is degenerated, and the area decreases that epidermic cell extends to corium, the integral body of upper epidermis layer are degenerated and reduced.The organ culture model provides a rapid and sensitive method, can be used for determining whether certain particular compound has the potentiality as anti-hyper-proliferative medicament.Abnormal structure learns feature and can improve when existing at IL-20, IL-22 antagonist, points out the validity of this medicament in the treatment psoriasis.

Embodiment 42

With neutrality monoclonal antibody R2.1.5F4.1 and R2.1.15E2.1 bonded mIL22RA (zCytoR11m)

The mapping location in zone

A. being neutralized property monoclonal antibody bonded epi-position on the mouse IL-22RA.

The purpose of experiment described below be determine in the aminoacid sequence of mouse IL-22RA soluble receptor body protein (SEQID NO:62) for receptor active or for antagonist or neutrality antibody in conjunction with one or more important zone.Mouse IL-22RA-Fc albumen excises Fc with zymoplasm earlier, holds the methionine(Met) sequence to excise C with cyanogen bromide (CNBr) incubation then.The peptide that produces through the CNBr excision is carried out fractional separation, and the combination that detects each fraction with ELISA is active, with Western analytical reaction (using in having in ELISA and Western and the monoclonal antibody-clone R2.1.5F4.1 and the R2.1.15E2.1 of character).

After the CNBr cutting, may produce following peptide (table 33) from non-reducing total length mIL-22RA.Under non-reduced condition, halfcystine is connected by disulfide linkage, may produce inner a connection in peptide 1, produces a connection between peptide 3 and 5.The residue of black matrix may participate in the aglucon combination, and these residues may participate in aglucon bonded residue corresponding to participating among the human il-22 RA in SEQ ID NO:2 or 3, described in embodiment 42B.Especially, SEQ IDNO:48 corresponding to the amino-acid residue 16 (His) of SEQ ID NO:42 to 83 (Met); SEQ ID NO:49 corresponding to the amino-acid residue 84 (Glu) of SEQ ID NO:42 to 109 (Met); SEQ ID NO:50 corresponding to the amino-acid residue 110 (Thr) of SEQ ID NO:42 to 137 (Met); SEQ ID NO:51 corresponding to the amino-acid residue 138 (Leu) of SEQ ID NO:42 to 177 (Met); SEQ ID NO:52 corresponding to the amino-acid residue 163 (His) of SEQ ID NO:42 to the amino-acid residue 163 (His) of 208 (Pro) or SEQ ID NO:62 to 212 (Arg).

Table 33

The peptide numbering	From	Arrive	Sequence
				CNBr peptide 1	1	68	HTTVDTSGLLQHVKFQSSNFENILTWDGGP ASTSDTVYSVEYKKYGERKWLAKAG CQRI TQKF CNLTM(SEQ ID NO：48)
Non-reduced: the halfcystine in the peptide 1 links to each other
				CNBr peptide 2	69	94	ETRNHTEFYYAKVTAVSAGGPPVTKM (SEQ ID NO：49)
CNBr peptide 3	95	122	TDRFSSLQHTTIKPPDVT CIPKVRSIQM (SEQ ID NO：50)
				Non-reduced: peptide 3-5 links to each other
CNBr peptide 4	123	162	LVHPTLTPVLSEDGHQLTLEEIFHDLFYRLE LHVNHTYQM (SEQ ID NO：51)
				CNBr peptide 5	163	212	HLEGKQREYEFLGLTPDTEFLGSITILTPILS KESAPYV CRVKTLPLVPR (SEQ ID NO：52)

1.CNBr separating of cutting and peptide fraction

With 50 microgram mIL22RA freeze-drying and heavy molten with 180 microlitre formic acid (70%).Add 1 microlitre and be dissolved in 5M CNBr in the acetonitrile.With the sample mixing, make it at room temperature dark place reaction 18 hours.The reaction mixture of getting 150 microlitres carries out fractional separation by the reversed-phase HPLC that is assemblied on the analytical Zorbax SB300-C8 chromatography column.The gradient separations elution peak of use from 25% acetonitrile (0.085%TFA) and 75% water (0.1%TFA) to 95% acetonitrile (0.085%TFA) and 5% water (0.1%TFA).UV analyzes and shows three main peaks and two small peaks, collects it.Each fraction is divided into two parts: a part is carried out ELISA and is detected, and another part freeze-drying is also heavy molten with 150 microlitre phosphate buffered saline buffers (PBS).The PBS fraction is carried out UV to be analyzed and to have proved conclusively all peaks and all be recovered from analytical chromatography column.The PBS fraction is carried out Western to be analyzed.

2.ELISA

The HPLC fraction that contains the peptide sequence after IL-22RA is cut by CNBr uses HPLC damping fluid (90% acetonitrile, 10% water, 0.09% trifluoroacetic acid) to be diluted to the concentration that approximately equates, sample is added in 4 holes on the 96 hole microtiter plates, every hole 100 microlitres, dried overnight under the room temperature in stink cupboard.((0.05%Tween-20) 37 ℃ were sealed 2 hours for PBS, 0.1%BSA to use ELISA B damping fluid then for PBS, 0.05%Tween-20) rinsing flat board with ELISA C damping fluid.(clone R2.1.5F4.1 and clone R2.1.15E2.1) is diluted to 2 μ g/ml with ELISA B damping fluid with two kinds of monoclonal antibodies at IL22RA.Every kind of monoclonal antibody all joins in each peptide sequence sample, and every hole 100 microlitres are hatched culture plate 60 minutes at 37 ℃.Rinsing is diluted to 1 μ g/ml with ELISA B damping fluid with two anti-(the mouse IgG of the goat Chinese People's Anti-Japanese Military and Political College (Jackson) that puted together horseradish peroxidase) to remove unconjugated antibody, adds 100 microlitres in every hole.Culture plate was hatched 60 minutes at 37 ℃.With each hole of ELISA C damping fluid rinsing, in TMB 1 Component HRP Microwell Substrate (BioFx), hatched 5 minutes.Add 450nm Stop Reagent for TMB Microwell (BioFx) termination reaction, read in the plate instrument (Molecular Devices) flat board to be read the absorbancy at 450nm place at Dynatech ELISA.

The result shows that the HPLC#4 fraction of monoclonal antibody R2.1.5F4.1 and mIL22RA CNBr reaction reacts, and has also produced a band simultaneously in the Western Blot experiment.

3.Western trace

To contain IL-22RA and be spent the night by freeze-drying under the HPLC fraction room temperature of the peptide sequence after the CNBr cutting, heavy molten with PBS.Then that sample and irreducibility sample buffer (Invitrogen) is mixed, boiled 10 minutes.Then with sample on the sample, and on 4-12%Bis-Tris gel (Invitrogen), carry out the SDS-PAGE electrophoresis, use 1x MES-SDS electrophoretic buffer (Invitrogen), and in 20% methyl alcohol transfering buffering liquid, transfer to nitrocellulose filter (0.2 μ m; Bio-Rad) on, all operations at room temperature carries out.Make filter membrane dried overnight at room temperature.Sealed filter membrane 30 minutes with the buffer A that contains 10% skim-milk (50mM Tris, pH 7.4,5mM EDTA, 0.05%Igepal CA-630,150mM NaCl, 0.25% gelatin) room temperature.To be diluted to 2 μ g/ml at the monoclonal antibody (clone R2.1.5F4.1) of IL-22RA with the buffer A that contains 2.5% skim-milk.With trace one anti-in incubated at room 1 hour.Hatch the back and wash film three times, hatched 1 hour by two anti-(the mouse IgG of the goat Chinese People's Anti-Japanese Military and Political College (Jackson Inc.) that puted together horseradish peroxidase) of dilution in 1: 5000 under the room temperature with the buffer A that contains 2.5% skim-milk with buffer A.The rinsing trace is used chemical luminous substrate (Lumi-Light Western Blotting Substrate then; Roche) colour developing is with luminescence imaging instrument (Mannheim Boehringer Lumi-Imager) exposure.

Under 30 minute time shutter, non-reduced gel shows that fraction 4 and fraction 5 have very strong band, and fraction 3 has only a weak band.Fraction 4 also is positive in ELIsA detects.

The N end order-checking of active fraction #4

5 CNBr peptide fractions of collecting from analytical reversed phase chromatography post, fraction 4 has shown active in ELISA and also has been positive in the Western trace.In order to identify the peptide in the active fraction 4, use well-known method that sample is carried out the Edman degraded.From this active fraction, identify 3 N ends, consistent with peptide 2 (SEQ ID NO:49), 3 (SEQ ID NO:50) and 5 (SEQ ID NO:52).These results show that antibody combines with peptide 2 (SEQ ID NO:49), 3 (SEQ ID NO:50) and 5 (SEQ ID NO:52).

Table 34

The Edman degraded	The N-terminal sequence	Peptide is identified
			The mIL22RA sequence that first sequence C NBr-that obtains from fraction 4 produces	HLEGK QREYE FLGLT PDTEF HLEGK QREYE FLGLT PDTEF LGSIT ILTPI LSKES APYV C RVKTL PLVPR (SEQ ID NO：53)	CNBr peptide 5 (SEQ ID NO:52)
The mIL22RA sequence that second the sequence C NBr-that obtains from fraction 4 produces	ETRNH TEFYY AKVTA VSAGG ETRNH TEFYY AKVTA VSAGG PPVTK M (SEQ ID NO：54)	CNBr peptide 2 (SEQ ID NO:49)
			The mIL22RA sequence that the 3rd the sequence C NBr-that obtains from fraction 4 produces	TDRFS XLQHT XIXPX DXXXI TDRFS SLQHT TIKPP DVT CI PKVRS IQM(SEQ ID NO：55)	CNBr peptide 3 (SEQ ID NO:50)

Discuss

From through the mixture of the mIL22RA peptide of CNBr cutting, isolating 5 fractions.Wherein have only the 4# fraction in ELISA, to have activity and the apparent positive in Western.Three N ends have been determined in the Edman degraded, and are corresponding with CNBr peptide 2 (SEQ ID NO:49), 3 (SEQID NO:50) and 5 (SEQ ID NO:52) among the fraction 4#.In these zones, six residues may participate in the aglucon combination potentially.These aglucons are Y93, R112, K210 and the E211 among the SEQ ID NO:42, and they are also corresponding to Y78, R97 among the SEQ ID NO:62, K195 and E196 residue.Y60 and F164 residue among the SEQ ID NO:42 also participate in the aglucon combination.

B. being neutralized property monoclonal antibody bonded epi-position on human il-22 RA.

The purpose of experiment described below be in the extracellular domain of determining in human il-22 RA albumen (the SEQ ID NO:2) aminoacid sequence for receptor active or for antagonist or neutralizing antibody in conjunction with one or more important zone.Then human soluble acceptor IL-22RA albumen (is for example comprised SEQ ID NO:3, for example with the IL-22RA-Fc behind the zymoplasm excision Fc) by hatching jointly C is held the methionine(Met) excision with cyanogen bromide (CNBr), perhaps use known in the art this albumen is cut into to determine segmental other reagent.The peptide that produces through CNBr cutting is carried out fractional separation, with ELISA detect each fraction in conjunction with active, with Western analytical reaction (use have in the monoclonal antibody of character).

Prediction has four halfcystines with disulfide linkage form Cheng Jian, i.e. Cys71-Cys79 and Cys204-Cys217 among the SEQ ID NO:2.After the CNBr cutting, may produce following peptide from non-reducing total length human il-22 RA: peptide 6 (SEQ ID NO:56), peptide 7 (SEQ ID NO:57), peptide 8 (SEQ ID NO:58), peptide 9 (SEQ ID NO:59), peptide 10 (SEQ ID NO:60) and peptide 11 (SEQ ID NO:61) (table 35).Halfcystine is connected by disulfide linkage, makes and produces a possible connection between peptide 7 (SEQ ID NO:57) and the peptide 10 (SEQ ID NO:60).Especially, SEQ ID NO:56 corresponding to the amino-acid residue 1 (Pro) of SEQ ID NO:3 to 92 (Met); SEQ ID NO:57 corresponding to the amino-acid residue 93 (Thr) of SEQ ID NO:3 to 120 (Met); SEQ ID NO:58 corresponding to the amino-acid residue 121 (Ile) of SEQ ID NO:3 to 160 (Met); SEQ ID NO:59 corresponding to the amino-acid residue 161 (His) of SEQ ID NO:3 to 185 (Met); SEQ ID NO:60 corresponding to the amino-acid residue 186 (Ile) of SEQ ID NO:3 to 199 (Met); SEQ ID NO:61 corresponding to the amino-acid residue 200 (Cys) of SEQ IDNO:3 to 211 (Thr).

Table 35

The peptide numbering	From	Arrive	Sequence
				CNBr peptide 6	1	92	Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val Lys Phe Gln Ser Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr Pro Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg Asp Trp Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn Leu Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala Arg Val Thr Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys Met (SEQ ID NO：56)
CNBr peptide 7	93	120	Thr Asp Arg Phe Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro Asp Val Thr Cys Ile Ser Lys Val Arg Ser Ile Gln Met (SEQ ID NO：57)
				CNBr peptide 8	121	160	Ile Val His Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn Arg Thr Tyr Gln Met (SEQ ID NO：58)
CNBr peptide 9	161	185	His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe Phe Gly Leu Thr Pro Asp Thr Glu Phe Leu Gly Thr Ile Met (SEQ ID NO：59)
				CNBr peptide 10	186	199	Ile Cys Val Pro Thr Trp Ala Lys Glu Ser Ala Pro Tyr Met (SEQ ID NO：60)
CNBr peptide 11	200	211	Cys Arg Val Lys Thr Leu Pro Asp Arg Thr Trp Thr (SEQ ID NO：61)

4.CNBr cutting and the separating of peptide fraction, Western and ELISA and N end check order

Lay equal stress on the people IL22RA freeze-drying of about 50 micrograms molten, fractional separation, collection and with analyzing as Western and the ELISA that describes among the embodiment 42A contain the fraction of anti-IL-22RA monoclonal antibody and are analyzed by ELISA and Western with evaluation and show and those fractions of IL-22RA bonded.To the CNBr peptide fraction ELISA detection of active of collecting from analytical reversed phase chromatography post, and positive with Western trace conclusive evidence.Use well-known method by the peptide in the positive fraction of Edman degraded evaluation.

Discuss

Mouse CNBr peptide #5 (SEQ ID NO:52) is corresponding to people CNBr peptide #9 and #10 (SEQID NO:59 and SEQ ID NO:60); Mouse CNBr peptide #2 (SEQ ID NO:49) is corresponding to people CNBr peptide #6 (SEQ ID NO:56); Mouse CNBr peptide #3 (SEQ ID NO:50) is corresponding to people CNBr peptide #7 (SEQ ID NO:57).From isolated fraction through the mixture of the people IL22RA peptide of CNBr cutting, six residues in Probability Area may participate in the aglucon combination potentially: comprise Tyr-60 and Phe-164, Tyr-93, Arg-112, Lys-210 and Glu-211 (and the corresponding residue among the SEQ ID NO:3) among the SEQ ID NO:2 for the important residue of aglucon-receptors bind (and the corresponding residue among the SEQ ID NO:3) among the SEQ ID NO:2.And, comprise Tyr-60 and Phe-164 (and the corresponding residue among the SEQ ID NO:3) among the SEQ ID NO:2 for the important elementary residue of aglucon-receptors bind (and the corresponding residue among the SEQ ID NO:3) among the SEQ ID NO:2, secondary residue comprises Tyr-93, Arg-112, Lys-210 and the Glu-211 (and the corresponding residue among the SEQ ID NO:3) among the SEQ ID NO:2.

Although be appreciated that from top embodiment illustrative purposes has been described special embodiment of the present invention in this article for example, under the prerequisite that does not break away from the present invention's spirit and category, can carry out various modification to embodiment.Therefore, except the restriction that is subjected to appending claims, the present invention is not restricted.

Sequence table

<110>ZymoGenetics，Inc.

＜120〉anti-IL-20 antibody and binding partners thereof and the method for in inflammation, using them

<130>04-04PC

<150>US 60/457,481

<151>2003-03-24

<150>US 60/523,295

<151>2003-11-17

<160>62

<170>FastSEQ for Windows Version 4.0

<210>1

<211>2831

<212>DNA

＜213〉human (Homo sapiens)

<220>

<221>CDS

<222>(34)...(1755)

<400>1

tagaggccaa gggagggctc tgtgccagcc ccg atg agg acg ctg ctg acc atc 54

Met Arg Thr Leu Leu Thr Ile

1 5

ttg act gtg gga tcc ctg gct gct cac gcc cct gag gac ccc tcg gat 102

Leu Thr Val Gly Ser Leu Ala Ala His Ala Pro Glu Asp Pro Ser Asp

10 15 20

ctg ctc cag cac gtg aaa ttc cag tcc agc aac ttt gaa aac atc ctg 150

Leu Leu Gln His Val Lys Phe Gln Ser Ser Asn Phe Glu Asn Ile Leu

25 30 35

acg tgg gac agc ggg cca gag ggc acc cca gac acg gtc tac agc atc 198

Thr Trp Asp Ser Gly Pro Glu Gly Thr Pro Asp Thr Val Tyr Ser Ile

40 45 50 55

gag tat aag acg tac gga gag agg gac tgg gtg gca aag aag ggc tgt 246

Glu Tyr Lys Thr Tyr Gly Glu Arg Asp Trp Val Ala Lys Lys Gly Cys

60 65 70

cag cgg atc acc cgg aag tcc tgc aac ctg acg gtg gag acg ggc aac 294

Gln Arg Ile Thr Arg Lys Ser Cys Asn Leu Thr Val Glu Thr Gly Asn

75 80 85

ctc acg gag ctc tac tat gcc agg gtc acc gct gtc agt gcg gga ggc 342

Leu Thr Glu Leu Tyr Tyr Ala Arg Val Thr Ala Val Ser Ala Gly Gly

90 95 100

cgg tca gcc acc aag atg act gac agg ttc agc tct ctg cag cac act 390

Arg Ser Ala Thr Lys Met Thr Asp Arg Phe Ser Ser Leu Gln His Thr

105 110 115

acc ctc aag cca cct gat gtg acc tgt atc tcc aaa gtg aga tcg att 438

Thr Leu Lys Pro Pro Asp Val Thr Cys Ile Ser Lys Val Arg Ser Ile

120 125 130 135

cag atg att gtt cat cct acc ccc acg cca atc cgt gca ggc gat ggc 486

Gln Met Ile Val His Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly

140 145 150

cac cgg cta acc ctg gaa gac atc ttc cat gac ctg ttc tac cac tta 534

His Arg Leu Thr Leu Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu

155 160 165

gag ctc cag gtc aac cgc acc tac caa atg cac ctt gga ggg aag cag 582

Glu Leu Gln Val Asn Arg Thr Tyr Gln Met His Leu Gly Gly Lys Gln

170 175 180

aga gaa tat gag ttc ttc ggc ctg acc cct gac aca gag ttc ctt ggc 630

Arg Glu Tyr Glu Phe Phe Gly Leu Thr Pro Asp Thr Glu Phe Leu Gly

185 190 195

acc atc atg att tgc gtt ccc acc tgg gcc aag gag agt gcc ccc tac 678

Thr Ile Met Ile Cys Val Pro Thr Trp Ala Lys Glu Ser Ala Pro Tyr

200 205 210 215

atg tgc cga gtg aag aca ctg cca gac cgg aca tgg acc tac tcc ttc 726

Met Cys Arg Val Lys Thr Leu Pro Asp Arg Thr Trp Thr Tyr Ser Phe

220 225 230

tcc gga gcc ttc ctg ttc tcc atg ggc ttc ctc gtc gca gta ctc tgc 774

Ser Gly Ala Phe Leu Phe Ser Met Gly Phe Leu Val Ala Val Leu Cys

235 240 245

tac ctg agc tac aga tat gtc acc aag ccg cct gca cct ccc aac tcc 822

Tyr Leu Ser Tyr Arg Tyr Val Thr Lys Pro Pro Ala Pro Pro Asn Ser

250 255 260

ctg aac gtc cag cga gtc ctg act ttc cag ccg ctg cgc ttc atc cag 870

Leu Asn Val Gln Arg Val Leu Thr Phe Gln Pro Leu Arg Phe Ile Gln

265 270 275

gag cac gtc ctg atc cct gtc ttt gac ctc agc ggc ccc agc agt ctg 918

Glu His Val Leu Ile Pro Val Phe Asp Leu Ser Gly Pro Ser Ser Leu

280 285 290 295

gcc cag cct gtc cag tac tcc cag atc agg gtg tct gga ccc agg gag 966

Ala Gln Pro Val Gln Tyr Ser Gln Ile Arg Val Ser Gly Pro Arg Glu

300 305 310

ccc gca gga gct cca cag cgg cat agc ctg tcc gag atc acc tac tta 1014

Pro Ala Gly Ala Pro Gln Arg His Ser Leu Ser Glu Ile Thr Tyr Leu

315 320 325

ggg cag cca gac atc tcc atc ctc cag ccc tcc aac gtg cca cct ccc 1062

Gly Gln Pro Asp Ile Ser Ile Leu Gln Pro Ser Asn Val Pro Pro Pro

330 335 340

cag atc ctc tcc cca ctg tcc tat gcc cca aac gct gcc cct gag gtc 1110

Gln Ile Leu Ser Pro Leu Ser Tyr Ala Pro Asn Ala Ala Pro Glu Val

345 350 355

ggg ccc cca tcc tat gca cct cag gtg acc ccc gaa gct caa ttc cca 1158

Gly Pro Pro Ser Tyr Ala Pro Gln Val Thr Pro Glu Ala Gln Phe Pro

360 365 370 375

ttc tac gcc cca cag gcc atc tct aag gtc cag cct tcc tcc tat gcc 1206

Phe Tyr Ala Pro Gln Ala Ile Ser Lys Val Gln Pro Ser Ser Tyr Ala

380 385 390

cct caa gcc act ccg gac agc tgg cct ccc tcc tat ggg gta tgc atg 1254

Pro Gln Ala Thr Pro Asp Ser Trp Pro Pro Ser Tyr Gly Val Cys Met

395 400 405

gaa ggt tct ggc aaa gac tcc ccc act ggg aca ctt tct agt cct aaa 1302

Glu Gly Ser Gly Lys Asp Ser Pro Thr Gly Thr Leu Ser Ser Pro Lys

410 415 420

cac ctt agg cct aaa ggt cag ctt cag aaa gag cca cca gct gga agc 1350

His Leu Arg Pro Lys Gly Gln Leu Gln Lys Glu Pro Pro Ala Gly Ser

425 430 435

tgc atg tta ggt ggc ctt tct ctg cag gag gtg acc tcc ttg gct atg 1398

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

440 445 450 455

gag gaa tcc caa gaa gca aaa tca ttg cac cag ccc ctg ggg att tgc 1446

Glu Glu Ser Gln Glu Ala Lys Ser Leu His Gln Pro Leu Gly Ile Cys

460 465 470

aca gac aga aca tct gac cca aat gtg cta cac agt ggg gag gaa ggg 1494

Thr Asp Arg Thr Ser Asp Pro Asn Val Leu His Ser Gly Glu Glu Gly

475 480 485

aca cca cag tac cta aag ggc cag ctc ccc ctc ctc tcc tca gtc cag 1542

Thr Pro Gln Tyr Leu Lys Gly Gln Leu Pro Leu Leu Ser Ser Val Gln

490 495 500

atc gag ggc cac ccc atg tcc ctc cct ttg caa cct cct tcc ggt cca 1590

Ile Glu Gly His Pro Met Ser Leu Pro Leu Gln Pro Pro Ser Gly Pro

505 510 515

tgt tcc ccc tcg gac caa ggt cca agt ccc tgg ggc ctg ctg gag tcc 1638

Cys Ser Pro Ser Asp Gln Gly Pro Ser Pro Trp Gly Leu Leu Glu Ser

520 525 530 535

ctt gtg tgt ccc aag gat gaa gcc aag agc cca gcc cct gag acc tca 1686

Leu Val Cys Pro Lys Agp Glu Ala Lys Ser Pro Ala Pro Glu Thr Ser

540 545 550

gac ctg gag cag ccc aca gaa ctg gat tct ctt ttc aga ggc ctg gcc 1734

Asp Leu Glu Gln Pro Thr Glu Leu Asp Ser Leu Phe Arg Gly Leu Ala

555 560 565

ctg act gtg cag tgg gag tcc tgaggggaat gggaaaggct tggtgcttcc 1785

Leu Thr Val Gln Trp Glu Ser

570

tccctgtccc tacccagtgt cacatccttg gctgtcaatc ccatgcctgc ccatgccaca 1845

cactctgcga tctggcctca gacgggtgcc cttgagagaa gcagagggag tggcatgcag 1905

ggcccctgcc atgggtgcgc tcctcaccgg aacaaagcag catgataagg actgcagcgg 1965

gggagctctg gggagcagct tgtgtagaca agcgcgtgct cgctgagccc tgcaaggcag 2025

aaatgacagt gcaaggagga aatgcaggga aactcccgag gtccagagcc ccacctccta 2085

acaccatgga ttcaaagtgc tcagggaatt tgcctctcct tgccccattc ctggccagtt 2145

tcacaatcta gctcgacaga gcatgaggcc cctgcctctt ctgtcattgt tcaaaggtgg 2205

gaagagagcc tggaaaagaa ccaggcctgg aaaagaacca gaaggaggct gggcagaacc 2265

agaacaacct gcacttctgc caaggccagg gccagcagga cggcaggact ctagggaggg 2325

gtgtggcctg cagctcattc ccagccaggg caactgcctg acgttgcacg atttcagctt 2385

cattcctctg atagaacaaa gcgaaatgca ggtccaccag ggagggagac acacaagcct 2445

tttctgcagg caggagtttc agaccctatc ctgagaatgg ggtttgaaag gaaggtgagg 2505

gctgtggccc ctggacgggt acaataacac actgtactga tgtcacaact ttgcaagctc 2565

tgccttgggt tcagcccatc tgggctcaaa ttccagcctc accactcaca agctgtgtga 2625

cttcaaacaa atgaaatcag tgcccagaac ctcggtttcc tcatctgtaa tgtggggatc 2685

ataacaccta cctcatggag ttgtggtgaa gatgaaatga agtcatgtct ttaaagtgct 2745

taatagtgcc tggtacatgg gcagtgccca ataaacggta gctatttaaa aaaaaaaaaa 2805

aaaaaaaaaa atagcggccg cctcga 2831

<210>2

<211>574

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＜213〉mankind

<400>2

Met Arg Thr Leu Leu Thr Ile Leu Thr Val Gly Ser Leu Ala Ala His

1 5 10 15

Ala Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val Lys Phe Gln Ser

20 25 30

Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr

35 40 45

Pro Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg Asp

50 55 60

Trp Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn

65 70 75 80

Leu Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala Arg Val

85 90 95

Thr Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys Met Thr Asp Arg

100 105 110

Phe Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro Asp Val Thr Cys

115 120 125

Ile Ser Lys Val Arg Ser Ile Gln Met Ile Val His Pro Thr Pro Thr

130 135 140

Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu Glu Asp Ile Phe

145 150 155 160

His Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn Arg Thr Tyr Gln

165 170 175

Met His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe Phe Gly Leu Thr

180 185 190

Pro Asp Thr Glu Phe Leu Gly Thr Ile Met Ile Cys Val Pro Thr Trp

195 200 205

Ala Lys Glu Ser Ala Pro Tyr Met Cys Arg Val Lys Thr Leu Pro Asp

210 215 220

Arg Thr Trp Thr Tyr Ser Phe Ser Gly Ala Phe Leu Phe Ser Met Gly

225 230 235 240

Phe Leu Val Ala Val Leu Cys Tyr Leu Ser Tyr Arg Tyr Val Thr Lys

245 250 255

Pro Pro Ala Pro Pro Asn Ser Leu Asn Val Gln Arg Val Leu Thr Phe

260 265 270

Gln Pro Leu Arg Phe Ile Gln Glu His Val Leu Ile Pro Val Phe Asp

275 280 285

Leu Ser Gly Pro Ser Ser Leu Ala Gln Pro Val Gln Tyr Ser Gln Ile

290 295 300

Arg Val Ser Gly Pro Arg Glu Pro Ala Gly Ala Pro Gln Arg His Ser

305 310 315 320

Leu Ser Glu Ile Thr Tyr Leu Gly Gln Pro Asp Ile Ser Ile Leu Gln

325 330 335

Pro Ser Asn Val Pro Pro Pro Gln Ile Leu Ser Pro Leu Ser Tyr Ala

340 345 350

Pro Asn Ala Ala Pro Glu Val Gly Pro Pro Ser Tyr Ala Pro Gln Val

355 360 365

Thr Pro Glu Ala Gln Phe Pro Phe Tyr Ala Pro Gln Ala Ile Ser Lys

370 375 380

Val Gln Pro Ser Ser Tyr Ala Pro Gln Ala Thr Pro Asp Ser Trp Pro

385 390 395 400

Pro Ser Tyr Gly Val Cys Met Glu Gly Ser Gly Lys Asp Ser Pro Thr

405 410 415

Gly Thr Leu Ser Ser Pro Lys His Leu Arg Pro Lys Gly Gln Leu Gln

420 425 430

Lys Glu Pro Pro Ala Gly Ser Cys Met Leu Gly Gly Leu Ser Leu Gln

435 440 445

Glu Val Thr Ser Leu Ala Met Glu Glu Ser Gln Glu Ala Lys Ser Leu

450 455 460

His Gln Pro Leu Gly Ile Cys Thr Asp Arg Thr Ser Asp Pro Asn Val

465 470 475 480

Leu His Ser Gly Glu Glu Gly Thr Pro Gln Tyr Leu Lys Gly Gln Leu

485 490 495

Pro Leu Leu Ser Ser Val Gln Ile Glu Gly His Pro Met Ser Leu Pro

500 505 510

Leu Gln Pro Pro Ser Gly Pro Cys Ser Pro Ser Asp Gln Gly Pro Ser

515 520 525

Pro Trp Gly Leu Leu Glu Ser Leu Val Cys Pro Lys Asp Glu Ala Lys

530 535 540

Ser Pro Ala Pro Glu Thr Ser Asp Leu Glu Gln Pro Thr Glu Leu Asp

545 550 555 560

Ser Leu Phe Arg Gly Leu Ala Leu Thr Val Gln Trp Glu Ser

565 570

<210>3

<211>211

<212>PRT

＜213〉mankind

<400>3

Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val Lys Phe Gln Ser Ser

1 5 10 15

Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr Pro

20 25 30

Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg Asp Trp

35 40 45

Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn Leu

50 55 60

Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala Arg Val Thr

65 70 75 80

Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys Met Thr Asp Arg Phe

85 90 95

Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro Asp Val Thr Cys Ile

100 105 110

Ser Lys Val Arg Ser Ile Gln Met Ile Val His Pro Thr Pro Thr Pro

115 120 125

Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu Glu Asp Ile Phe His

130 135 140

Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn Arg Thr Tyr Gln Met

145 150 155 160

His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe Phe Gly Leu Thr Pro

165 170 175

Asp Thr Glu Phe Leu Gly Thr Ile Met Ile Cys Val Pro Thr Trp Ala

180 185 190

Lys Glu Ser Ala Pro Tyr Met Cys Arg Val Lys Thr Leu Pro Asp Arg

195 200 205

Thr Trp Thr

210

<210>4

<211>541

<212>PRT

＜213〉artificial sequence

<220>

＜223〉solubility IL-22RA-Fc fusion polypeptide

<400>4

Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val Lys Phe Gln Ser Ser

1 5 10 15

Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr Pro

20 25 30

Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg Asp Trp

35 40 45

Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn Leu

50 55 60

Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala Arg Val Thr

65 70 75 80

Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys Met Thr Asp Arg Phe

85 90 95

Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro Asp Val Thr Cys Ile

100 105 110

Ser Lys Val Arg Ser Ile Gln Met Ile Val His Pro Thr Pro Thr Pro

115 120 125

Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu Glu Asp Ile Phe His

130 135 140

Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn Arg Thr Tyr Gln Met

145 150 155 160

His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe Phe Gly Leu Thr Pro

165 170 175

Asp Thr Glu Phe Leu Gly Thr Ile Met Ile Cys Val Pro Thr Trp Ala

180 185 190

Lys Glu Ser Ala Pro Tyr Met Cys Arg Val Lys Thr Leu Pro Asp Arg

195 200 205

Thr Trp Thr Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

210 215 220

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

225 230 235 240

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

245 250 255

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

260 265 270

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

275 280 285

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

290 295 300

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

305 310 315 320

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

325 330 335

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

340 345 350

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

355 360 365

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

435 440 445

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

515 520 525

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

530 535 540

<210>5

<211>1116

<212>DNA

＜213〉mankind

<220>

<221>CDS

<222>(21)...(557)

tcgagttaga attgtctgca atg gcc gcc ctg cag aaa tct gtg agc tct ttc 53

Met Ala Ala Leu Gln Lys Ser Val Ser Ser Phe

1 5 10

ctt atg ggg acc ctg gcc acc agc tgc ctc ctt ctc ttg gcc ctc ttg 101

Leu Met Gly Thr Leu Ala Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu

15 20 25

gta cag gga gga gca gct gcg ccc atc agc tcc cac tgc agg ctt gac 149

Val Gln Gly Gly Ala Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp

30 35 40

aag tcc aac ttc cag cag ccc tat atc acc aac cgc acc ttc atg ctg 197

Lys Ser Asn Phe Gln Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu

45 50 55

gct aag gag gct agc ttg gct gat aac aac aca gac gtt cgt ctc att 245

Ala Lys Glu Ala Ser Leu Ala Asp Asn Asn Thr Asp Val Arg Leu Ile

60 65 70 75

ggg gag aaa ctg ttc cac gga gtc agt atg agt gag cgc tgc tat ctg 293

Gly Glu Lys Leu Phe His Gly Val Ser Met Ser Glu Arg Cys Tyr Leu

80 85 90

atg aag cag gtg ctg aac ttc acc ctt gaa gaa gtg ctg ttc cct caa 34l

Met Lys Gln Val Leu Asn Phe Thr Leu Glu Glu Val Leu Phe Pro Gln

95 100 105

tct gat agg ttc cag cct tat atg cag gag gtg gtg ccc ttc ctg gcc 389

Ser Asp Arg Phe Gln Pro Tyr Met Gln Glu Val Val Pro Phe Leu Ala

110 115 120

agg ctc agc aac agg cta agc aca tgt cat att gaa ggt gat gac ctg 437

Arg Leu Ser Asn Arg Leu Ser Thr Cys His Ile Glu Gly Asp Asp Leu

125 130 135

cat atc cag agg aat gtg caa aag ctg aag gac aca gtg aaa aag ctt 485

His Ile Gln Arg Asn Val Gln Lys Leu Lys Asp Thr Val Lys Lys Leu

140 145 150 155

gga gag agt gga gag atc aaa gca att gga gaa ctg gat ttg ctg ttt 533

Gly Glu Ser Gly Glu Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe

160 165 170

atg tct ctg aga aat gcc tgc att tgaccagagc aaagctgaaa aatgaataac 587

Met Ser Leu Arg Asn Ala Cys Ile

175

taaccccctt tccctgctag aaataacaat tagatgcccc aaagcgattt tttttaacca 647

aaaggaagat gggaagccaa actccatcat gatgggtgga ttccaaatga acccctgcgt 707

tagttacaaa ggaaaccaat gccacttttg tttataagac cagaaggtag actttctaag 767

catagatatt tattgataac atttcattgt aactggtgtt ctatacacag aaaacaattt 827

attttttaaa taattgtctt tttccataaa aaagattact ttccattcct ttaggggaaa 887

aaacccctaa atagcttcat gtttGcataa tcagtacttt atatttataa atgtatttat 947

tattattata agactgcatt ttatttatat cattttatta atatggattt atttatagaa 1007

acatcattcg atattgctac ttgagtgtaa ggctaatatt gatatttatg acaataatta 1067

tagagctata acatgtttat ttgacctcaa taaacacttg gatatccta 1116

<210>6

<211>179

<212>PRT

＜213〉mankind

<400>6

Met Ala Ala Leu Gln Lys Ser Val Ser Ser Phe Leu Met Gly Thr Leu

1 5 10 15

Ala Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu Val Gln Gly Gly Ala

20 25 30

Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp Lys Ser Asn Phe Gln

35 40 45

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

50 55 60

Leu Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys Leu Phe

65 70 75 80

His Gly Val Ser Met Ser Glu Arg Cys Tyr Leu Met Lys Gln Val Leu

85 90 95

Asn Phe Thr Leu Glu Glu Val Leu Phe Pro Gln Ser Asp Arg Phe Gln

100 105 110

Pro Tyr Met Gln Glu Val Val Pro Phe Leu Ala Arg Leu Ser Asn Arg

115 120 125

Leu Ser Thr Cys His Ile Glu Gly Asp Asp Leu His Ile Gln Arg Asn

130 135 140

Val Gln Lys Leu Lys Asp Thr Val Lys Lys Leu Gly Glu Ser Gly Glu

145 150 155 160

Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn

165 170 175

Ala Cys Ile

<210>7

<211>926

<212>DNA

＜213〉mankind

<220>

<221>CDS

<222>(45)...(575)

＜221〉change

<222>(188)...(188)

＜223〉can be C or G at the 188th Nucleotide

<400>7

ctttgaattc ctagctcctg tggtctccag atttcaggcc taag atg aaa gcc tct 56

Met Lys Ala Ser

1

agt ctt gcc ttc agc ctt ctc tct gct gcg ttt tat ctc cta tgg act 104

Ser Leu Ala Phe Ser Leu Leu Ser Ala Ala Phe Tyr Leu Leu Trp Thr

5 10 15 20

cct tcc act gga ctg aag aca ctc aat ttg gga agc tgt gtg atc gcc 152

Pro Ser Thr Gly Leu Lys Thr Leu Asn Leu Gly Ser Cys Val Ile Ala

25 30 35

aca aac ctt cag gaa ata cga aat gga ttt tct gas ata cgg ggc agt 200

Thr Asn Leu Gln Glu Ile Arg Asn Gly Phe Ser Xaa Ile Arg Gly Ser

40 45 50

gtg caa gcc aaa gat gga aac att gac atc aga atc tta agg agg act 248

Val Gln Ala Lys Asp Gly Asn Ile Asp Ile Arg Ile Leu Arg Arg Thr

55 60 65

gag tct ttg caa gac aca aag cct gcg aat cga tgc tgc ctc ctg cgc 296

Glu Ser Leu Gln Asp Thr Lys Pro Ala Asn Arg Cys Cys Leu Leu Arg

70 75 80

cat ttg cta aga ctc tat ctg gac agg gta ttt aaa aac tac cag acc 344

His Leu Leu Arg Leu Tyr Leu Asp Arg Val Phe Lys Asn Tyr Gln Thr

85 90 95 100

cct gac cat tat act ctc cgg aag atc agc agc ctc gcc aat tcc ttt 392

Pro Asp His Tyr Thr Leu Arg Lys Ile Ser Ser Leu Ala Asn Ser Phe

105 110 115

ctt acc atc aag aag gac ctc cgg ctc tgt cat gcc cac atg aca tgc 440

Leu Thr Ile Lys Lys Asp Leu Arg Leu Cys His Ala His Met Thr Cys

120 125 130

cat tgt ggg gag gaa gca atg aag aaa tac agc cag att ctg agt cac 488

His Cys Gly Glu Glu Ala Met Lys Lys Tyr Ser Gln Ile Leu Ser His

135 140 145

ttt gaa aag ctg gaa cct cag gca gca gtt gtg aag gct ttg ggg gaa 536

Phe Glu Lys Leu Glu Pro Gln Ala Ala Val Val Lys Ala Leu Gly Glu

150 155 160

cta gac att ctt ctg caa tgg atg gag gag aca gaa tag gaggaaagtg 585

Leu Asp Ile Leu Leu Gln Trp Met Glu Glu Thr Glu *

165 170 175

atgctgctgc taagaatatt cgaggtcaag agctccagtc ttcaatacct gcagaggagg 645

catgacccca aaccaccatc tctttactgt actagtcttg tgctggtcac agtgtatctt 705

atttatgcat tacttgcttc cttgcatgat tgtctttatg catccccaat cttaattgag 765

accatacttg tataagattt ttgtaatatc tttctgctat tggatatatt tattagttaa 825

tatatttatt tattttttgc tattaatgta tttaattttt tacttgggca tgaaacttta 885

aaaaaaattc acaagattat atttataacc tgactagagc a 926

<210>8

<211>176

<212>PRT

＜213〉mankind

<220>

＜221〉variant

<222>(48)...(48)

＜223〉the 48th amino acid can be D (Asp) or E (Glu)

＜221〉variant

<222>48

＜223〉any amino acid of xaa=

<400>8

Met Lys Ala Ser Ser Leu Ala Phe Ser Leu Leu Ser Ala Ala Phe Tyr

1 5 10 15

Leu Leu Trp Thr Pro Ser Thr Gly Leu Lys Thr Leu Asn Leu Gly Ser

20 25 30

Cys Val Ile Ala Thr Asn Leu Gln Glu Ile Arg Asn Gly Phe Ser Xaa

35 40 45

Ile Arg Gly Ser Val Gln Ala Lys Asp Gly Asn Ile Asp Ile Arg Ile

50 55 60

Leu Arg Arg Thr Glu Ser Leu Gln Asp Thr Lys Pro Ala Asn Arg Cys

65 70 75 80

Cys Leu Leu Arg His Leu Leu Arg Leu Tyr Leu Asp Arg Val Phe Lys

85 90 95

Asn Tyr Gln Thr Pro Asp His Tyr Thr Leu Arg Lys Ile Ser Ser Leu

100 105 110

Ala Asn Ser Phe Leu Thr Ile Lys Lys Asp Leu Arg Leu Cys His Ala

115 120 125

His Met Thr Cys His Cys Gly Glu Glu Ala Met Lys Lys Tyr Ser Gln

130 135 140

Ile Leu Ser His Phe Glu Lys Leu Glu Pro Gln Ala Ala Val Val Lys

145 150 155 160

Ala Leu Gly Glu Leu Asp Ile Leu Leu Gln Trp Met Glu Glu Thr Glu

165 170 175

<210>9

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide linker

<400>9

Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>10

<211>1050

<212>DNA

<213>Mus musculus

<220>

<221>CDS

<222>(5)...(589)

<400>10

aaca ggc tct cct ctc act tat caa ctt ttg aca ctt gtg cga tcg gtg 49

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

1 5 10 15

atg gct gtc ctg cag aaa tct atg agt ttt tcc ctt atg ggg act ttg 97

Met Ala Val Leu Gln Lys Ser Met Ser Phe Ser Leu Met Gly Thr Leu

20 25 30

gcc gcc agc tgc ctg ctt ctc att gcc ctg tgg gcc cag gag gca aat 145

Ala Ala Ser Cys Leu Leu Leu Ile Ala Leu Trp Ala Gln Glu Ala Asn

35 40 45

gcg ctg ccc atc aac acc cgg tgc aag ctt gag gtg tcc aac ttc cag 193

Ala Leu Pro Ile Asn Thr Arg Cys Lys Leu Glu Val Ser Asn Phe Gln

50 55 60

cag ccg tac atc gtc aac cgc acc ttt atg ctg gcc aag gag gcc agc 241

Gln Pro Tyr Ile Val Asn Arg Thr Phe Met Leu Ala Lys Glu Ala Ser

65 70 75

ctt gca gat aac aac aca gac gtc cgg ctc atc ggg gag aaa ctg ttc 289

Leu Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys Leu Phe

80 85 90 95

cga gga gtc agt gct aag gat cag tgc tac ctg atg aag cag gtg ctc 337

Arg Gly Val Ser Ala Lys Asp Gln Cys Tyr Leu Met Lys Gln Val Leu

100 105 110

aac ttc acc ctg gaa gac att ctg ctc ccc cag tca gac agg ttc cgg 385

Asn Phe Thr Leu Glu Asp Ile Leu Leu Pro Gln Ser Asp Arg Phe Arg

115 120 125

ccc tac atg cag gag gtg gtg cct ttc ctg acc aaa ctc agc aat cag 433

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

130 135 140

ctc agc tcc tgt cac atc agt ggt gac gac cag aac atc cag aag aat 481

Leu Ser Ser Cys His Ile Ser Gly Asp Asp Gln Asn Ile Gln Lys Asn

145 150 155

gtc aga agg ctg aag gag aca gtg aaa aag ctt gga gag agc gga gag 529

Val Arg Arg Leu Lys Glu Thr Val Lys Lys Leu Gly Glu Ser Gly Glu

160 165 170 175

atc aaa gcg atc ggg gaa ctg gac ctg ctg ttt atg tct ctg aga aat 577

Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn

180 185 190

gct tgc gtc tga gcgagaagaa gctagaaaac gaagaactgc tccttcctgc 629

Ala Cys Val *

cttctaaaaa gaacaataag atccctgaat ggactttttt actaaaggaa agtgagaagc 689

taacgtccac catcattaga agatttcaca tgaaacctgg ctcagttgaa agagaaaata 749

gtgtcaagtt gtccatgaga ccagaggtag acttgataac cacaaagatt cattgacaat 809

attttattgt cattgataat gcaacagaaa aagtatgtac tttaaaaaat tgtttgaaag 869

gaggttacct ctcattcctc tagaagaaaa gcctatgtaa cttcatttcc ataaccaata 929

ctttatatat gtaagtttat ttattataag tatacatttt atttatgtca gtttattaat 989

atggatttat ttatagaaaa attatctgat gttgatattt gagtataaag caaataatat 1049

t 1050

<210>11

<211>194

<212>PRT

<213>Mus musculus

<400>11

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

1 5 10 15

Ala Val Leu Gln Lys Ser Met Ser Phe Ser Leu Met Gly Thr Leu Ala

20 25 30

Ala Ser Cys Leu Leu Leu Ile Ala Leu Trp Ala Gln Glu Ala Asn Ala

35 40 45

Leu Pro Ile Asn Thr Arg Cys Lys Leu Glu Val Ser Asn Phe Gln Gln

50 55 60

Pro Tyr Ile Val Asn Arg Thr Phe Met Leu Ala Lys Glu Ala Ser Leu

65 70 75 80

Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys Leu Phe Arg

85 90 95

Gly Val Ser Ala Lys Asp Gln Cys Tyr Leu Met Lys Gln Val Leu Asn

100 105 110

Phe Thr Leu Glu Asp Ile Leu Leu Pro Gln Ser Asp Arg Phe Arg Pro

115 120 125

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

130 135 140

Ser Ser Cys His Ile Ser Gly Asp Asp Gln Asn Ile Gln Lys Asn Val

145 150 155 160

Arg Arg Leu Lys Glu Thr Val Lys Lys Leu Gly Glu Ser Gly Glu Ile

165 170 175

Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn Ala

180 185 190

Cys Val

<210>12

<211>2149

<212>DNA

＜213〉mankind

<220>

<221>CDS

<222>(1)...(693)

<400>12

atg atg cct aaa cat tgc ttt cta ggc ttc ctc atc agt ttc ttc ctt 48

Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu

1 5 10 15

act ggt gta gca gga act cag tca acg cat gag tct ctg aag cct cag 96

Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln

20 25 30

agg gta caa ttt cag tcc cga aat ttt cac aac att ttg caa tgg cag 144

Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln

35 40 45

cct ggg agg gca ctt act ggc aac agc agt gtc tat ttt gtg cag tac 192

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

50 55 60

aaa ata tat gga cag aga caa tgg aaa aat aaa gaa gac tgt tgg ggt 240

Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly

65 70 75 80

act caa gaa ctc tct tgt gac ctt acc agt gaa acc tca gac ata cag 288

Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln

85 90 95

gaa cct tat tac ggg agg gtg agg gcg gcc tcg gct ggg agc tac tca 336

Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser

100 105 110

gaa tgg agc atg acg ccg cgg ttc act ccc tgg tgg gaa aca aaa ata 384

Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys Ile

115 120 125

gat cct cca gtc atg aat ata acc caa gtc aat ggc tct ttg ttg gta 432

Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu Leu Val

130 135 140

att ctc cat gct cca aat tta cca tat aga tac caa aag gaa aaa aat 480

Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn

145 150 155 160

gta tct ata gaa gat tac tat gaa cta cta tac cga gtt ttt ata att 528

Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile

165 170 175

aac aat tca cta gaa aag gag caa aag gtt tat gaa ggg gct cac aga 576

Asn Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg

180 185 190

gcg gtt gaa att gaa gct cta aca cca cac tcc agc tac tgt gta gtg 624

Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val

195 200 205

gct gaa ata tat cag ccc atg tta gac aga aga agt cag aga agt gaa 672

Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser Glu

210 215 220

gag aga tgt gtg gaa att cca tgacttgtgg aatttggcat tcagcaatgt 723

Glu Arg Cys Val Glu Ile Pro

225 230

ggaaattcta aagctccctg agaacaggat gactcgtgtt tgaaggatct tatttaaaat 783

tgtttttgta ttttcttaaa gcaatattca ctgttacacc ttggggactt ctttgtttat 843

ccattctttt atcctttata tttcatttta aactatattt gaacgacatt ccccccgaaa 903

aattgaaatg taaagatgag gcagagaata aagtgttcta tgaaattcag aactttattt 963

ctgaatgtaa catccctaat aacaaccttc attcttctaa tacagcaaaa taaaaattta 1023

acaaccaagg aatagtattt aagaaaatgt tgaaataatt tttttaaaat agcattacag 1083

actgaggcgg tcctgaagca atggtttttc actctcttat tgagccaatt aaattgacat 1143

tgctttgaca atttaaaact tctataaagg tgaatatttt tcatacattt ctattttata 1203

tgaatatact ttttatatat ttattattat taaatatttc tacttaatga atcaaaattt 1263

tgttttaaag tctactttat gtaaataaga acaggttttg gggaaaaaaa tcttatgatt 1323

tctggattga tatctgaatt aaaactatca acaacaagga agtctactct gtacaattgt 1383

ccctcattta aaagatatat taagcttttc ttttctgttt gtttttgttt tgtttagttt 1443

ttaatcctgt cttagaagaa cttatcttta ttctcaaaat taaatgtaat ttttttagtg 1503

acaaagaaga aaggaaacct cattactcaa tccttctggc caagagtgtc ttgcttgtgg 1563

cgccttcctc atctctatat aggaggatcc catgaatgat ggtttattgg gaactgctgg 1623

ggtcgacccc atacagagaa ctcagcttga agctggaagc acacagtggg tagcaggaga 1683

aggaccggtg ttggtaggtg cctacagaga ctatagagct agacaaagcc ctccaaactg 1743

gcccctcctg ctcactgcct ctcctgagta gaaatctggt gacctaaggc tcagtgcggt 1803

caacagaaag ctgccttctt cacttgaggc taagtcttca tatatgttta aggttgtctt 1863

tctagtgagg agatacatat cagagaacat ttgtacaatt ccccatgaaa attgctccaa 1923

agttgataac aatatagtcg gtgcttctag ttatatgcaa gtactcagtg ataaatggat 1983

taaaaaatat tcagaaatgt attggggggt ggaggagaat aagaggcaga gcaagagcta 2043

gagaattggt ttccttgctt ccctgtatgc tcagaaaaca ttgatttgag catagacgca 2103

gagactgaaa aaaaaaaaat gctcgagcgg ccgccatatc cttggt 2149

<210>13

<211>231

<212>PRT

＜213〉mankind

<400>13

Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu

1 5 10 15

Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln

20 25 30

Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln

35 40 45

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

50 55 60

Lys Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly

65 70 75 80

Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln

85 90 95

Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser

100 105 110

Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys Ile

115 120 125

Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu Leu Val

130 135 140

Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn

145 150 155 160

Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile

165 170 175

Asn Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg

180 185 190

Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val

195 200 205

Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser Glu

210 215 220

Glu Arg Cys Val Glu Ile Pro

225 230

<210>14

<211>699

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the terminal Fc4 label of C-

<400>14

gagcccagat cttcagacaa aactcacaca tgcccaccgt gcccagcacc tcaagccgag 60

ggggcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 180

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 240

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc catcctccat cgagaaaacc 360

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 540

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 600

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 660

tacacgcaga agagcctctc cctgtctccg ggtaaataa 699

<210>15

<211>6

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Glu-Glu (CEE) peptide tag

<400>15

Glu Tyr Met Pro Met Glu

1 5

<210>16

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉have Glu-Glu (CEE) peptide tag of introns

<400>16

Gly Ser Gly Gly Glu Tyr Met Pro Met Glu

1 5 10

<210>17

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC39289

<400>17

tccgaggagt caatgctaag 20

<210>18

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC39290

<400>18

tccaagcttt ttcactgtct 20

<210>19

<211>16

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC39776

<400>19

gggcccgcta gcacct 16

<210>20

<211>16

<212>DMA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC39777

<400>20

gggtgatccg ctggca 16

<210>21

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the TaqMan probe ZC38752 of IL-20 FAM/TAMRA mark

<400>21

ccagccactt tctctctccg tatttcttat attcca 36

<210>22

<211>16

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer ZC42459

<400>22

tggccaggct cagcaa 16

<210>23

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer ZC42458

<400>23

gcacattcct ctggatatgc a 21

<210>24

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IL-22 TaqMan probe ZC42460

<400>24

aggctaagca catgtcatat tgaaggtgat g 31

<210>25

<21l>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer ZC40541

<400>25

tcgccaattc ctttcttacc a 21

<210>26

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer ZC40542

<400>26

cccacaatgg catgtcatgt 20

<210>27

<21l>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉IL-20 TaqMan  probe ZC40544

<400>27

agaaggacct ccggctctgt catgc 25

<210>28

<211>57

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC45,593

<400>28

caggaaatcc atgccgagtt gagacgcttc cgtagacacg cccctgagga cccctcg 57

<210>29

<211>63

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC45,592

<400>29

tctgggctca ccgcttccag acccgcttcc agacccgctt cctgtccggt ctggcagtgt 60

ctt 63

<210>30

<211>63

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC45,591

<400>30

gaccggacag gaagcgggtc tggaagcggg tctggaagcg gtgagcccag aggccccaca 60

atc 63

<210>31

<211>57

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC45,594

<400>31

agagctgttt taaggcgcgc ctctagatta tttttattta cccggagtcc gggagaa 57

<210>32

<211>531

<212>DNA

<213>Mus musculus

<220>

<221>CDS

<222>(1)...(531)

<400>32

atg aaa ggc ttt ggt ctt gcc ttt gga ctg ttc tcc cct gtg ggt ttt 48

Met Lys Gly Phe Gly Leu Ala Phe Gly Leu Phe Ser Ala Val Gly Phe

1 5 10 15

ctt ctc tgg act cct tta act ggg ctc aag acc ctc cat ttg gga agc 96

Leu Leu Trp Thr Pro Leu Thr Gly Leu Lys Thr Leu His Leu Gly Ser

20 25 30

tgt gtg att act gca aac cta cag gca ata caa aag gaa ttt tct gag 144

Cys Val Ile Thr Ala Asn Leu Gln Ala Ile Gln Lys Glu Phe Ser Glu

35 40 45

att cgg gat agt gtg caa gct gaa gat aca aat att gac atc aga att 192

Ile Arg Asp Ser Val Gln Ala Glu Asp Thr Asn Ile Asp Ile Arg Ile

50 55 60

tta agg acg act gag tct ttg aaa gac ata aag tct ttg gat agg tgc 240

Leu Arg Thr Thr Glu Ser Leu Lys Asp Ile Lys Ser Leu Asp Arg Cys

65 70 75 80

tgc ttc ctt cgt cat cta gtg aga ttc tat ctg gac agg gta ttc aaa 288

Cys Phe Leu Arg His Leu Val Arg Phe Tyr Leu Asp Arg Val Phe Lys

85 90 95

gtc tac cag acc cct gac cac cat acc ctg aga aag atc agc agc ctc 336

Val Tyr Gln Thr Pro Asp His His Thr Leu Arg Lys Ile Ser Ser Leu

100 105 110

gcc aac tcc ttt ctt atc atc aag aag cac ctc tca gtc tgt cat tct 384

Ala Asn Ser Phe Leu Ile Ile Lys Lys Asp Leu Ser Val Cys His Ser

115 120 125

cac atg gca tgt cat tgt ggg gaa gaa gca atg gag aaa tac aac caa 432

His Met Ala Cys His Cys Gly Glu Glu Ala Met Glu Lys Tyr Asn Gln

130 135 140

att ctg agt cac ttc ata gag ttg gaa ctt cag gca gcg gtg gta aag 480

Ile Leu Ser His Phe Ile Glu Leu Glu Leu Gln Ala Ala Val Val Lys

145 150 155 160

gct ttg gga gaa cta ggc att ctt ctg aga tgg atg gag gag atg cta 528

Ala Leu Gly Glu Leu Gly Ile Leu Leu Arg Trp Met Glu Glu Met Leu

165 170 175

tag 531

*

<210>33

<211>176

<212>PRT

<213>Mus musculus

<400>33

Met Lys Gly Phe Gly Leu Ala Phe Gly Leu Phe Ser Ala Val Gly Phe

1 5 10 15

Leu Leu Trp Thr Pro Leu Thr Gly Leu Lys Thr Leu His Leu Gly Ser

20 25 30

Cys Val Ile Thr Ala Asn Leu Gln Ala Ile Gln Lys Glu Phe Ser Glu

35 40 45

Ile Arg Asp Ser Val Gln Ala Glu Asp Thr Asn Ile Asp Ile Arg Ile

50 55 60

Leu Arg Thr Thr Glu ser Leu Lys Asp Ile Lys Ser Leu Asp Arg Cys

65 70 75 80

Cys Phe Leu Arg His Leu Val Arg Phe Tyr Leu Asp Arg Val Phe Lys

85 90 95

Val Tyr Gln Thr Pro Asp His His Thr Leu Arg Lys Ile Ser Ser Leu

100 105 110

Ala Asn Ser Phe Leu Ile Ile Lys Lys Asp Leu Ser Val Cys His Ser

115 120 125

His Met Ala Cys His Cys Gly Glu Glu Ala Met Glu Lys Tyr Asn Gln

130 135 140

Ile Leu Ser His Phe Ile Glu Leu Glu Leu Gln Ala Ala Val Val Lys

145 150 155 160

Ala Leu Gly Glu Leu Gly Ile Leu Leu Arg Trp Met Glu Glu Met Leu

165 170 175

<2l0>34

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC22901

<400>34

catcaaaccg cctgatgtga c 21

<210>35

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC45039

<400>35

attaggcttg ggagggaatg g 21

<210>36

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC38573

<400>36

tggcgatgcc tgcttgccga ata 23

<210>37

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC25223

<400>37

gtcttcctca catctgttat cg 22

<210>38

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Oligonucleolide primers ZC40128

<400>38

ggcttgaact ttgagaaagg cagt 24

<210>39

<211>1473

<212>DNA

＜213〉artificial sequence

<220>

＜223〉have the IL-22RA ectodomain of tPA leader sequence,

It merges mutually with mouse gamma 2a heavy chain Fc district (mG2a)

<221>CDS

<222>(1)...(1473)

<400>39

atg gat gca atg aag aga ggg ctc tgc tgt gtg ctg ctg ctg tgt ggc 48

Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

gcc gtc ttc gtt tcg ctc agc cag gaa atc cat gcc cag ttg aga cgc 96

Ala Val Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu Leu Arg Arg

20 25 30

ttc cgt aga cac gcc cct gag gac ccc tcg gat ctg ctc cag cac gtg 144

Phe Arg Arg His Ala Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val

35 40 45

aaa ttc cag tcc agc aac ttt gaa aac atc ctg acg tgg gac agc ggg 192

Lys Phe Gln Ser Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly

50 55 60

cca gag ggc acc cca gac acg gtc tac agc atc gag tat aag acg tac 240

Pro Glu Gly Thr Pro Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr

65 70 75 80

gga gag agg gac tgg gtg gca aag aag ggc tgt cag cgg atc acc cgg 288

Gly Glu Arg Asp Trp Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg

85 90 95

aag tcc tgc aac ctg acg gtg gag acg ggc aac ctc acg gag ctc tac 336

Lys Ser Cys Asn Leu Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr

100 105 110

tat gcc agg gtc acc gct gtc agt gcg gga ggc cgg tca gcc acc aag 384

Tyr Ala Arg Val Thr Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys

115 120 125

atg act gac agg ttc agc tct ctg cag cac act acc ctc aag cca cct 432

Met Thr Asp Arg Phe Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro

130 135 140

gat gtg acc tgt atc tcc aaa gtg aga tcg att cag atg att gtt cat 480

Asp Val Thr Cys Ile Ser Lys Val Arg Ser Ile Gln Met Ile Val His

145 150 155 160

cct acc ccc acg cca atc cgt gca ggc gat ggc cac cgg cta acc ctg 528

Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu

165 170 175

gaa gac atc ttc cat gac ctg ttc tac cac tta gag ctc cag gtc aac 576

Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn

180 185 190

cgc acc tac caa atg cac ctt gga ggg aag cag aga gaa tat gag ttc 624

Arg Thr Tyr Gln Met His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe

195 200 205

ttc ggc ctg acc cct gac aca gag ttc ctt ggc acc atc atg att tgc 672

Phe Gly Leu Thr Pro Asp Thr Glu Phe Leu Gly Thr Ile Met Ile Cys

210 215 220

gtt ccc acc tgg gcc aag gag agt gcc ccc tac atg tgc cga gtg aag 720

Val Pro Thr Trp Ala Lys Glu Ser Ala Pro Tyr Met Cys Arg Val Lys

225 230 235 240

aca ctg cca gac cgg aca gga agc ggg tct gga agc ggg tct gga agc 768

Thr Leu Pro Asp Arg Thr Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

245 250 255

ggt gag ccc aga ggc ccc aca atc aag ccc tgt cct cca tgc aaa tgc 816

Gly Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

260 265 270

cca gca cct aac ctc ttg ggt gga cca tcc gtc ttc atc ttc cct cca 864

Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro

275 280 285

aag atc aag gat gta ctc atg atc tcc ctg agc ccc ata gtc aca tgt 912

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

290 295 300

gtg gtg gtg gat gtg agc gag gat gac cca gat gtc cag atc agc tgg 960

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

305 310 315 320

ttt gtg aac aac gtg gaa gta cac aca gct cag aca caa acc cat aga 1008

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

325 330 335

gag gat tac aac agt act ctc cgg gtg gtc agt gcc ctc ccc atc cag 1056

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

340 345 350

cac cag gac tgg atg agt ggc aag gag ttc aaa tgc aag gtc aac aac 1104

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

355 360 365

aaa gac ctc cca gcg ccc atc gag aga acc atc tca aaa ccc aaa ggg 1152

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

370 375 380

tca gta aga gct cca cag gta tat gtc ttg cct cca cca gaa gaa gag 1200

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu

385 390 395 400

atg act aag aaa cag gtc act ctg acc tgc atg gtc aca gac ttc atg 1248

Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met

405 410 415

cct gaa gac att tac gtg gag tgg acc aac aac ggg aaa aca gag cta 1296

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

420 425 430

aac tac aag aac act gaa cca gtc ctg gac tct gat ggt tct tac ttc 1344

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

435 440 445

atg tac agc aag ctg aga gtg gaa aag aag aac tgg gtg gaa aga aat 1392

Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

450 455 460

agc tac tcc tgt tca gtg gtc cac gag ggt ctg cac aat cac cac acg 1440

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

465 470 475 480

act aag agc ttc tcc cgg act ccg ggt aaa taa 1473

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys *

485 490

<210>40

<211>490

<212>PRT

＜213〉artificial sequence

<220>

＜223〉have the IL-22RA ectodomain of tPA leader sequence,

It merges mutually with mouse gamma 2a heavy chain Fc district (mG2a)

<400>40

Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu Leu Arg Arg

20 25 30

Phe Arg Arg His Ala Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val

35 40 45

Lys Phe Gln Ser Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly

50 55 60

Pro Glu Gly Thr Pro Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr

65 70 75 80

Gly Glu Arg Asp Trp Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg

85 90 95

Lys Ser Cys Asn Leu Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr

100 105 110

Tyr Ala Arg Val Thr Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys

115 120 125

Met Thr Asp Arg Phe Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro

130 135 140

Asp Val Thr Cys Ile Ser Lys Val Arg Ser Ile Gln Met Ile Val His

145 150 155 160

Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu

165 170 175

Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu Glu Leu Gln Val Asn

180 185 190

Arg Thr Tyr Gln Met His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe

195 200 205

Phe Gly Leu Thr Pro Asp Thr Glu Phe Leu Gly Thr Ile Met Ile Cys

210 215 220

Val Pro Thr Trp Ala Lys Glu Ser Ala Pro Tyr Met Cys Arg Val Lys

225 230 235 240

Thr Leu Pro Asp Arg Thr Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

245 250 255

Gly Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys

260 265 270

Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro

275 280 285

Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys

290 295 300

Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp

305 310 315 320

Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg

325 330 335

Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln

340 345 350

His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn

355 360 365

Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly

370 375 380

Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu

385 390 395 400

Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met

405 410 415

Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu

420 425 430

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

435 440 445

Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn

450 455 460

Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr

465 470 475 480

Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

485 490

<210>41

<211>1834

<212>DNA

<213>Mus musculus

<220>

<221>CDS

<222>(43)...(1788)

<400>41

ttggtccaga gccgaggccc gaaggggccc tggagggacc ca atg aag aca cta 54

Met Lys Thr Leu

1

ctg acc atc ctg acg gtg gga tcc ctg gcc gct cac acc act gtg gac 102

Leu Thr Ile Leu Thr Val Gly Ser Leu Ala Ala His Thr Thr Val Asp

5 10 15 20

aca tcc ggt ctc ctt caa cac gtg aaa ttc cag tcc agc aac ttt gag 150

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

25 30 35

aac atc ttg acg tgg gat ggt ggg ccc gct agc acc tct gac acc gtc 198

Asn Ile Leu Thr Trp Asp Gly Gly Pro Ala Ser Thr Ser Asp Thr Val

40 45 50

tac agt gtg gaa tat aag aaa tac gga gag aga aag tgg ctg gcc aag 246

Tyr Ser Val Glu Tyr Lys Lys Tyr Gly Glu Arg Lys Trp Leu Ala Lys

55 60 65

gcg ggc tgc cag cgg atc acc cag aag ttc tgc aac ctg act atg gag 294

Ala Gly Cys Gln Arg Ile Thr Gln Lys Phe Cys Asn Leu Thr Met Glu

70 75 80

acc cgc aac cac act gag ttt tac tac gcc aag gtc acg gca gtc agc 342

Thr Arg Asn His Thr Glu Phe Tyr Tyr Ala Lys Val Thr Ala Val Ser

85 90 95 100

gca gga ggc cca cca gtc aca aag atg act gat cgt ttc agc tcg ctg 390

Ala Gly Gly Pro Pro Val Thr Lys Met Thr Asp Arg Phe Ser Ser Leu

105 110 115

cag cac act acc atc aaa ccg cct gat gtg acc tgt atc ccc aaa gtg 438

Gln His Thr Thr Ile Lys Pro Pro Asp Val Thr Cys Ile Pro Lys Val

120 125 130

agg tcc att cag atg ctg gtc cac ccc aca ctc aca ccg gtc ctc tcg 486

Arg Ser Ile Gln Met Leu Val His Pro Thr Leu Thr Pro Val Leu Ser

135 140 145

gaa gat ggc cac cag cta acc ctg gag gag att ttc cat gac ctg ttc 534

Glu Asp Gly His Gln Leu Thr Leu Glu Glu Ile Phe His Asp Leu Phe

150 155 160

tac cgc tta gag ctc cac gtc aac cac acc tac cag atg cac ctt gaa 582

Tyr Arg Leu Glu Leu His Val Asn His Thr Tyr Gln Met His Leu Glu

165 170 175 180

ggc aaa cag aga gaa tac gag ttc ctt ggc ctg act ccc gac aca gag 630

Gly Lys Gln Arg Glu Tyr Glu Phe Leu Gly Leu Thr Pro Asp Thr Glu

185 190 195

ttc ctc ggc tcc atc aca att ttg act ccg ata ttg tcc aag gaa agt 678

Phe Leu Gly Ser Ile Thr Ile Leu Thr Pro Ile Leu Ser Lys Glu Ser

200 205 210

gcc ccc tac gtg tgc cga gtg aag acg ctg ccc gat cgg acg tgg gcc 726

Ala Pro Tyr Val Cys Arg Val Lys Thr Leu Pro Asp Arg Thr Trp Ala

215 220 225

tac tcc ttc tcg ggc gcc gtg ctc ttt tcc atg ggt ttc ctc gtc ggc 774

Tyr Ser Phe Ser Gly Ala Val Leu Phe Ser Met Gly Phe Leu Val Gly

230 235 240

ttg ctc tgt tat ctg ggc tac aaa tac atc acc aag cca cct gta cct 822

Leu Leu Cys Tyr Leu Gly Tyr Lys Tyr Ile Thr Lys Pro Pro Val Pro

245 250 255 260

cct aac tcc ctg aac gtc caa cgt gtc ctg acc ttt caa ccc cta cgc 870

Pro Asn Ser Leu Asn Val Gln Arg Val Leu Thr Phe Gln Pro Leu Arg

265 270 275

ttc atc caa gaa cac gta ctg atc cct gtc ttg gac ctc agt ggc ccc 918

Phe Ile Gln Glu His Val Leu Ile Pro Val Leu Asp Leu Ser Gly Pro

280 285 290

agc agt ctg cct cag ccc atc cag tac tcc caa gtg gtg gtg tct ggg 966

Ser Ser Leu Pro Gln Pro Ile Gln Tyr Ser Gln Val Val Val Ser Gly

295 300 305

ccc agg gag cct cct gga gct gtg tgg cgg cag agc ctg tct gac ctc 1014

Pro Arg Glu Pro Pro Gly Ala Val Trp Arg Gln Ser Leu Ser Asp Leu

310 315 320

acc tac gta ggg cag tca gat gtc tcc atc ctg caa cct acc aac gtg 1062

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

325 330 335 340

cca gct cag cag aca ctg tcc cca cca tcc tac gct ccg aag gct gtc 1110

Pro Ala Gln Gln Thr Leu Ser Pro Pro Ser Tyr Ala Pro Lys Ala Val

345 350 355

cct gag gtc cag ccc cct tcc tat gcg cct cag gta gcc tcg gat gcc 1158

Pro Glu Val Gln Pro Pro Ser Tyr Ala Pro Gln Val Ala Ser Asp Ala

360 365 370

aaa gct ctg ttc tac tca cca caa cag ggg atg aag acc agg cct gcc 1206

Lys Ala Leu Phe Tyr Ser Pro Gln Gln Gly Met Lys Thr Arg Pro Ala

375 380 385

acc tat gac ccg cag gac att ctg gac agc tgc cct gct tct tat gct 1254

Thr Tyr Asp Pro Gln Asp Ile Leu Asp Ser Cys Pro Ala Ser Tyr Ala

390 395 400

gtg tgt gtg gaa gac tct ggc aaa gac tct acc cca ggc atc ctc tcc 1302

Val Cys Val Glu Asp Ser Gly Lys Asp Ser Thr Pro Gly Ile Leu Ser

405 410 415 420

act ccc aaa tac ctc aag aca aaa ggt cag ctc cag gaa gac aca ctt 1350

Thr Pro Lys Tyr Leu Lys Thr Lys Gly Gln Leu Gln Glu Asp Thr Leu

425 430 435

gtt aga agc tgt ctc cca ggg gac ctt tct cta cag aaa gtc acc tcc 1398

Val Arg Ser Cys Leu Pro Gly Asp Leu Ser Leu Gln Lys Val Thr Ser

440 445 450

tta ggt gaa ggg gag aca cag aga cca aaa tca ctc ccc tca cct ctg 1446

Leu Gly Glu Gly Glu Thr Gln Arg Pro Lys Ser Leu Pro Ser Pro Leu

455 460 465

gga ttt tgc aca gac aga gga cct gac ctt cac aca ctg cgc agt gag 1494

Gly Phe Cys Thr Asp Arg Gly Pro Asp Leu His Thr Leu Arg Ser Glu

470 475 480

gaa cca gag aca cca cgg tac ctg aag ggg gcg ctg tct ctc ctg tcc 1542

Glu Pro Glu Thr Pro Arg Tyr Leu Lys Gly Ala Leu Ser Leu Leu Ser

485 490 495 500

tct gtg cag atc gag ggc cac cct gtc tcc ctc cct ttg cac gtc cat 1590

Ser Val Gln Ile Glu Gly His Pro Val Ser Leu Pro Leu His Val His

505 510 515

tct gtc tca tgt tcc ccc tca gac gag gga cca agt ccc tgg ggc ctg 1638

Ser Val Ser Cys Ser Pro Ser Asp Glu Gly Pro Ser Pro Trp Gly Leu

520 525 530

ctg gac tcc ctt gtg tgt cca aag gat gag ggt ccc gcg gtt gag act 1686

Leu Asp Ser Leu Val Cys Pro Lys Asp Glu Gly Pro Ala Val Glu Thr

535 540 545

gag gcc atg tgc ccc agt gct gca gcc tct gag ctg gag cag tcc aca 1734

Glu Ala Met Cys Pro Ser Ala Ala Ala Ser Glu Leu Glu Gln Ser Thr

550 555 560

gaa ctg gac tct ctt ttc aaa ggc ttg gcc ctg act gtg cag tgg gaa 1782

Glu Leu Asp Ser Leu Phe Lys Gly Leu Ala Leu Thr Val Gln Trp Glu

565 570 575 580

tcc tga agggagatcg gagcaagcag gcctaagttt cctcccgccc caccta 1834

Ser *

<210>42

<211>581

<212>PRT

<213>Mus musculus

<400>42

Met Lys Thr Leu Leu Thr Ile Leu Thr Val Gly Ser Leu Ala Ala His

1 5 10 15

Thr Thr Val Asp Thr Ser Gly Leu Leu Gln His Val Lys Phe Gln Ser

20 25 30

Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Gly Gly Pro Ala Ser Thr

35 40 45

Ser Asp Thr Val Tyr Ser Val Glu Tyr Lys Lys Tyr Gly Glu Arg Lys

50 55 60

Trp Leu Ala Lys Ala Gly Cys Gln Arg Ile Thr Gln Lys Phe Cys Asn

65 70 75 80

Leu Thr Met Glu Thr Arg Asn His Thr Glu Phe Tyr Tyr Ala Lys Val

85 90 95

Thr Ala Val Ser Ala Gly Gly Pro Pro Val Thr Lys Met Thr Asp Arg

100 105 110

Phe Ser Ser Leu Gln His Thr Thr Ile Lys Pro Pro Asp Val Thr Cys

115 120 125

Ile Pro Lys Val Arg Ser Ile Gln Met Leu Val His Pro Thr Leu Thr

130 135 140

Pro Val Leu Ser Glu Asp Gly His Gln Leu Thr Leu Glu Glu Ile Phe

145 150 155 160

His Asp Leu Phe Tyr Arg Leu Glu Leu His Val Asn His Thr Tyr Gln

165 170 175

Met His Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu Gly Leu Thr

180 185 190

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

195 200 205

Ser Lys Glu Ser Ala Pro Tyr Val Cys Arg Val Lys Thr Leu Pro Asp

210 215 220

Arg Thr Trp Ala Tyr Ser Phe Ser Gly Ala Val Leu Phe Ser Met Gly

225 230 235 240

Phe Leu Val Gly Leu Leu Cys Tyr Leu Gly Tyr Lys Tyr Ile Thr Lys

245 250 255

Pro Pro Val Pro Pro Asn Ser Leu Asn Val Gln Arg Val Leu Thr Phe

260 265 270

Gln Pro Leu Arg Phe Ile Gln Glu His Val Leu Ile Pro Val Leu Asp

275 280 285

Leu Ser Gly Pro Ser Ser Leu Pro Gln Pro Ile Gln Tyr Ser Gln Val

290 295 300

Val Val Ser Gly Pro Arg Glu Pro Pro Gly Ala Val Trp Arg Gln Ser

305 310 315 320

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

325 330 335

Pro Thr Asn Val Pro Ala Gln Gln Thr Leu Ser Pro Pro Ser Tyr Ala

340 345 350

Pro Lys Ala Val Pro Glu Val Gln Pro Pro Ser Tyr Ala Pro Gln Val

355 360 365

Ala Ser Asp Ala Lys Ala Leu Phe Tyr Ser Pro Gln Gln Gly Met Lys

370 375 380

Thr Arg Pro Ala Thr Tyr Asp Pro Gln Asp Ile Leu Asp Ser Cys Pro

385 390 395 400

Ala Ser Tyr Ala Val Cys Val Glu Asp Ser Gly Lys Asp Ser Thr Pro

405 410 415

Gly Ile Leu Ser Thr Pro Lys Tyr Leu Lys Thr Lys Gly Gln Leu Gln

420 425 430

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

435 440 445

Lys Val Thr Ser Leu Gly Glu Gly Glu Thr Gln Arg Pro Lys Ser Leu

450 455 460

Pro Ser Pro Leu Gly Phe Cys Thr Asp Arg Gly Pro Asp Leu His Thr

465 470 475 480

Leu Arg Ser Glu Glu Pro Glu Thr Pro Arg Tyr Leu Lys Gly Ala Leu

485 490 495

Ser Leu Leu Ser Ser Val Gln Ile Glu Gly His pro Val Ser Leu Pro

500 505 510

Leu His Val His Ser Val Ser Cys Ser Pro Ser Asp Glu Gly Pro Ser

515 520 525

Pro Trp Gly Leu Leu Asp Ser Leu Val Cys Pro Lys Asp Glu Gly Pro

530 535 540

Ala Val Glu Thr Glu Ala Met Cys Pro Ser Ala Ala Ala Ser Glu Leu

545 550 555 560

Glu Gln Ser Thr Glu Leu Asp Ser Leu Phe Lys Gly Leu Ala Leu Thr

565 570 575

ValGln Trp Glu Ser

580

<210>43

<211>660

<212>DNA

＜213〉mankind

<220>

<221>CDS

<222>(1)...(660)

<400>43

atg gcg tgg agt ctt ggg agc tgg ctg ggt ggc tgc ctg ctg gtg tca 48

Met Ala Trp Ser Leu Gly Ser Trp Leu Gly Gly Cys Leu Leu Val Ser

1 5 10 15

gca ttg gga atg gta cca cct ccc gaa aat gtc aga atg aat tct gtt 96

Ala Leu Gly Met Val Pro Pro Pro Glu Asn Val Arg Met Asn Ser Val

20 25 30

aat ttc aag aac att cta cag tgg gag tca cct gct ttt gcc aaa ggg 144

Asn Phe Lys Asn Ile Leu Gln Trp Glu Ser Pro Ala Phe Ala Lys Gly

35 40 45

aac ctg act ttc aca gct cag tac cta agt tat agg ata ttc caa gat 192

Asn Leu Thr Phe Thr Ala Gln Tyr Leu Ser Tyr Arg Ile Phe Gln Asp

50 55 60

aaa tgc atg aat act acc ttg acg gaa tgt gat ttc tca agt ctt tcc 240

Lys Cys Met Asn Thr Thr Leu Thr Glu Cys Asp Phe Ser Ser Leu Ser

65 70 75 80

aag tat ggt gac cac acc ttg aga gtc agg gct gaa ttt gca gat gag 288

Lys Tyr Gly Asp His Thr Leu Arg Val Arg Ala Glu Phe Ala Asp Glu

85 90 95

cat tca gac tgg gta aac atc acc ttc tgt cct gtg gat gac acc att 336

His Ser Asp Trp Val Asn Ile Thr Phe cys Pro Val Asp Asp Thr Ile

100 105 110

att gga ccc cct gga atg caa gta gaa gta ctt gat gat tct tta cat 384

Ile Gly Pro Pro Gly Met Gln Val Glu Val Leu Asp Asp Ser Leu His

115 120 125

atg cgt ttc tta gcc cct aaa att gag aat gaa tac gaa act tgg act 432

Met Arg Phe Leu Ala Pro Lys Ile Glu Asn Glu Tyr Glu Thr Trp Thr

130 135 140

atg aag aat gtg tat aac tca tgg act tat aat gtg caa tac tgg aaa 480

Met Lys Asn Val Tyr Asn Ser Trp Thr Tyr Asn Val Gln Tyr Trp Lys

145 150 155 160

aac ggt act gat gaa aag ttt caa att act ccc cag tat gac ttt gag 528

Asn Gly Thr Asp Glu Lys Phe Gln Ile Thr Pro Gln Tyr Asp Phe Glu

165 170 175

gtc ctc aga aac ctg gag cca tgg aca act tat tgt gtt caa gtt cga 576

Val Leu Arg Asn Leu Glu Pro Trp Thr Thr Tyr Cys Val Gln Val Arg

180 185 190

ggg ttt ctt cct gat cgg aac aaa gct ggg gaa tgg agt gag cct gtc 624

Gly Phe Leu Pro Asp Arg Asn Lys Ala Gly Glu Trp Ser Glu Pro Val

195 200 205

tgt gag caa aca acc cat gac gaa acg gtc ccc tcc 660

Cys Glu Gln Thr Thr His Asp Glu Thr Val Pro Ser

210 215 220

<210>44

<211>220

<212>PPT

＜213〉mankind

<400>44

Met Ala Trp Ser Leu Gly Ser Trp Leu Gly Gly Cys Leu Leu Val Ser

1 5 10 15

Ala Leu Gly Met Val Pro Pro Pro Glu Asn Val Arg Met Asn Ser Val

20 25 30

Asn Phe Lys Asn Ile Leu Gln Trp Glu Ser Pro Ala Phe Ala Lys Gly

35 40 45

Asn Leu Thr Phe Thr Ala Gln Tyr Leu Ser Tyr Arg Ile Phe Gln Asp

50 55 60

Lys Cys Met Asn Thr Thr Leu Thr Glu Cys Asp Phe Ser Ser Leu Ser

65 70 75 80

Lys Tyr Gly Asp His Thr Leu Arg Val Arg Ala Glu Phe Ala Asp Glu

85 90 95

His Ser Asp Trp Val Asn Ile Thr Phe Cys Pro Val Asp Asp Thr Ile

100 105 110

Ile Gly Pro Pro Gly Met Gln Val Glu Val Leu Asp Asp Ser Leu His

115 120 125

Met Arg Phe Leu Ala Pro Lys Ile Glu Asn Glu Tyr Glu Thr Trp Thr

130 135 140

Met Lys Asn Val Tyr Asn Ser Trp Thr Tyr Asn Val Gln Tyr Trp Lys

145 150 155 160

Asn Gly Thr Asp Glu Lys Phe Gln Ile Thr Pro Gln Tyr Asp Phe Glu

165 170 175

Val Leu Arg Asn Leu Glu Pro Trp Thr Thr Tyr Cys Val Gln Val Arg

180 185 190

Gly Phe Leu Pro Asp Arg Asn Lys Ala Gly Glu Trp Ser Glu Pro Val

195 200 205

Cys Glu Gln Thr Thr His Asp Glu Thr Val Pro Ser

210 215 220

<210>45

<211>199

<212>PRT

＜213〉mankind

<400>45

Met Val Pro Pro Pro Glu Asn Val Arg Met Asn Ser Val Asn Phe Lys

1 5 10 15

Asn Ile Leu Gln Trp Glu Ser Pro Ala Phe Ala Lys Gly Asn Leu Thr

20 25 30

Phe Thr Ala Gln Tyr Leu Ser Tyr Arg Ile Phe Gln Asp Lys Cys Met

35 40 45

Asn Thr Thr Leu Thr Glu Cys Asp Phe Ser Ser Leu Ser Lys Tyr Gly

50 55 60

Asp His Thr Leu Arg Val Arg Ala Glu Phe Ala Asp Glu His Ser Asp

65 70 75 80

Trp Val Asn Ile Thr Phe Cys Pro Val Asp Asp Thr Ile Ile Gly Pro

85 90 95

Pro Gly Met Gln Val Glu Val Leu Ala Asp Ser Leu His Met Arg Phe

100 105 110

Leu Ala Pro Lys Ile Glu Asn Glu Tyr Glu Thr Trp Thr Met Lys Asn

115 120 125

Val Tyr Asn Ser Trp Thr Tyr Asn Val Gln Tyr Trp Lys Asn Gly Thr

130 135 140

Asp Glu Lys Phe Gln Ile Thr Pro Gln Tyr Asp Phe Glu Val Leu Arg

145 150 155 160

Asn Leu Glu Pro Trp Thr Thr Tyr Cys Val Gln Val Arg Gly Phe Leu

165 170 175

Pro Asp Arg Asn Lys Ala Gly Glu Trp Ser Glu Pro Val Cys Glu Gln

180 185 190

Thr Thr His Asp Glu Thr Val

195

<2l0>46

<211>211

<212>PRT

＜213〉mankind

<400>46

Ser Asp Ala His Gly Thr Glu Leu Pro Ser Pro Pro Ser Val Trp Phe

1 5 10 15

Glu Ala Glu Phe Phe His His Ile Leu His Trp Thr Pro Ile Pro Asn

20 25 30

Gln Ser Glu Ser Thr Cys Tyr Glu Val Ala Leu Leu Arg Tyr Gly Ile

35 40 45

Glu Ser Trp Asn Ser Ile Ser Asn Cys Ser Gln Thr Leu Ser Tyr Asp

50 55 60

Leu Thr Ala Val Thr Leu Asp Leu Tyr His Ser Asn Gly Tyr Arg Ala

65 70 75 80

Arg Val Arg Ala Val Asp Gly Ser Arg His Ser Asn Trp Thr Val Thr

85 90 95

Asn Thr Arg Phe Ser Val Asp Glu Val Thr Leu Thr Val Gly Ser Val

100 105 110

Asn Leu Glu Ile His Asn Gly Phe Ile Leu Gly Lys Ile Gln Leu Pro

115 120 125

Arg Pro Lys Met Ala Pro Ala Asn Asp Thr Tyr Glu Ser Ile Phe Ser

130 135 140

His Phe Arg Glu Tyr Glu Ile Ala Ile Arg Lys Val Pro Gly Asn Phe

145 150 155 160

Thr Phe Thr His Lys Lys Val Lys His Glu Asn Phe Ser Leu Leu Thr

165 170 175

Ser Gly Glu Val Gly Glu Phe Cys Val Gln Val Lys Pro Ser Val Ala

180 185 190

Ser Arg Ser Asn Lys Gly Met Trp Ser Lys Glu Glu Cys Ile Ser Leu

195 200 205

Thr Arg Gln

210

<210>47

<211>201

<212>PRT

＜213〉mankind

<400>47

Asp Glu Val Ala Ile Leu Pro Ala Pro Gln Asn Leu Ser Val Leu Ser

1 5 10 15

Thr Asn Met Lys His Leu Leu Met Trp Ser Pro Val Ile Ala Pro Gly

20 25 30

Glu Thr Val Tyr Tyr Ser Val Glu Tyr Gln Gly Glu Tyr Glu Ser Leu

35 40 45

Tyr Thr Ser His Ile Trp Ile Pro Ser Ser Trp Cys Ser Leu Thr Glu

50 55 60

Gly Pro Glu Cys Asp Val Thr Asp Asp Ile Thr Ala Thr Val Pro Tyr

65 70 75 80

Asn Leu Arg Val Arg Ala Thr Leu Gly Ser Gln Thr Ser Ala Trp Ser

85 90 95

Ile Leu Lys His Pro Phe Asn Arg Asn Ser Thr Ile Leu Thr Arg Pro

100 105 110

Gly Met Glu Ile Thr Lys Asp Gly Phe His Leu Val Ile Glu Leu Glu

115 120 125

Asp Leu Gly Pro Gln Phe Glu Phe Leu Val Ala Tyr Trp Arg Arg Glu

130 135 140

Pro Gly Ala Glu Glu His Val Lys Met Val Arg Ser Gly Gly Ile Pro

145 150 155 160

Val His Leu Glu Thr Met Glu Pro Gly Ala Ala Tyr Cys Val Lys Ala

165 170 175

Gln Thr Phe Val Lys Ala Ile Gly Arg Tyr Ser Ala Phe Ser Gln Thr

180 185 190

Glu Cys Val Glu Val Gln Gly Glu Ala

195 200

<210>48

<211>68

<212>PRT

<213>Mus musculus

<400>48

His Thr Thr Val Asp Thr Ser Gly Leu Leu Gln His Val Lys Phe Gln

1 5 10 15

Ser Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Gly Gly Pro Ala Ser

20 25 30

Thr Ser Asp Thr Val Tyr Ser Val Glu Tyr Lys Lys Tyr Gly Glu Arg

35 40 45

Lys Trp Leu Ala Lys Ala Gly Cys Gln Arg Ile Thr Gln Lys Phe Cys

50 55 60

Asn Leu Thr Met

65

<210>49

<211>26

<212>PRT

<213>mus musculus

<400>49

Glu Thr Arg Asn His Thr Glu Phe Tyr Tyr Ala Lys Val Thr Ala Val

1 5 10 15

Ser Ala Gly Gly Pro Pro Val Thr Lys Met

20 25

<210>50

<211>28

<212>PRT

<213>mus musculus

<400>50

Thr Asp Arg Phe Ser Ser Leu Gln His Thr Thr Ile Lys Pro Pro Asp

1 5 10 15

Val Thr Cys Ile Pro Lys Val Arg Ser Ile Gln Met

20 25

<210>51

<211>40

<212>PRT

<213>Mus musculus

<400>51

Leu Val His Pro Thr Leu Thr Pro Val Leu Ser Glu Asp Gly His Gln

1 5 10 15

Leu Thr Leu Glu Glu Ile Phe His Asp Leu Phe Tyr Arg Leu Glu Leu

20 25 30

His Val Asn His Thr Tyr Gln Met

35 40

<210>52

<211>50

<212>PRT

<213>Mus musculus

<400>52

His Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu Gly Leu Thr Pro

1 5 10 15

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

20 25 30

Lys Glu Ser Ala Pro Tyr Val Cys Arg Val Lys Thr Leu Pro Leu Val

35 40 45

Pro Arg

50

<210>53

<211>70

<212>PRT

<213>Mus musculus

<400>53

His Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu Gly Leu Thr Pro

1 5 10 15

Asp Thr Glu Phe His Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu

20 25 30

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

35 40 45

Pro Ile Leu Ser Lys Glu Ser Ala Pro Tyr Val Cys Arg Val Lys Thr

50 55 60

Leu Pro Leu Val Pro Arg

65 70

<210>54

<211>46

<212>PRT

<213>Mus musculus

<400>54

Glu Thr Arg Asn His Thr Glu Phe Tyr Tyr Ala Lys Val Thr Ala Val

1 5 10 15

Ser Ala Gly Gly Glu Thr Arg Asn His Thr Glu Phe Tyr Tyr Ala Lys

20 25 30

Val Thr Ala Val Ser Ala Gly Gly Pro Pro Val Thr Lys Met

35 40 45

<210>55

<211>48

<212>PRT

<213>mus musculus

<220>

＜221〉variant

<222>6，11，13，

＜223〉any amino acid of xaa=

<400>55

Thr Asp Arg Phe Ser Xaa Leu Gln His Thr Xaa Ile Xaa Pro Xaa Asp

1 5 10 15

Xaa Xaa Xaa Ile Thr Asp Arg Phe Ser Ser Leu Gln His Thr Thr Ile

20 25 30

Lys Pro Pro Asp Val Thr Cys Ile Pro Lys Val Arg Ser Ile Gln Met

35 40 45

<210>56

<211>92

<212>PRT

＜213〉mankind

<400>56

Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val Lys Phe Gln Ser Ser

1 5 10 15

Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr Pro

20 25 30

Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg Asp Trp

35 40 45

Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn Leu

50 55 60

Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala Arg Val Thr

65 70 75 80

Ala Val Ser Ala Gly Gly Arg Ser Ala Thr Lys Met

85 90

<210>57

<211>28

<212>PRT

＜213〉mankind

<400>57

Thr Asp Arg Phe Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro Asp

1 5 10 15

Val Thr Cys Ile Ser Lys Val Arg Ser Ile Gln Met

20 25

<210>58

<211>40

<212>PRT

＜213〉mankind

<400>58

Ile Val His Pro Thr Pro Thr Pro Ile Arg Ala Gly Asp Gly His Arg

1 5 10 15

Leu Thr Leu Glu Asp Ile Phe His Asp Leu Phe Tyr His Leu Glu Leu

20 25 30

Gln Val Asn Arg Thr Tyr Gln Met

35 40

<210>59

<211>25

<212>PRT

＜213〉mankind

<400>59

His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe Phe Gly Leu Thr Pro

1 5 10 15

Asp Thr Glu Phe Leu Gly Thr Ile Met

20 25

<210>60

<211>14

<212>PRT

＜213〉mankind

<400>60

Ile Cys Val Pro Thr Trp Ala Lys Glu Ser Ala Pro Tyr Met

1 5 10

<210>61

<211>12

<212>PRT

＜213〉mankind

<400>61

Cys Arg Val Lys Thr Leu Pro Asp Arg Thr Trp Thr

1 5 10

<210>62

<211>212

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the C-end has kept the mouse IL-22RA soluble receptors of cleavage site (Leu Val Pro Arg)

<400>62

His Thr Thr Val Asp Thr Ser Gly Leu Leu Gln His Val Lys Phe Gln

1 5 10 15

Ser Ser Asn Phe Glu Asn Ile Leu Thr Trp Asp Gly Gly Pro Ala Ser

20 25 30

Thr Ser Asp Thr Val Tyr Ser Val Glu Tyr Lys Lys Tyr Gly Glu Arg

35 40 45

Lys Trp Leu Ala Lys Ala Gly Cys Gln Arg Ile Thr Gln Lys Phe Cys

50 55 60

Asn Leu Thr Met Glu Thr Arg Asn His Thr Glu Phe Tyr Tyr Ala Lys

65 70 75 80

Val Thr Ala Val Ser Ala Gly Gly Pro Pro Val Thr Lys Met Thr Asp

85 90 95

Arg Phe Ser Ser Leu Gln His Thr Thr Ile Lys Pro Pro Asp Val Thr

100 105 110

Cys Ile Pro Lys Val Arg Ser Ile Gln Met Leu Val His Pro Thr Leu

115 120 125

Thr Pro Val Leu Ser Glu Asp Gly His Gln Leu Thr Leu Glu Glu Ile

130 135 140

Phe His Asp Leu Phe Tyr Arg Leu Glu Leu His Val Asn His Thr Tyr

145 150 155 160

Gln Met His Leu Glu Gly Lys Gln Arg Glu Tyr Glu Phe Leu Gly Leu

165 170 175

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

180 185 190

Leu Ser Lys Glu Ser Ala Pro Tyr Val Cys Arg Val Lys Thr Leu Pro

195 200 205

Leu Val Pro Arg

210

Claims (39)

1. preparation comprises at the method for the antibody of polypeptide:

Be selected from down the group in peptide vaccination in animal:

(a) polypeptide of forming to 102 (Asp) by the amino-acid residue 42 (Ile) of SEQ ID NO:8;

(b) polypeptide of forming to 60 (Ile) by the amino-acid residue 42 (Ile) of SEQ ID NO:8;

(c) polypeptide of forming to 69 (Glu) by the amino-acid residue 42 (Ile) of SEQ ID NO:8;

(d) polypeptide of forming to 81 (Cys) by the amino-acid residue 42 (Ile) of SEQ ID NO:8;

(e) polypeptide of forming to 96 (Lys) by the amino-acid residue 42 (Ile) of SEQ ID NO:8;

(f) polypeptide of forming to 102 (Asp) by the amino-acid residue 42 (Ile) of SEQ ID NO:8;

(g) polypeptide of forming to 69 (Glu) by the amino-acid residue 60 (Ile) of SEQ ID NO:8;

(h) polypeptide of forming to 81 (Cys) by the amino-acid residue 60 (Ile) of SEQ ID NO:8;

(i) polypeptide of forming to 96 (Lys) by the amino-acid residue 60 (Ile) of SEQ ID NO:8;

(j) polypeptide of forming to 102 (Asp) by the amino-acid residue 60 (Ile) of SEQ ID NO:8;

(k) polypeptide of forming to 81 (Cys) by the amino-acid residue 69 (Glu) of SEQ ID NO:8;

(l) polypeptide of forming to 96 (Lys) by the amino-acid residue 69 (Glu) of SEQ ID NO:8;

(m) polypeptide of forming to 102 (Asp) by the amino-acid residue 69 (Glu) of SEQ ID NO:8;

(n) polypeptide of forming to 96 (Lys) by the amino-acid residue 81 (Cys) of SEQ ID NO:8;

(o) polypeptide of forming to 102 (Asp) by the amino-acid residue 81 (Cys) of SEQ ID NO:8; With

(p) polypeptide of forming to 102 (Asp) by the amino-acid residue 96 (Lys) of SEQ ID NO:8;

Wherein said polypeptide brings out immunne response in animal, produce described antibody; And

Separation antibody from animal;

Wherein said antibody is specifically in conjunction with IL-20 polypeptide (SEQ ID NO:8).

2. according to the process of claim 1 wherein that described antibody can reduce the short scorching active of IL-20 (SEQ IDNO:8).

3. the process of claim 1 wherein the antibody that produces by this method can in and the interaction of IL-20 (SEQ ID NO:8) and IL-22RA (SEQ ID NO:2).

4. the method for claim 3, the neutralization that wherein said antibody produces is measured by in based on the cells in vitro neutralization test IL-20 (SEQ ID NO:8) being shown to neutralize.

5. the process of claim 1 wherein that described antibody can reduce the two short scorching active of IL-20 (SEQ ID NO:8) and IL-22 (SEQ ID NO:6).

6. the antibody that produces according to the method for claim 1, it with polypeptide shown in the SEQ ID NO:8 can in conjunction with.

7. the antibody of claim 2, wherein said antibody is (a) polyclonal antibody, (b) mouse monoclonal antibody, (c) derived from the humanized antibodies of (b), (d) antibody fragment, perhaps (e) human monoclonal antibodies.

8. the antibody of claim 2, wherein said antibody also comprises radionuclide, enzyme, substrate, cofactor, fluorescent mark, chemiluminescent labeling, peptide tag, magnetic-particle or toxin.

9. the antibody of claim 7, wherein said antibody also comprises PEGization.

10.-plant antibody or antibody fragment, it can combine with the polypeptide that comprises amino acid residue sequence shown in the SEQ ID NO:8, and reduces the short scorching active of IL-20 (SEQ ID NO:8).

11. the antibody of claim 11 or antibody fragment, wherein this antibody or antibody fragment can reduce the short scorching active of IL-20 (SEQ ID NO:8) or IL-22 (SEQ ID NO:6).

12. according to the antibody or the antibody fragment of claim 10, wherein said antibody or antibody fragment are (a) polyclonal antibodies, (b) mouse monoclonal antibody, and (c) derived from the humanized antibodies of (b), (d) antibody fragment, perhaps (e) human monoclonal antibodies.

13. the antibody of claim 10 or antibody fragment, wherein said antibody also comprises radionuclide, enzyme, substrate, cofactor, fluorescent mark, chemiluminescent labeling, peptide tag, magnetic-particle, medicine or toxin.

14. the antibody of claim 10, wherein said antibody also comprises PEGization.

15. one kind is used to reduce or suppress the propagation of IL-20 inductive hematopoietic cell and hematopoiesis progenitor cell or the method for differentiation, this method comprises cultivates marrow or peripheral blood cells with the composition that comprises the described antibody of a certain amount of claim 3, the marrow or the peripheral blood cells of cultivating during with this antibody are not compared, and the amount of described antibody is enough to reduce the propagation or the differentiation of the hematopoietic cell in marrow or the peripheral blood cells.

16. the method for claim 15, wherein said hematopoietic cell and hematopoiesis progenitor cell are lymphoidocytes.

17. the method for claim 16, wherein said lymphoidocyte are scavenger cell or T cell.

18. a method that reduces IL-20 inductive inflammation, this method comprises the composition that gives the described antibody of a certain amount of claim 3 to the Mammals with inflammation, and the amount of said composition is enough to reduce inflammation.

19. a method that reduces IL-20 inductive inflammation, this method comprise the composition that gives described antibody of a certain amount of claim 10 or antibody fragment to the Mammals with inflammation, this amount is enough to reduce inflammation.

20. an inhibition has the method for inflammatory reaction in the Mammals of inflammation, this method comprises:

(1) determines the proteic level of serum amyloid sample A;

(2) be included in the composition of the described antibody of claim 3 in the acceptable drug carrier;

(3) determine the proteic level of serum amyloid sample A after the administration;

(4) the proteic level of serum amyloid sample A in comparison step (1) and the step (3);

Wherein the proteic level of serum amyloid sample A does not raise or decline occurs and shows that inflammatory reaction is suppressed.

21. an inhibition has the method for inflammatory reaction in the Mammals of inflammation, this method comprises:

(1) determines the proteic level of serum amyloid sample A;

(2) be included in the composition of the described antibody of claim 5 in the acceptable drug carrier;

(3) determine the proteic level of serum amyloid sample A after the administration;

(4) the proteic level of serum amyloid sample A in comparison step (1) and the step (3);

Wherein the proteic level of serum amyloid sample A does not raise or decline occurs and shows that inflammatory reaction is suppressed.

22. an inhibition has the method for inflammatory reaction in the Mammals of inflammation, this method comprises:

(1) determines the proteic level of serum amyloid sample A;

(2) be included in the composition of the described antibody of claim 16 in the acceptable drug carrier;

(3) determine the proteic level of serum amyloid sample A after the administration;

(4) the proteic level of serum amyloid sample A in comparison step (1) and the step (3);

Wherein the proteic level of serum amyloid sample A does not raise or decline occurs and shows that inflammatory reaction is suppressed.

23. a treatment is subjected to the mammiferous method that inflammatory disease that IL-20 works therein torments, this methods of treatment comprises:

Give the antagonist of IL-20 to Mammals, inflammation alleviated,

Wherein said antagonist comprises with antibody, the antibody fragment of the polypeptide of IL-22RA (SEQ ID NO:3) or polypeptide fragment specific combination or combines polypeptide, or polypeptide or the polypeptide fragment of IL-22RA (SEQ IDNO:3); And wherein the inflammatory activity of IL-20 (SEQ ID NO:8) is lowered.

24. the method for claim 23, wherein said disease is a chronic inflammatory diseases.

25. the method for claim 24, wherein said disease is a chronic inflammatory diseases, comprises inflammatory bowel, ulcerative colitis, Crohn disease, sacroiliitis, atopic dermatitis or psoriasis.

26. the method for claim 23, wherein said disease are the acute inflammation diseases.

27. the method for claim 26, wherein said disease is the acute inflammation disease, comprises endotoxemia, septicemia, toxic shock syndrome, TSS or infectious diseases.

28. the method for claim 23, wherein said antibody also comprises radionuclide, enzyme, substrate, cofactor, fluorescent mark, chemiluminescent labeling, peptide tag, magnetic-particle, medicine or toxin.

29. comprise can with human IL-2 0 (SEQ ID NO:8) in be selected from down the antibody of the monoclonal antibody that combines of antigenic epitopes of group:

A) polypeptide of forming to 102 (Asp) by the amino-acid residue 42 (Ile) of SEQ ID NO:8;

(b) polypeptide of forming to 60 (Ile) by the amino-acid residue 42 (Ile) of SEQ ID NO:8;

(c) polypeptide of forming to 69 (Glu) by the amino-acid residue 42 (Ile) of SEQ ID NO:8;

(d) polypeptide of forming to 81 (Cys) by the amino-acid residue 42 (Ile) of SEQ ID NO:8;

(e) polypeptide of forming to 96 (Lys) by the amino-acid residue 42 (Ile) of SEQ ID NO:8;

(f) polypeptide of forming to 102 (Asp) by the amino-acid residue 42 (Ile) of SEQ ID NO:8;

(g) polypeptide of forming to 69 (G1u) by the amino-acid residue 60 (Ile) of SEQ ID NO:8;

(h) polypeptide of forming to 81 (Cys) by the amino-acid residue 60 (Ile) of SEQ ID NO:8;

(i) polypeptide of forming to 96 (Lys) by the amino-acid residue 60 (Ile) of SEQ ID NO:8;

(j) polypeptide of forming to 102 (Asp) by the amino-acid residue 60 (Ile) of SEQ ID NO:8;

(k) polypeptide of forming to 81 (Cys) by the amino-acid residue 69 (Glu) of SEQ ID NO:8;

(l) polypeptide of forming to 96 (Lys) by the amino-acid residue 69 (Glu) of SEQ ID NO:8;

(m) polypeptide of forming to 102 (Asp) by the amino-acid residue 69 (Glu) of SEQ ID NO:8;

(n) polypeptide of forming to 96 (Lys) by the amino-acid residue 81 (Cys) of SEQ ID NO:8;

(o) polypeptide of forming to 102 (Asp) by the amino-acid residue 81 (Cys) of SEQ ID NO:8; With

(p) polypeptide of forming to 102 (Asp) by the amino-acid residue 96 (Lys) of SEQ ID NO:8;

Wherein said antibody can reduce or in and human IL-2's 0 (SEQ ID NO:8) activity.

30. the antibody of claim 29, wherein said antibody can reduce or in and human IL-2's 0 (SEQ ID NO:8) activity.

31. the antibody of claim 29, wherein said antibody are selected from down in the group: (a) mouse monoclonal antibody, (b) derived from the humanized antibodies of (a), (c) antibody fragment and (d) human monoclonal antibodies.

32. the antibody of claim 30, wherein said antibody also comprises PEGization.

33. the antibody of claim 30, wherein said antibody are selected from down in the group: (a) mouse monoclonal antibody, (b) derived from the humanized antibodies of (a), (c) antibody fragment and (d) human monoclonal antibodies.

34. the antibody of claim 33, wherein said antibody also comprises PEGization.

35. a method for the treatment of pathological condition relevant with the activity of IL-20 in the study subject comprises the described antibody of the claim 29 that gives significant quantity, treats described pathological condition thus.

36. the method for claim 35, wherein said pathological condition are the chronic inflammatory diseases patient's condition.

37. the method for claim 36, the wherein said chronic inflammatory diseases patient's condition comprises inflammatory bowel, ulcerative colitis, Crohn disease, sacroiliitis, atopic dermatitis or psoriasis.

38. the method for claim 36, wherein said pathological condition are the acute inflammation patient's condition.

39. the method for claim 38, the wherein said acute inflammation patient's condition comprises endotoxemia, septicemia, toxic shock syndrome, TSS or infectious diseases.