CN101166757A - A novel chimeric polypeptide and use thereof - Google Patents

Abstract

A chimeric polypeptide comprises: a tumor necrosis factor neutralizing agent structural domain, a interleukin-1 receptor antagonists structural domain and a dimerization structural domain, wherein the three structural domains is operational connected each other. The present invention range is: (i) decoding the polypeptide nucleic acid; (ii) an expression vector and host cells with the nucleic acid; (iii) corresponding medicinal composition; and (iv) corresponding preparation method and therapeutic method.

Description

New chimeric polyeptides and application thereof

Related application

The application requires the right of priority of No. the 60/497th, 988, the U.S. Provisional Application of filing an application on August 26th, 2003, and the full content of this application is incorporated herein by reference at this.

Invention field

The present invention relates to a kind of chimeric protein therapeutical agent that can be used for treating as the disease of acute and chronic inflammatory diseases.

Background of invention

Inflammation is a health for the defensive raction such as those damages that caused by mechanicalness injury, infection or antigenic stimulation.When inflammation one is stimulated [as self antigen, its mode with a kind of exaggeration is expressed or also still retained afterwards removing damage former (injurious agents)] to bring out improperly, inflammatory reaction can by pathology show.

Two kinds of important inflammatory response mediators are tumour necrosis factor (TNF) and il-1 (IL-1).TNF neutralizing agent (neutralizer) has been used to treat inflammation dependent form disease (inflammation-dependent diseases) with the IL-1 antagonist.

Tumor necrosis factor-alpha (TNF-α) is the Mammals excretory protein that can bring out extensive diversified effectiveness to the cell type of vast number with tumour necrosis factor-β (TNF-β).It is " TNF " that the very big similarity of these two kinds of cytokines on structure and functional performance causes their communality to describe.

Pair cell starts their biological effectiveness to TNF protein by being bonded to specificity T NF acceptor (TNFR) protein on the cytolemma that is expressed in the reactive cell of TNF-.Knownly have two kinds of dissimilar TNFR:I type TNFR (TNFRI), it has the molecular weight that is about 75kDa: and II type TNFR (TNFRII), it has the molecular weight that is about 55kDa.TNFRI combines with TNF-β with TNF-α separately with TNFRII.

TNF antagonist (as soluble TNF R and TNF conjugated protein) is bonded to TNF, and prevents that TNF is bonded to cytolemma bonded TNF acceptor.This protein is used to suppress by the caused biological activity of TNF.

The role of TNF on the diseases associated with inflammation of being regulated is by clear and definite foundation.For the indication (indications) of TNF dependent form disease [as rheumatoid arthritis and psoriasis], it is safe and efficient clinically that TNFRII has been proved.

One of the most powerful struvite cytokine is IL-1.IL-1 is generated by the cell that monocytes/macrophages is, and can be generated as two kinds of forms: IL-1 α and IL-1 β.Pair cell starts their biological effectiveness to IL-1 protein by binding specificity IL-1 acceptor (IL-1R) (being expressed in the protein on the cytolemma of the reactive cell of a kind of IL-1).

IL-1 receptor antagonist (IL-1ra) is a kind of human protein, and it is used as a kind of natural inhibitor of IL-1.IL-1ra is in conjunction with cytolemma bonded IL-1 acceptor, and prevents that IL-1 is in conjunction with identical IL-1 acceptor.This protein has been used to suppress by the caused biological activity of IL-1.

In theory, neutralize simultaneously or block two kinds of important inflammatory mediators (such as TNF and IL-1), for inflammation dependent form treatment of diseases, should have best therapeutic value.Yet, use a kind of soluble TNF RII and a kind of non-glycosylated IL-1ra that is issued by Immunex company and Amgen company in the time of 242 patients' clinical trial and 24 weeks, do not improve validity, can cause the higher infection and the generation of neutropenia when using a kind of soluble TNF RII and IL-1ra to make single therapy on the contrary.

Brief summary of the invention

The present invention relates to the new chimeric polyeptides of a kind of TNF of being used for the treatment of and IL-1 dependent form disease.This chimeric polyeptides comprises: (1) TNF neutralizing agent structural domain, (2) IL-1 receptor antagonist structural domains, and (3) dimerization structural domains.These three structural domains are operationally connected each other.This TNF neutralizing agent structural domain can comprise the extracellular domain of a human TNFRII: this IL-1 receptor antagonist structural domain can comprise IL-1ra; And this dimerization structural domain can comprise a human IgG1 Fc fragment or human immunoglobulin CH territory.Especially, this IL-1ra is glycosylated Mammals polypeptide.

In one embodiment, this chimeric polyeptides holds the C end to comprise a TNF neutralizing agent structural domain, a dimerization structural domain and an IL-1 receptor antagonist structural domain from N.For example, this chimeric polyeptides can comprise extracellular domain, IgG 1 Fc and the IL-1ra (for example, SEQ ID NO:2) of a human TNF RII.

On the other hand, the present invention is characterised in that the polynucleotide of the sequence that comprises the chimeric polyeptides of the present invention of encoding, and a kind of cell that produces these polynucleotide.For example, this cell can be a kind of mammalian cell, as Chinese hamster ovary celI, NS0 cell and SP2/0 cell.Polynucleotide of the present invention and cell can be used to produce chimeric polyeptides of the present invention.

" polynucleotide " or " nucleic acid " mean a kind of dna molecular (for example, cDNA or genomic dna), a kind of RNA molecule (for example, mRNA), or the analogue of a kind of DNA or RNA.The analogue of DNA or RNA can be synthesized from nucleotide analog.This nucleic acid molecule can be strand or two strands, but double-stranded DNA preferably." isolating nucleic acid " is any segmental nucleic acid of the genomic nucleic acids of a kind of textural nucleic acid that is different from any natural generation or natural generation.Therefore, this term is contained, for example: (a) a kind of DNA, it has the partial sequence of the genomic dna molecule of natural generation, but the position is not the encoding sequence of both sides of the part of this molecule of position in the genome of the organism of natural generation in the both sides of this partial sequence; (b) a kind of nucleic acid, it is integrated in a carrier or prokaryotic organism or the Eukaryotic genomic dna in one way, and makes that the molecule that is produced is inequality in the carrier or the genomic dna of any natural generation; (c) a kind of isolating molecule, the fragment that is produced as cDNA, genomic fragment, by polymerase chain reaction (PCR), or restricted fragment; And (d) a kind of recombinant nucleotide sequence, it is the part of heterozygous genes (that is, the gene of encoding fusion protein matter).Being excluded at the outer of this definition especially is the nucleic acid that appears in the mixture with following different substances: (i) dna molecular, (ii) transfectional cell, or (iii) cell clone, for example, similarly be to be present in the DNA library (as cDNA or genome dna library) these.Nucleic acid described above can be used to express fused protein of the present invention.For reaching this purpose, this nucleic acid can be may be operably coupled to suitable adjusting sequence, to produce expression vector.

The present invention further provides a kind of composition, it comprises: chimeric polyeptides of the present invention or polynucleotide, and pharmaceutically acceptable carrier.Said composition can be used to treat TNF and IL-1 dependent form disease.

Fall equally within the scope of the invention be a kind of method for the treatment of TNF and IL-1 dependent form disease by the present composition that has the individuality that needs to use significant quantity to.For example, this disease can be a kind of inflammatory disease, as rheumatoid arthritis or psoriasis.

Below the details of one or more embodiments of the present invention is described in the appended accompanying drawing of literary composition with describe in detail.Other technologies feature of the present invention, purpose and advantage become from this detailed description and claims obviously as can be known.

Description of drawings

Fig. 1: the chimeric Chinese hamster ovary celI clone of TNFRII-Fc that the 1st generation produced and TNFRII-Fc-IL-1ra: the 24-hole culture plate in the substratum that does not contain serum is expressed; Direct Coomassie blue (Coomasie blue) protein staining; All recombinant proteins in the scope of 0.5 to 1.0 μ g are can be observable; Sample 10 to 15 μ L on each swimming lane;

The chimeric affinity purifying of Fig. 2: TNFRII-Fc-IL-1ra: the reduction of SDS-PAGE and non-reduced condition; The Coomassie blue protein staining;

Fig. 3 a: example of our problem-solving ability: reduce one and do completely to analyze with the chimeric HPLC of the TNFRII-Fc-IL-1ra part degraded at the chimeric degradation problem of TNFRII-Fc-IL-1ra-utilize TNFRII-Fc (control group) via changing first purification step;

Fig. 4: the SEC-HPLC of TNFRII-Fc-IL-1ra mosaic after preparation (formulation) and lyophilize analyzes;

Fig. 5: the IL-1 receptor binding assay demonstrates the TNFRII-Fc-IL-1ra mosaic to have than the taller avidity of non-glycosylated IL-1ra (Kineret) of going on the market in conjunction with human IL-1 acceptor;

Fig. 6: TNF α binding analysis demonstrates, when with-when ve control group Tie2-Fc compared, TNFRH-Fc (homemade) (+ve control group) was specifically in conjunction with TNF α;

Fig. 7: TNF α binding analysis demonstrates, and is similar to TNFRII-Fc (Fig. 6), and the TNFRII-Fc-IL-1ra mosaic is specifically in conjunction with TNF α;

Fig. 8: based among the TNF α of cell and the test (neutralization test) demonstrate, be similar to the TNFRII-Fc (Enbrel) of listing, in the TNFRII-Fc-IL-1ra mosaic and TNF α kill activity (killing activity) for the L979 cell; And

Fig. 9: based among the IL-1 of cell and test demonstrate, the IL-1ra of listing (Kineret) and TNFRII-Fc-IL-1ra mosaic the two can in and IL-1 for the biological activity of D10 cell proliferation.As expected, glycosylation IL-1ra has the external activity that the non-glycosylated IL-1ra (Kineret) that produced than intestinal bacteria (E.coli) also will be low.

Detailed Description Of The Invention

The present invention is based on and finds a kind of chimeric polyeptides that can be used to treat TNF and IL-1 dependent form disease. This chimeric polyeptides comprises: (1) TNF nertralizer domain, (2) IL-1 receptor antagonist domains, and (3) dimerization domains. These three domains are operationally connected each other. Surprisingly, with the TNFRII-Fc level of production as a reference, this kind chimeric molecule is manufactured in mammalian hosts on the commodity production level that is in (commercial production level). It contains among complete TNF and the IL-1 after packing interaction and activity. In other words, a large TNFRII-Fc molecule is merged among the IL-1 receptors bind that can not disturb IL-1ra to the N-of IL-1ra end and the IL-1 and activity. Unlike using simultaneously TNFRII-Fc and IL-1ra, this kind chimeric molecule via general use the path in the inflammation position by more extensive distribution, make it to become the TNFRII-Fc of inflammation position guiding. Our result shows that further this kind chimeric molecule of manufacturing in mammalian hosts contains glycosylated IL-1ra, and has one than the two large molecular weight also of TNFRII-Fc and IL-1ra. Therefore, it has the long biological existence phase (biological life), and the effective ID of less frequency. Because its inflammation position guiding character and less effective dose and dose frequency, therefore when compared to TNFRII-Fc and when using simultaneously TNFRII-Fc and IL-1ra, this kind chimeric molecule can have less side effect.

" TNF nertralizer domain " mean one can in and the domain of TNF, that is, suppress the activity of TNF (for example, TNF-α). For example, TNF nertralizer domain can comprise the extracellular domain of extracellular domain, the IL-6 acceptor of human TNF RII, for the antibody of TNF or TNF acceptor, or for the antibody of IL-6 or IL-6 acceptor.

TNF nertralizer domain is to know clearly in this area to know. For example, United States Patent (USP) the 5th, 605, disclosing TNF acceptor (TNFR) in No. 690, to have amino acid sequence be basically similar in appearance to natural mammal TNF acceptor or the amino acid sequence of TNF conjugated protein, and these TNF acceptors can and suppress TNF in conjunction with the TNF molecule and are bonded to TNFR in conjunction with cell membrane.

As first mentioned, known two kinds of dissimilar TNFR:I type TNFR (TNFRI) and the II type TNFR (TNFRII) of having. The preferred TNFRs of the present invention is TNFRI and the TNFRII of soluble form, and the TNF conjugated protein of solubility. Soluble TNF R molecule comprises for example, having analog or the subunit of at least 20 amino acid whose native proteins, and it represents at least some biologically active similar to TNFRI, TNFRII or TNF conjugated protein. Soluble TNF R construct does not have cross-film zone (and being secreted) from cell, but possesses the ability in conjunction with TNF. Different bioequivalence (bioequivalent) protein and amino acids analog corresponding to a natural TNFR (for example has the monoamino-acid sequence, huTNFRI.DELTA.235, huTNFRI.DELTA.185 and huTNFRI.DELTA.163) zone, extracellular all or part of, or has amino acid sequence basically similar in appearance to United States Patent (USP) the 5th, 605, the sequence of amino acid/11-163, amino acid/11-185 or the amino acid/11-235 of the SEQ ID NO:1 that discloses in No. 690, and when they are bonded to tnf ligand be have bioactive. The soluble TNF R of equivalence comprises by one or more replacement, disappearance or adds the polypeptide that changes from these sequences, and it is possessed in conjunction with TNF or suppresses the ability of TNF signaling activity via the TNF receptor protein of cell surface combination, huTNFRI.DELTA.x for example, wherein x is selected from by United States Patent (USP) the 5th, any one group that consists of among the amino acid/11 63-235 of the SEQ ID NO:1 that discloses in 605, No. 690. Similar disappearance can cause muTNFR. The inhibition of TNF signaling activity can be out determined by cell transfecting is obtained the recombinant receptor expression with recombinant type TNFR DNAs. These cell nows are touched with TNF, and the metabolism effectiveness that causes is detected. If effectiveness generation is the effect owing to part, this recombinant receptor has signaling activity so. The exemplary method that whether has signaling activity for mensuration one polypeptide is disclosed in following document: Idzerda et al., J.Exp.Med 171:861 (1990); Curtis et al., Proc.Natl.Acad.Sci.U.S.A.86:3045 (1989): Prywes et al., EMBO be (1986) and Chou et.al. J.5:2179, J.Biol.Chem. 262:1842 (1987). Perhaps, express endogenous TNF acceptor and also can be utilized for primary cell or clone that TNF has a detectable biologically.

For the name of as used herein " TNFR analog " according to the routine name at protein (for example, TNFR) add " hu " (representing human) or " mu " (representing mouse) before, and add afterwards the number of a DELTA. (in order to point out a disappearance) and C-terminal amino acid. For example, huTNFR.DELTA.235 means to have Asp.sup.235 as the human TNF R of C-terminal amino acid (that is one has at United States Patent (USP) the 5th, 605, the polypeptide of the sequence of the amino acid/11-235 of the SEQ ID NO:1 that discloses in No. 690). Without any the title of the mankind or mouse species (species) time, TNFR generally means mammal TNFR. Similarly, without any for the special title of deletion mutant the time, this word of TNFR means all types of TNFR, comprises having the bioactive routants of TNFR and analog.

As employed in this case specification, one feature of TNF acceptor, " biologically active " mean a special molecule therewith embodiment of the present invention of disclosing of place enjoy enough amino acid sequence similarities and can in conjunction with detectable TNF quantity, transmit TNF stimulate to cell [for example, one composition of picture heterozygosis acceptor construct] or carry out cross reaction (cross-reacting) with the anti-TNF R antibody of the TNFR of anti-natural source (that is, non-recombinant type). Preferably; the biologically active TNF acceptor that drops in protection scope of the present invention can be in conjunction with the TNF greater than 0.1 nanomole (nmoles) in the acceptor of every nanomole; and most preferably, the acceptor of every nanomole is greater than the TNF of 0.5 nanomole in the binding analysis of standard.

Of the present invention preferred aspect, the TNF nertralizer is to be selected from the group that is made of the human TNFRI of solubility and TNFRII. The pCAV/NOT-TNFR carrier that comprises human TNF RI cDNA clone 1 is used to the human TNFRI of expression and purification solubility. PCAV/NOT-TNFR with " pCAV/NOT-TNFR " this title be preserved in American type culture collection (ATCC, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.) (preserving number: 68088).

As most mammalian genes, it is coded by multiple exon genes (multi-exon gene) that Mammals TNF acceptor is inferred.Owing to mRNA splicing results different after transcribing, and with United States Patent (USP) the 5th, 605, the different mRNA construct in the total most of same or analogous zone of the cDNA that is disclosed in No. 690 also can be used.

Other Mammals TNFR cDNA can be separated by using a suitable human TNF RDNA sequence to screen specific Mammals cDNA library as probe via cross species hybridization (cross-specieshybridization).Employed in the present invention Mammals TNFR comprises, for example, and the TNFR of primates, the mankind, mouse, dog, cat, ox, sheep, horse and pig.Mammals TNFRs can pass through the cross species hybridization, and the use one strand cDNA derived from human TNFR dna sequence dna comes to isolate TNFR cDNA from Mammals cDNA library as hybridization probe and obtains.

Can use the functional equivalents of TNFR in the present invention." functional equivalents " means a polypeptide derived from the TNFR polypeptide, for example, and fused protein or have the protein of one or more point mutation, insertion, disappearance, brachymemma (truncations) or these combinations.It possesses the activity of TNFR polypeptide basically, that is, be bonded to the ability of TNF.Polypeptide separated can comprise the fragment of SEQ ID NO:3 or SEQ ID NO:3, maybe can be the fragment of SEQ ID NO:3 or SEQ ID NO:3.

The one-level amino acid structure can by with other chemical parts (such as; glycosylation group, lipid, phosphoric acid, acetyl group and similar thing) form covalency or assemble conjugate (aggregative conjugates), or modified by producing the aminoacid sequence mutant.Covalence derivative is produced out by special functional groups being connected to TNFR amino acid side chain or N-or C-end.Other TNFR derivative comprises that TNFR or its fragment add other the protein or the covalency of polypeptide or assemble conjugate, as by in the reorganization culture become the N-end or C-holds fusions.For example, (conjugated) peptide of being puted together can be a signal at proteinic N-end regions (or leading) peptide sequence, it translates ground (co-translationally) altogether or this protein is guided from its synthetic position transfer to its position of function in the inside or the outside of cytolemma or cell walls (for example, yeast α-factor leader sequence) in translation ground, back (post-translationally).TNFR protein merges to comprise and is added with the purifying that the promotes TNFR peptide (for example, gathering His) with discriminating.The aminoacid sequence of TNF acceptor also can be connected to peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (Hopp et al., Bio/Technology 6:1204,1988).The sequence of back is high antigenic, and provides one by special monoclonal antibody bonded epi-position and can analyze the recombinant protein of being expressed with easy purifying fast reversibly.This sequence is also specifically cut by ox mucous membrane enteropeptidase (bovine mucosal enterokinase) at the residue place afterwards immediately in Asp-Lys pairing (pairing).Being added cap can also be resistive with the fused protein of this peptide for degraded in the cell in intestinal bacteria (E.coli).

Relevant with the glycosylation of natural pattern or irrelevant TNFR can also be used.The TNFR that is expressed in yeast or mammalian expression system (for example, COS-7 cell) may be similar to natural molecule according to this expression system and on molecular weight and glycosylation pattern or slightly different.The expression of TNFR DNAs in bacterium (such as intestinal bacteria) provides non-glycosylated molecule.The functional mutants analogue of Mammals TNFR with N-glycosylation site of inactivation can be produced with ligation or by site-directed mutagenesis effect technology by oligonucleotide is synthetic.These analogue protein can use expression system with one isostructural (homogeneous), reductibility carbohydrate form, present good productive rate and produced, the N-glycosylation site in eukaryotic protein is characterised in that amino acid triplet Asn-A ₁-Z, wherein A ₁Be any amino acid except Pro, and Z is Ser or Thr.In this sequence, Asn provides a pendant amino group to be used for the covalent attachment of carbohydrate.This site can replace Asn or residue Z, disappearance Asn or Z by the amino acid with other or at A ₁And insert the amino acid of a non--Z between the Z, or at Asn and A ₁Between insert the amino acid of a non-Asn and be removed.

The TNFR derivative also can be obtained by the sudden change of TNFR or its subunit.So the place means, and the TNFR mutant is a kind of and TNFR homologous (homologous) polypeptide, but its because of a disappearance, insert or replacement has an aminoacid sequence that is different from natural TNFR.

The proteinic bioequivalence analogue of TNFR can pass through, and for example, makes the different residues or the replacement of sequence, or disappearance unwanted end or inner residue or sequence and be fabricated out for biological activity.For example, cysteine residues can be by disappearance (for example, Cys ¹⁷⁸) or replace and not needing or incorrect intramolecular disulfide bond owing to renaturation forms to avoid with other amino acid.Other mutafacient system comprise that binary (dibasic) amino-acid residue of modifying vicinity strengthens the expression in the yeast system, wherein exists the KEX2 protease activity.Generally speaking, replacement should by conservative property finish, that is most preferred substituted amino acid is to have those of the plysiochemical characteristic similar to wanting substituted residue.Similarly, when disappearance or when inserting strategy and being used, disappearance or insert should be taken into account for bioactive potential impact.Though for asking the C-end brachymemma that reaches the purpose that makes up soluble TNF R will contain less aminoacid sequence, as top defined, similar basically peptide sequence generally comprises the aminoacid sequence of similar number.In order to keep the biological activity of TNFR, disappearance preferably will cause homology or conservative property to replace sequence with replacement, mean a set residue and be replaced by biological similarity residue.The example that conservative property replaces comprises with an aliphatic residue and replaces another person, replaces each other as Ile, Val, Leu or Ala; Or replace another person with a polar residues, and as at Lys and Arg, Glu and Asp, or between Gln and the Asn.Other this kind conservative propertys replace, and for example, whole replacement with zone of similar hydrophobic property is well known.What is more, the special acid difference between the mankind, mouse and other Mammals TNFR is to mean that extra conservative property replaces, and it can be caused at the following of the basic biological nature that does not change TNFR.

The subunit of TNFR can be fabricated out by residue or the sequence that lacks end or inside.Particularly preferred sequence comprises that stride diaphragm area and the cell intracellular domain of TNFR are lacked or replace with the wetting ability residue and be beneficial to secrete acceptor those to the cell culture medium.The protein that is produced is called as soluble TNF R molecule, and it possesses the ability in conjunction with TNF.Particularly preferred soluble TNF R construction is TNFRI.DELTA.235 (at a United States Patent (USP) the 5th, 605, the sequence of the amino acid/11-235 of the SEQ ID NO:1 that is disclosed in No. 690), and it comprises the zone, extracellular of whole TNFRI, stops with Asp ²³⁵Adjoining span diaphragm area immediately.Extra amino acid can be lacked from stride diaphragm area, but still possesses TNF in conjunction with activity.For example, be included in United States Patent (USP) the 5th, 605, the huTNFRI.DELTA.183 of the sequence of the amino acid/11-183 of the SEQ ID NO:1 that is disclosed in No. 690, and be included in United States Patent (USP) the 5th, the TNFRI.DELTA.163 of the sequence of the amino acid/11-163 of the SEQ ID NO:1 that is disclosed in 605, No. 690 possesses the ability in conjunction with tnf ligand.Yet TNFRI.DELTA.142 does not keep the ability in conjunction with tnf ligand.This means Cys ¹⁵⁷With Cys ¹⁶³In the middle of one or the two be needs for forming an intramolecular disulfide bond for being used for suitable TNFRI folding.Cys ¹⁷⁸(its lacked soluble TNF RI in conjunction with the ability of TNF without any tangible disadvantageous effect) seems not necessarily for suitable TNFRI is folding.Therefore, any C-holds to Cys ¹⁶³Disappearance will can be caused bioactive soluble TNF RI by expection.This invention is intended to use this kind corresponding to all or part of at Cys ¹⁶³Afterwards with the soluble TNF R construction in the zone, extracellular of any amino acid terminated TNFR.The mode that other C-end disappearance (such as TNFRI.DELTA.157) can be fit to is via cutting TNFR cDNA with suitable restriction enzyme, and if necessary, makes up special sequence again and is done with the synthetic oligonucleotide joint.Soluble TNF R with N-end disappearance also can be used among the present invention.For example, do not having influence on significantly under the ability that TNFRI serves as a TNF antagonist effectively, the N-of TNFRI end can Leu ¹, Pro ², or Ala ³Initial.The soluble TNF R construction that is produced then is inserted into and is expressed in the suitable expression, and analyzes the ability in conjunction with TNF.

Sudden change in being fabricated the nucleotide sequence of expressing analogue TNFR must be possessed the frame stage of encoding sequence certainly, and preferably will can not produce can heterozygosis and produce secondary mRNA structure [as, the ring or hairpin structure] complementary region, this kind secondary mRNA structure will have influence on the translation of receptor mrna unfriendly.Though can be scheduled come out in the sudden change position, the character itself of suddenling change be do not need scheduled.For example, in order to pick out the mutant of optimal performance in a set position, random mutagenesis can carry out at target codon place, and the TNFR mutant of expressing is at active screened come out of being desired.

Be not that all sudden changes in the nucleotide sequence of coding TNFR will be expressed on end product, for example, can produce Nucleotide replaces and strengthens expression, main is avoids secondary structure ring in the mRNA that transcribes (referring to EPA 75,444A, incorporate this case at this and think bibliography), or provide and pass through the host who is selected and the codon of more easily being translated (for example, the preferred codon of known intestinal bacteria at escherichia coli expression).

Sudden change can comprise the oligonucleotide of mutant sequence and is imported at special genes seat (loci) by synthetic, and described mutant sequence flank is to be connected to the segmental restriction site of native sequences.After ligation, formed reconstruction sequence coding one analogue with aminoacid insertion, replacement or disappearance of being desired.

Perhaps, oligonucleotide-fixed point specificity mutagenesis step can be used to provide a kind of modifying gene with special password of doing to change according to needed replacement, disappearance or insertion.The exemplary method that causes transformation recited above is by people such as Walder (Gene 42:133,1986); People such as Bauer (Gene 37:73,1985); (12-19): people such as Smith (Genetic Engineering:Principles and Methods, Plenum Press, 1981) disclose Craik for Bio Techniques, Jan.1985; And the technology that United States Patent (USP) is fit to the 4th, 737, No. 462 announcements for the 4th, 518, No. 584, and incorporate this case at this and think bibliography.

Can be used as the Antybody therapy agent of treatment TNF dependent form disease at the antibody of TNF or TNFRs.These antibody comprise the IgG Fc fragment similar immunoglobulin heavy chain constant region territory Dimerized at its C-end, and comprise two TNF or TNFR in conjunction with the Fab structural domain at its N-end.

The activity of these antibody is can be by using TNF dependent form cell [as L979 cell (ATTC)] determined comes out.TNF-dependent form cell can be killed via the recombinant type TNF α that adds effective dose.This kind TNF-dependent form activity can be neutralized to reaction by adding these antibody.The activity of these antibody can also be by using the external binding analysis of TNF in the culture plate in 96-hole determined come out.

The IL-6 acceptor of solubility or at the antibody of IL-6, or can be used to replace the TNFRII of this kind chimeric molecule at the IL-6R that its C-end has the IgG CH territory similar to the IgG Fc fragment of a dimerization.The activity of these molecules can be by using the external binding analysis of IL-6 acceptor in the culture plate of 96-hole determined come out.This activity is can also be by using recombinant type IL-6 and IL-6-dependent form D10 cell determined comes out.

" interleukin-1 receptor antagonist structural domain " means one and can be bonded to the IL-1 acceptor specifically and prevent the structural domain of cell receptor for the activation of IL-1.The example of interleukin-1 receptor antagonist comprises IL-1ra (United States Patent (USP) the 6th, 096, No. 728) with the monoclonal antibody (EP 623674) of anti--IL-1 acceptor, and their functional equivalents (that is, polypeptide derived from the monoclonal antibody of IL-1ra or anti--IL-1 acceptor), for example, fused protein or have the protein of one or more point mutation, insertion, disappearance, brachymemma, or its combination.They possess the activity that is bonded to the IL-1 acceptor specifically basically, and prevent the activation of cell receptor for IL-1.They can comprise the fragment of SEQ ID NO:5 or SEQ ID NO:5.Preferably, this IL-1ra is a kind of by glycosylated Mammals polypeptide.The activity of interleukin-1 receptor antagonist can by use IL-1 dependent form D10 cell based on the IL-1 neutralization analysis of cell and determined come out (referring to embodiment 4).

Preferred IL-1ra protein is disclosed in United States Patent (USP) the 5th, 075, among No. 222 (being called ' 222 patent at this), WO 91/08285, WO 91/17184, AU 9173636, WO92/16221 and the WO 96/22793, the disclosure of its grade is incorporated this case at this and is thought bibliography.These protein comprise glycosylation and nonglycosylated IL-1 receptor antagonist.

Especially, in revealed and ' 222 patent that is described in this of three kinds of useful IL-1ra types and its variant.In the middle of these the first, IL-1ra. α is characterized as the 22-23 kD molecule on SDS-PAGE, and iso-electric point is about 4.8, and under about 52 mM NaCl (be assigned in the Tris damping fluid, pH 7.6), wash-out comes out from Mono Q FPLC post.The two, IL-1ra. β is characterized as 22-23 kD protein, wash-out comes out from Mono Q post under 48mM NaCl.IL-1ra. α and IL-1ra. β the two all by glycosylated.The third party, IL-1rax is characterized as 20kD protein, and wash-out comes out from Mono Q post under 48mM NaCl, and is nonglycosylated.Whole threes in these inhibitor hold similar function and immunologic competence.

The present invention also comprises the modified types of IL-1ra.As used herein, the modified types of IL-1ra comprises variant polypeptide, wherein amino acid the residue in the aminoacid sequence of IL-1ra (1) lacked (" disappearance variant "), (2) are inserted into (" interpolation variant "), or (3) are substituted (" replacement variant ").

With regard to IL-1ra disappearance variant, each polypeptide typically can have a variation from about 1 to 30 residue, more typically about 1 to 10 residue, and the sequential amino acid deletion of the scope of the most about 1 to 5 continuous residue.N-end, C-end and inner sequence interior (intrasequence) disappearance are expected.Disappearance in this IL-1ra aminoacid sequence can with IL-1 family in other members' sequence have in the zone of low homology and produced.Disappearance in this IL-1ra aminoacid sequence can basically with IL-1 family in other members' sequence have in the zone of homology and produced, and modified biological activity more significantly.

With regard to IL-1ra added variant, each polypeptide can comprise that a kind of length variations merges with carboxyl terminal from the amino of the scope of 1 residue to 100 or more a plurality of residues, and insertion in the sequence of the inside of single or multiple amino-acid residue.Inner interpolation typically can change from about 1 to 10 amino-acid residue more typically about 1 to 5 amino-acid residue, and the scope of the most about 1 to 3 amino-acid residue.

Aminoterminal adds variant and comprises and add methionine(Met) [for example, as a kind of in bacterium recombinant type cell culture the directly artefact of marking protein or artificial construction (artifact)] or extra amino-acid residue or sequence.The further example that aminoterminal inserts comprise merge signal sequence with and/or former sequence (pre-pro sequence) before other, be beneficial to from recombinant host cell, secrete protein.Each polypeptide can comprise a kind of be selected to allow identification of this host cell and the signal sequence of handling (that is, cut by signal peptidase).With regard to the prokaryotic organism host cell, its nonrecognition with handle natural IL-1ra signal sequence, each polypeptide can comprise and for example is selected from the prokaryotic organism signal sequence of the group of alkaline phosphatase, penicillinase or thermostability enterotoxin 1 I leader sequence.With regard to yeast cell, each polypeptide can have and for example is selected from yeast invertase, the signal sequence of the group of alpha factor or acid phosphatase leader sequence.With regard to the expression of mammalian cell, each polypeptide can have natural IL-1ra signal sequence, though other mammalian signal sequence perhaps be fit to, for example, derived from other IL-1 family members' sequence.

With regard to IL-1ra replaced variant, each this peptide species can have at least one amino-acid residue that is removed and one and be inserted in its locational different residue in IL-1ra.Replace variant and comprise equipotential (allelic) variant, it is characterized in that may or may not can causing amino acid change, abiogenous nucleotide sequence changes in species colony.Those skilled in the art can utilize and anyly knownly select possible mutational site relevant for the combination of this polypeptide or the information of avtive spot.Exemplary replacement variant by teaching in WO 91/17184, WO92/16221 and WO 96/09323.

A kind ofly be used for differentiating that the method in the amino-acid residue that is used for protein mutagenesis or zone is called as " alanine scanning mutagenesis (alanine scanning mutagcnesis) " (Cunningham andWells (1989), Science, 244:1081-1085, its disclosure is incorporated this case at this and is thought bibliography).In this method, the amino-acid residue of proteinic target residue or group are (for example, charged residue, such as Arg, Asp, His, Lys and Glu) differentiated out, and replaced by neutral or electronegative amino acid (most preferably L-Ala or poly-L-Ala), to reach in these amino acid and the cell or the extracellular interaction of aqueous environments on every side.Those residues that show function sensitive (functional sensitivity) for replacement are then modified by importing residue extra or that select in addition in the position that replaces.Therefore, come out as the position that imports amino acid sequence modifications is scheduled, and the execution of optimizing sudden change on set position, L-Ala scanning or random mutagenesis can be carried out, and formed variant polypeptide is desired the combination of activity and level of activity and screened come out at the institute of the best.

Appeal to most become that the position that replaces mutagenesis is included among the IL-ra to be found, according to side chain size, electric charge with and/or hydrophobicity be different from basically IL-1ra sample protein (such as, other different plant species of IL-1ra or other IL-1 family member) amino acid whose position.Other attractive positions comprise those positions that wherein special IL-1ra residue is identical with these IL-1ra-sample protein.Generally speaking these positions are very important for proteinic biological activity.Originally, these positions are modified via replacement in conservative relatively mode.These conservative propertys replacements are shown in table 1, and title is the below of " the preferred replacement ".If if these replacements cause a change on biological activity, the so more change of essence (exemplary replacement) is imported into, with and/or other interpolation/disappearance can be produced and formed polypeptide is screened comes out.

Table 1

Modify for the conservative property of the aminoacid sequence of IL-1ra (and modify for the correspondence of nucleic acid sequence encoding) and can be produced protein with identity function and chemical property by expection.On the contrary, the function of IL-1ra with and/or chemical property on the modification of essence can be done by being chosen in for keeping replacements different significantly on the following effectiveness: (a) replacing in the zone, the structure of polypeptide main chain (for example, folding or the helical conformation of picture), (b) in the proteinic electric charge and the hydrophobicity of target position, or (c) size of side chain.Each group below abiogenous residue is divided into based on common side chain character:

(1) hydrophobic: nor-leucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr;

(3) tart: Asp, Glu;

(4) alkalescence: Asn, Gln, His, Lys, Arg;

(5) aromatic: Trp, Tyr, Phe; And

(6) influence the residue of chain orientation (chain orientation): Gly, Pro.

Non-conservation replaces may relate to a member who exchanges another group with a member of one group in these groups.These substituted residues can be fed to the IL-1ra zone of other IL-1 family members for homology or non-homology in.

Special sudden change in the IL-1ra sequence may relate at adorned proteinic N-end, C-end or the non-natural aminoacid replacement in any position via the carbohydrate that adds that N-connects or O-connection.These modifications may have particular utility, as, adding amino acid (for example, halfcystine), its polymkeric substance that helps connecting water soluble forms derivative.Further, the sequence of IL-1ra can be modified to add the glycosylation position that glycosylation position or disappearance N-connection or O-connect.The glycosylation identifying position that l-asparagine connects comprises one three peptide (tripeptide) sequence, and it is come out by suitable cell glycosylase identification specifically.These three peptide sequences are Asn-Xaa-Thr or Asn-Xaa-Ser, and wherein Xaa can be the non-any amino acid of Pro that is.

In a special embodiment, the amino acid of variant and IL-1ra (SEQ ID NO:5) is homologous basically.As used herein, " homologous basically " this speech means the degree of a homology, and it preferably surpasses 70%, more preferably surpasses 80%, be more preferably to surpass 90%, or most preferably or even 95%.As described herein, in the time in 100 amino acid, can introducing 4 gaps with the assistance contrast, the percentage ratio of the amino-acid residue that aligns according to the identical amino-acid residue with in the sequence that is compared in less in two sequences sequence calculates percent homology, for example Dayhoff is at Atlas of Protein Sequence and Structure, 5:124 (1972), National Biochemical Research Foundation, Washington is described in the D.C. (its disclosure is incorporated this case at this and thought bibliography).The variant of the IL-1ra that those can be separated for the cross reactivity of the aminoacid sequence of SEQ ID NO:5 by antibody, or its gene can be considered to homologous basically equally via the variant of the IL-1ra that is separated with the fragment hybridization of the DNA of coding SEQ ID NO:5 or this DNA.

The IL-1ra variant can be by changing suitable Nucleotide among the DNA that is directed into coding IL-1ra variant, or be produced out by the IL-1ra variant of desiring in external chemosynthesis.To those skilled in the art, what can recognize is, the combination of many disappearances, insertion and replacement can be reached, the final IL-1ra variant that is provided be have bioactive.

About the induced-mutation technique of replacement, insertion or the disappearance of one or more selecteed amino-acid residues are (for example, United States Patent (USP) the 4th, 518, No. 584, its disclosure is incorporated this case at this and thought bibliography) well-known to those skilled in the art.Two main parameters are arranged: the place of sudden change position and the essence of sudden change on each aminoacid sequence variant of structure.When each variant of design, the place of each sudden change position and the essence of each sudden change will depend on wants adorned biochemical characteristic.Each sudden change position can for example be passed through, (1) at first, replacement is with the conserved amino acid kind, and then look the result that will reach, do to select more completely, (2) disappearance target amino acid residue, or (3) are inserted and one or morely are adjacent to the amino-acid residue of the position of being found out and individually or are continuously modified.

" dimerization structural domain " means a kind of structural domain that the polypeptide of the present invention of two copies can be bonded into a part.For example, a dimerization structural domain can comprise a Dimerized IgG Fc fragment or IgG CH territory.The segmental example of the Fc of this class comprises SEQ ID NO:4.

IgG Fc fragment forms interchain (inter-chain) disulfide linkage (covalency) via its cysteine residues and comes Dimerized.Sometimes non-covalent dimerisation also can take place under the situation that does not relate to disulfide linkage.Dimerized IgG Fc fragment in this chimeric molecule can present two kinds of functional TNFRII molecules at its N-end, and can present two kinds of functional IL-1ra molecules at its C-end.This kind arrangement has increased (in vivo) in vivo and has been used for and the two receptor/ligand bonded chance of TNF α and IL-1 acceptor.

Come out via the activity of the covalency dimerisation of disulfide linkage is can be by using reductive and non-reducing SDS-PAGE electrophoresis determined.When reductive condition was used, proteinic molecular weight should be able to be reduced to half.Non-covalent dimerisation can naturally be come electrophoresis with condition sex change and determined come out by using.When the sex change condition was used, proteinic molecular weight should be able to be reduced to half.

In a polypeptide of the present invention, TNF neutralizing agent structural domain, dimerization structural domain and IL-1 receptor antagonist structural domain are operably connected.As used herein, the node configuration that " by being operably connected " means this polypeptide can not disturb the activity of each structural domain, that is this TNF neutralizing agent structural domain is possessed in it and the ability of TNF; This interleukin-1 receptor antagonist structural domain is possessed it specifically in conjunction with the IL-1 acceptor and prevent the ability of cell receptor for the activation of IL-1; And this dimerization structural domain possesses its polypeptide of the present invention with two copies and is bonded into a part, and can present two kinds of functional TNFRII molecules at its N-end, and can present the ability of two kinds of functional IL-1ra molecules at its C-end.

For example, a chimeric polyeptides of the present invention is held the end to C-from N-, can comprise a TNF neutralizing agent structural domain, a dimerization structural domain, and an IL-1 receptor antagonist structural domain.An example of the TNF neutralizing agent structural domain of this class comprises SEQ ID NO:3.Especially, this chimeric polyeptides comprises extracellular domain, IgG 1 Fc of a human TNF RII, and IL-1ra, and an example of the chimeric polyeptides of this class comprises SEQ ID NO:2.

The present invention also provides a kind of isolating polynucleotide that comprise the dna sequence dna of the chimeric polyeptides of the present invention of encoding.The recombinant DNA technology that the polynucleotide of this class can use this area to know clearly to know and be fabricated out.For example, polynucleotide of the present invention can be a kind of carriers that comprises the dna sequence dna of the chimeric polyeptides of the present invention of encoding.This carrier can be used to make this polypeptide.

As used herein, " carrier " this speech means and a kind ofly can transport the polynucleotide that another has been connected to polynucleotide wherein.Dissimilar carriers is to know clearly in this area to know.Referring to, for example, United States Patent (USP) the 6th, 756, No. 196.The carrier of one type is a kind of " plasmid ", and it means a kind of circular double stranded DNA ring that has wherein connected extra dna fragmentation.The carrier of another type is a kind of virus vector, and wherein extra dna fragmentation can be connected in the viral genome.Some carrier can be in its host cell that is imported into self-replicating (bacteria carrier and additive type (episomal) the Mammals carrier that for example, have a bacterium replication orgin).Other carrier (for example, non-add type (non-episomal) Mammals carrier) is integrated in the genome of this host cell after importing a host cell, and along with this host genome is duplicated.What is more, some carrier (expression vector) can be guided the expression of gene that is operably connected to it.Generally speaking, in the recombinant type dna technique, useful expression vector is the form of plasmid (carrier) usually.Yet, this invention is intended to comprise those other forms of expression vectors, such as virus vector (for example, replication defect type retrovirus, adenovirus and adeno associated virus), its grade represents the function of equivalence.

Recombinant type expression vector of the present invention comprises polynucleotide of the present invention with the form that is adapted at expression polynucleotide in the host cell, it means this recombinant type expression vector and comprises one or more adjusting sequences (based on the host cell that will be used to express and selected come out), and it is operably connected on the polynucleotide sequence that will be expressed.In the recombinant type expression vector, " be operably connected " and be intended to mean this interesting nucleotide sequence and allow mode that this nucleotide sequence expresses (for example with a kind of, in external transcribing/translation system, maybe when this carrier is fed in the host cell, in this host cell) and be connected to the adjusting sequence." adjusting sequence " this speech is intended to comprise promotor, enhanser and other expression controlling elements [for example, polyadenylation signal].These are regulated sequence and are described in, for example, and Goeddel; Gene ExpressionTechnology:Methods in Enzymology 185, Academic Press, San Diego, Calf. (1990).Regulate those of constitutive expression of guiding one nucleotide sequence in the host cell that sequence is included in numerous species, and only in some host cell the expression of this nucleotide sequence of guiding those (for example, tissue-specific adjusting sequences).To those skilled in the art, what can recognize is, the design of expression vector can be depended on as will be by transformed host cells, the factors of being desired such as protein expression level.Expression vector of the present invention can be fed in the host cell, thereby produces by polynucleotide as described herein coded protein or polypeptide.

Recombinant type expression vector of the present invention at protokaryon or eukaryotic cell [for example can be designed to come, bacterial cell (such as intestinal bacteria), insect cell (use rhabdovirus expression vector), yeast cell or mammalian cell] a middle expression polypeptide of the present invention.The host cell that is fit to is further discussed: Goeddel, Gene Expression Technology:Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990).Perhaps, this recombinant type expression vector can be transcribed and be translated external, for example uses the T7 promotor to regulate sequence and T7 polysaccharase.

In one embodiment, polynucleotide of the present invention use mammalian expression vector to be expressed in mammalian cell.The example of mammalian expression vector comprises pCDM8[Seed (1987) Nature 329:840], pCI (Promega), and pMT2PC[Kaufman et al. (1987) EMBO J.6:187-195].In the time of in being used in mammalian cell, the controlled function of this expression vector is provided by viral regulatory element usually.For example, the promotor of often using is derived from polyomavirus (polyoma), adenovirus 2, cytomegalovirus and simian virus 40 (Simian Virus 40).About the two expression system of other suitable protokaryons and eukaryotic cell referring to 16 and 17 chapters, Sambrook et al.eds., Molecular Cloning:A Laboratory Manual.2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant type mammalian expression vector can the preferentially expression [for example, the tissue specificity regulatory element is used to express polynucleotide] of guiding polynucleotide in specific cell type.The tissue specificity regulatory element is to know clearly in this area to know.The unrestriced example of the tissue-specific promoter that is fit to comprises: albumin promoter [liver-specificity: Pinkert et al. (1987) Genes Dev.1:268-277]; Lymph-specificity promoter [Calame and Eaton (1988) Adv.Immunol.43:235-275], especially, TXi Baoshouti [Winoto and Baltimore (1989) EMBO J.8:729-733] and immunoglobulin (Ig) [Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748] promotor; Neurone-specificity promoter [for example, neurofilament promotor; Byrne and Ruddle (1989) Proc.Natl.Acad.Sci.USA 86:5473-5477]; Pancreas-specificity promoter [Edlund et al. (1985) Science 230:912-916]; And mammary gland-specificity promoter [for example, milk whey promotor; United States Patent (USP) the 4th, 873, No. 316 with No. the 264th, 166, European Application Publication case].Grow in modulability promotor (developmentally-regulated promoters) also is comprised in, for example hox promotor [Kesse] and Gruss (1990) the Science 249:374-379 of mouse] and afp promoter [Campes and Tilghman (1989) Genes Dev.3:537-546].

Another aspect of the present invention is about being imported into the recombinant expression vector of the present invention or the host cell of isolating polynucleotide molecule of the present invention." host cell " alternately used herein with " recombinant host cell " these terms.What recognized is that these terms not only mean specific individual cells, also represent the filial generation of this cell or possible filial generation (potentialprogeny).Because modifying, some may in the filial generation (succeeding generations) of inheriting, can take place owing to the relation of sudden change or environmental influence, in fact, this kind filial generation is may not can identical with parental cell, but still is included in the scope of this speech as used herein.

Host cell can be any protokaryon or eukaryotic cell.For example, polypeptide of the present invention can be at bacterial cell (such as E.coli), is expressed in insect cell, yeast or the mammalian cell [such as Chinese hamster ovary cell cell (CHO) or NS0 cell].Other host cells that are fit to are that those skilled in the art know clearly and know.

Carrier DNA of the present invention or isolating polynucleotide molecule can be via traditional conversion or rotaring dyeing technologies and are imported in protokaryon or the eukaryotic cell.As used herein, " conversion " is intended to mean various with external polynucleotide (for example being used for " transfection " these terms, DNA) be directed into the technology (art-recognizedtechniques) that the skill in the host cell is confirmed, comprise transfection, lipofection (lipofection) or the electroporation of calcium phosphate or calcium chloride coprecipitation method, DEAE-dextran-mediation.Be fit to be used for transforming or the method for transfection host cell can find in people such as Sambrook (on seeing) and other laboratory manual.

With regard to stable transfection mammalian cell, known is according to employed expression vector and rotaring dyeing technology, only has the cell of small part can integrate external DNA to its genome.In some case, carrier DNA is kept by host cell.In other case, host cell does not keep carrier DNA, and only keeps the separation polynucleotide molecule of being brought into by this carrier of the present invention.In some case, separation polynucleotide molecule of the present invention is used to transformant under the situation of not using carrier.

In order to identify and select these intasomies (integrants), the gene of coding selective marker (for example, for antibiotic resistance) is usually along with interesting gene is fed in the host cell together.Preferred selective marker comprises gives for those of medicine [such as G418, Totomycin and methotrexate] resistance.The polynucleotide of coding selective marker can be fed in the host cell in the identical carrier as the polypeptide of the present invention of encoding, and perhaps can be imported in the carrier of opening in a minute.Be stabilized the ground transfection and can be identified out (for example, having incorporated the cell that selectable marker gene is arranged into will survive, and other cell is then dead) by medicament selection with the cell of these importing polynucleotide.

Host cell of the present invention (such as a protokaryon or the eukaryotic host cell in culture) can be used to produce (that is, express) polypeptide of the present invention.Therefore, the present invention further provides the method for using host cell of the present invention to produce polypeptide of the present invention.In one embodiment, this method comprises cultivates host cell of the present invention (the separation polynucleotide molecule of the recombinant expression vector or the polypeptide of the present invention of encoding is imported into wherein) in the substratum that is fit to, and by this, this polypeptide is generated.In another embodiment, this method further comprises this polypeptide is separated in this substratum or this host cell.

Polypeptide of the present invention can be incorporated in the pharmaceutical composition that is suitable for using.These compositions typically comprise this polypeptide and pharmaceutically acceptable carrier.As used herein, " pharmaceutically acceptable carrier " this speech is intended to comprise any and whole solvent, dispersion medium (dispersion media), coating (coatings), antibiotic and anti-mycotic agent, isotonicity (isotonic) and absorption delay agent (delaying agents) that pharmacy is used that be suitable for, and similar thing.Using these media (media) and reagent (agents) conduct active substance pharmaceutically is to know clearly in this area to know.Except any and inconsistent traditional sucrose of active compound or reagent, its use in said composition is expected.Additional active compound can also be incorporated in the said composition.

It is compatible that pharmaceutical composition of the present invention is formulated into the route of administration that is intended to it.The example of route of administration comprises parenteral (for example, intravenously, intracutaneous, subcutaneous), oral (for example, sucking), through skin (partial), through (transmucosal) of mucous membrane, and rectal administration.Can comprise following ingredients for the solution or the suspension that are used for parenteral, intracutaneous or subcutaneous administration: aseptic thinner, such as water, salt brine solution, stationary state oils (fixed oils), polyoxyethylene glycol, glycerine, propylene glycol or other the synthetic of injection; Antiseptic-germicide is such as phenylcarbinol or methyl benzoic acid ester class (methyl parabens); Antioxidant is such as xitix or sodium bisulfite; Sequestrant is such as ethylenediamine tetraacetic acid (EDTA); Damping fluid is such as acetate, Citrate trianion or phosphoric acid salt; And the reagent that is used to regulate isotonicity (tonicity), such as sodium-chlor or dextrose.PH can adjust with acid or alkali, such as spirit of salt or sodium hydroxide.Parenteral goods can be installed in ampoule (ampoules), disposable syringe or by in the made multiple dose jar (multiple dose vials) of glass or plastics.

The pharmaceutical composition that is fit to injection comprises aseptic aqueous solution (it is a water soluble) or dispersion agent, and confession is used for the sterilized powder of the temporary preparation of aseptic Injectable solution or dispersion agent.With regard to intravenously was used, the carrier that is fit to comprised normal saline solution, bacteriostatic water (bacteriostatic water), Cremophor EL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS).Under any circumstance, said composition must be aseptic, and should be mobile so that can easily inject.It must be stable under the situation of making and storing, and should protectedly prevent the pollution of microorganism (such as bacterium and fungi).Carrier can be solvent or dispersion medium, and it for example comprises, water, ethanol, polyvalent alcohol (polyol) [for example, glycerine, propylene glycol and liquid polyethylene glycol, and similar thing], with and the mixture that is fit to.Suitable fluidity (fluidity) can be kept, for example, by use coating (such as Yelkin TTS), by keeping needed size of particles (if situation of dispersion agent), and by using tensio-active agent.Prevent that action of microorganisms from can be reached by different antibiotic and anti-mycotic agent [for example, benzoates (parabens), butylene-chlorohydrin, phenol, xitix, lucky Metro sand (thimerosal), and similar thing].In many cases, preferably in said composition, comprise isotonic agent (isotonic agents), for example, carbohydrate, polyvalent alcohol (such as N.F,USP MANNITOL, Sorbitol Powder), sodium-chlor.The absorption that prolongs Injectable composition can be caused by comprise the reagent [for example, aluminum monostearate and gelatin] that postpones absorption in said composition.

Aseptic Injectable solution can prepare by a kind of or its combination in the polypeptide of the aequum in the appropriate solvent and the mentioned component is merged, if necessary, and then filtration sterilization.Generally speaking, dispersion liquid is produced out by this polypeptide being incorporated into to containing in the sterile carrier that basic dispersion medium and required other come from composition listed above.In the case for the sterilized powder that is used for preparing aseptic Injectable solution, preferred manufacturing procedure is vacuum-drying and lyophilize, and this method produces from its previous sterile filtration solution to be had activeconstituents and add any extra powder of desiring composition.

Oral compositions generally includes inert diluent or edible carrier.They can be encapsulated in the gelatine capsule or be compressed into tablet.Reach the purpose that oral administration is used for asking, this polypeptide can mix vehicle and be used with tablet, tablet (troches) or capsular form.Oral compositions also can use liquid vehicle and be produced out and be provided as collutory (mouthwash), and wherein the compound in this liquid vehicle is used orally and send rustle, and spues or swallow.Pharmaceutically the tackiness agent of Shi Heing (binding agents) with and/or Adjuvanting material can be included a part that becomes said composition.Tablet, pill, capsule, tablet and similar thing can comprise any one in the following composition, or compound with similar quality: tackiness agent (binder), as crystallite shape Mierocrystalline cellulose (microcrystallinecellulose), tragacanth gum (gum tragacanth) or gelatin; Vehicle is as starch or lactose; Disintegrating agent is such as alginic acid, Primogel or W-Gum; Lubricant is as Magnesium Stearate or Sterotes; Glidant (glidant) is as colloidal silicon-dioxide (colloidal silicondioxide); Sweeting agent (sweetening agent) is as sucrose or asccharin; Or seasonings, as peppermint (peppermint), wintergreen oil (methyl salicylate) or orange fragrance (orangeflavoring).In order to use via sucking, this compound with one come from the pressurized vessel that comprises suitable propelling agent (propellant) [for example, gas (as carbonic acid gas)] or atomizer (nebulizer) or divider aerosol spray (aerosol spray) form and be transferred.

Using of general also can be by reaching through mucous membrane or through the method for skin.For through mucous membrane or applied dermally, the permeate agent (penetrant) that is suitable for desiring the barrier that permeated is used in this prescription.These permeate agents normally in this area know clearly and know, and, for example comprise stain remover, cholate and fudidic acid (fusidic acid) derivative for the purpose of mucosal administration.Mucosal administration can be reached via using nasal spray or suppository (suppositories).For applied dermally, active compound is usually known as knowing clearly in this area is formulated into ointment (ointment), ointment (salves), gel or newborn white (creams).

Said composition also [for example can be prepared to suppository, contain traditional suppository base, such as theobroma oil (cocoa butter) and other glyceryl ester] or the form of stable enema (retentionenemas) carry (rectal delivery) for being used for rectum.

In one embodiment, prepare this polypeptide can protect polypeptide to resist the carrier of getting rid of fast from health (as the sustained release prescription), it comprises implant and microcapsule delivery system (microencapsulated delivery systems).

Biology is decomposable, the polymkeric substance of biocompatibility, such as ethylene vinyl acetate (ethylene vinyl acetate), polyanhydrides (polyanhydrides), polyoxyethylene glycol acid (polyglycolic acid), collagen protein, poe (polyorthoesters), and poly(lactic acid) can be used.The method for preparing these prescriptions will be obviously as can be known for those skilled in the art.These materials also can be obtained from Alza company and NovaPharmaceuticals company commercially.Liposome suspension (comprise target and have liposome at the cells infected of the monoclonal antibody of virus antigen) also can be used as pharmaceutically acceptable carrier.These can be according to the know clearly method known (for example, at United States Patent (USP) the 4th, 522, described in No. 811) and be produced out of those skilled in the art.

With oral or parenteral composition be mixed with dosage unit form (dosage unit form) for easily use with the dosage consistence be particularly advantageous.As used herein, " dosage unit form " means the unit that physically separates of the single dose (unitary dosages) that is suitable as the individuality that will be treated; What each unit comprised pre-determined quantity is calculated the active compound produce the treatment effectiveness of being desired with needed pharmacy supporting agent.Specified and directly depended on the peculiar property of this polypeptide and the special treatment effectiveness of desiring to be reached as the specification of dosage unit form of the present invention, and the compound technical inherent limitations that is used for the treatment of this individual peptide species.

Polypeptide of the present invention can be inserted in the carrier and be used as gene therapy vector.Gene therapy vector can pass through, for example, intravenous injection, topical application (United States Patent (USP) the 5th, 328, No. 470) or three-dimensional location (stereotactic) injection (referring to, for example, Chen et al. (1994) Proc.Natl.Acad.Sci.USA 91:3054-3057) be transported in the individuality.The pharmacy goods of gene therapy vector can be included in the gene therapy vector in the acceptable diluent, maybe can comprise to embed the sustained-release matrix that gene delivery carrier is arranged.Perhaps, wherein complete gene delivery carrier (for example, retrovirus vector) can be complete from reconstitution cell manufactured come out, these pharmacy goods can comprise the cell of one or more these gene delivery systems of generation.

This pharmaceutical composition can be included in container, packing or divider with using indication.

The present invention further provides the method for a kind of TNF of treatment and IL-1 dependent form disease, it comprises has the individuality of needs to use the composition of the present invention of significant quantity to one." TNF and IL-1 dependent form disease " means the active diseases associated of a kind of and unusual gene expression dose or TNF or IL-1.The example of this kind disease comprises, but non-being limited to, acute and chronic inflammatory diseases [for example, the inflammatory conditions in joint, such as osteoarthritis, chronic eczema sacroiliitis (psoriatic arthritis) with and/or rheumatoid arthritis]; Psoriasis; Acute hepatitis; Cardiovascular disorder; The brain injury that wound caused; Epilepsy; Hemorrhage or apoplexy; And graft resistivity disease.

It is to need treatment TNF and IL-1 dependent form disease that the individuality that will be treated can be accredited as.The individuality of this kind of needs of evaluation treatment can be nursed professional's suggestion according to individual or health, and can be subjective (for example, suggestion) or objective (for example, can measure by test or diagnostic method)." treatment " this speech be defined as with cure (cure), alleviate (alleviate), relax (relieve), correct (remedy), prevention (prevent) or improve (ameliorate) disease, this disease symptom, give individual application of substances inferior to the morbid state of this disease or the purpose that is tending towards the tendency (predisposition) of this disease." significant quantity " is the amount that can produce the result who is medically desired as described herein in the individuality of being treated.This result who is medically desired can be objectively (that is, can measure by some test or mark) or subjective (that is individuality gives the indication or the impression of an effectiveness).

The significant quantity of composition of the present invention is per two weeks 1 to 4 time, everyone 2.5 and 300mg between.This significant quantity can be any specific quantity (specific amount) that drops in the above-mentioned scope.This significant quantity can be applicable to single therapy (monotherapy) or combined therapy (combination therapy) is treated TNF and IL-1 dependent form disease.What those skilled in the art will understand that is possible need to be below or above dosage listed above.[for example, a Mammals (such as the mankind)] significant quantity and treatment plan will be decided on various factor, comprise the activity of special extract or its employed composition at the individuality of any one uniqueness; Age; Body weight; The general health situation; Sex; Diet; Time of application; Discharge rate; Drug regimen; Disease, the severity of symptom or symptom and development; Individual processing for disease, symptom or symptom; And treatment doctor or veterinarian's suggestion.

Following embodiment only is for illustrating, but not limits remaining disclosure by any way.Do not need further to describe in detail, believe that those skilled in the art can utilize the present invention to reach most complete degree based on description herein.All are put forward the open case of drawing herein and incorporate this case into its integral body in view of the above and think bibliography.

Embodiment

Embodiment 1

The construction and expression of TNFRII-Fc-IL-1ra and control group TNFRII-Fc

Construction described in SEQ ID NO:1,6 is successfully made up, is checked order and expressed in mammalian cell strain.Natural signals-human TNF RII extracellular domain-IgG 1 Fc gene-human IL-1ra (SEQ ID NO:1) and the natural signals-expression of human TNF RII extracellular domain-IgG 1 Fc gene (SEQ ID NO:6) in the substratum that does not contain serum of 24-hole culture plate tired (titers) respectively for 50mg to 100mg/L (Fig. 1).TNFRII-Fc-IL-1ra has higher expression [estimating out by the direct Coomassie blue protein staining to conditioned medium] to be found in our nearest production colony screening experiment than TNFRII-Fc in the CHOK1 cell.

Sequence table

(1) general information:

(i) applicant: favour look for cosmos (Hui, Mizhou)

(ii) denomination of invention: the method for a kind of treatment TNF of novelty and IL-1 dependent form related application.

(iii) sequence number: 7

(iv) mailing address:

(a) Amprotien company

(b) street: 355N Lantana Street#220

(c) city: Camarillo

(d) state: California

(e) country: the U.S.

(f)ZIP：93010

(2) information of SEQ ID NO:1:

(i) sequence signature: (A) length: 1923bp; (B) type: nucleic acid; (C) chain (Strandedness); Strand; (D) topology (Topology): wire.

(ii) molecule type (Molecular Type): cDNA

(iii) antisense: not

(iv) primary source: human (homo sapiens)

(v) direct sources: synthetic

(vi) feature: the encoding sequence of total length

(vii) feature: signalase 11-66

(viii) sequence explanation: SEQ ID NO:1 (full length nucleotide that does not have the coding TNFRII-Fc-IL-Lra sequence of terminator codon)

atggcgcccgtcgccgtctgggccgcgctggccgtcggactggagctctgggctgcggcgcacgccttgcccgcc

caggtggcatttacaccctacgccccggagcccgggagcacatgccggctcagagaatactatgaccagacagctc

agatgtgctgcagcaaatgctcgccgggccaacatgcaaaagtcttctgtaccaagacctcggacaccgtgtgtgact

cctgtgaggacagcacatacacccagctctggaactgggttcccgagtgcttgagctgtggctcccgctgtagctctg

accaggtggaaactcaagcctgcactcgggaacagaaccgcatctgcacctgcaggcccggctggtactgcgcgc

tgagcaagcaggaggggtgccggctgtgcgcgccgctgcgcaagtgccgcccgggcttcggcgtggccagacca

ggaactgaaacatcagacgtggtgtgcaagccctgtgccccggggacgttctccaacacgacttcatccacggatatt

tgcaggccccaccagatctgtaacgtggtggccatccctgggaatgcaagcatggatgcagtctgcacgtccacgtc

ccccacccggagtatggccccaggggcagtacacttaccccagccagtgtccacacgatcccaacacacgcagcc

aactccagaacccagcactgctccaagcacctccttcctgctcccaatgggccccagccccccagctgaagggagc

actggcgacgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggac

cgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtgg

tggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagac

aaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggct

gaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcc

aaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcag

cctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaa

caactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaaga

gcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagag

cctctccctgtctccgggtaaacgaccctctgggagaaaatccagcaagatgcaagccttcagaatctgggatgttaa

ccagaagaccttctatctgaggaacaaccaactagttgctggatacttgcaaggaccaaatgtcaatttaaaagaaaag

atagatgtggtacccattgagcctcatgctctgttcttgggaatccatggagggaagatgtgcctgtcctgtgtcaagtct

ggtgatgagaccagactccagctggaggcagttaacatcactgacctgagcgagaacagaaagcaggacaagcgc

ttcgccttcatccgctcagacagtggccccaccaccagttttgagtctgccgcctgccccggttggttcctctgcacag

cgatggaagctgaccagcccgtcagcctcaccaatatgcctgacgaaggcgtcatggtcaccaaattctacttccag

gaggacgag

(2) information of SEQ ID NO:2:

(i) sequence signature: (A) length: 619 amino acid; (B) type: amino acid; (C) topology: wire

(ii) molecule type: protein

(iii) sequence explanation: SEQ ID NO:2 (through the sophisticated TNFRII-Fc-IL-1ra aminoacid sequence of translation)

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTV

CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYC

ALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDIC

RPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPE

PSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDLAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKRPSGRK

SSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLKEKIDVVPIEPHALFL

GIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSF

ESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE

(2) information of SEQ ID NO:3: (aminoacid sequence of sophisticated TNFRII extracellular domain)

(i) sequence signature: (A) length: 235 amino acid; (B) type: amino acid; (C) topology: wire

(ii) molecule type: protein

(iii) sequence explanation: SEQ ID NO:3 (aminoacid sequence of sophisticated TNFRII extracellular domain)

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTV

CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYC

ALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDIC

RPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPE

PSTAPSTSFLLPMGPSPPAEGSTGD

(2) information of SEQ ID NO:4: (the segmental aminoacid sequence of human immunoglobulin Fc)

(i) sequence signature: (A) length: 232 amino acid; (B) type: amino acid; (C) topology: wire

(ii) molecule type: protein

(iii) sequence explanation: SEQ ID NO:4 (the segmental aminoacid sequence of human immunoglobulin Fc)

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE

DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPG

(2) information of SEQ ID NO:5: (sophisticated IL-1ra aminoacid sequence)

(i) sequence signature: (A) length: 152 amino acid; (B) type: amino acid; (C) topology: wire

(ii) molecule type: protein

(iii) sequence explanation: SEQ ID NO:5 (sophisticated IL-1ra aminoacid sequence)

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLKEKIDVVPIE

PHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDS

GPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE

(2) information of SEQ ID NO:6:

(i) sequence signature: (A) length: 1467bp; (B) type: nucleic acid; (C) chain (Strandedness): strand; (D) topology: wire

(ii) molecule type: cDNA

(iii) antisense: not

(iv) primary source: human (homo sapiens)

(v) direct sources: synthetic

(vi) feature: the encoding sequence of total length

(vii) feature: signalase 11-66

(viii) sequence explanation: SEQ ID NO:6 (full length nucleotide that does not have the coding TNFRII-Fc sequence of terminator codon)

atggcgcccgtcgccgtctgggccgcgctggccgtcggactggagctctgggctgcggcgcacgccttgcccgcccag

gtggcatttacaccctacgccccggagcccgggagcacatgccggctcagagaatactatgaccagacagctcagatgtg

ctgcagcaaatgctcgccgggccaacatgcaaaagtcttctgtaccaagacctcggacaccgtgtgtgactcctgtgagga

cagcacatacacccagctctggaactgggttcccgagtgcttgagctgtggctcccgctgtagctctgaccaggtggaaac

tcaagcctgcactcgggaacagaaccgcatctgcacctgcaggcccggctggtactgcgcgctgagcaagcaggaggg

gtgccggctgtgcgcgccgctgcgcaagtgccgcccgggcttcggcgtggccagaccaggaactgaaacatcagacgt

ggtgtgcaagccctgtgccccggggacgttctccaacacgacttcatccacggatatttgcaggccccaccagatctgtaa

cgtggtggccatccctgggaatgcaagcatggatgcagtctgcacgtccacgtcccccacccggagtatggccccaggg

gcagtacacttaccccagccagtgtccacacgatcccaacacacgcagccaactccagaacccagcactgctccaagca

cctccttcctgctcccaatgggccccagccccccagctgaagggagcactggcgacgagcccaaatcttgtgacaaaact

cacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacac

cctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaact

ggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtg

gtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctccc

agcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccg

ggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtggg

agagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagc

aagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaacca

ctacacgcagaagagcctctccctgtctccgggtaaa

(2) information of SEQ ID NO:7:

(i) sequence signature: (A) length: 467 amino acid; (B) type: amino acid; (C) topology: wire

(ii) molecule type: protein

(iii) sequence explanation: SEQ ID NO:7 (through the sophisticated TNFRII-Fc aminoacid sequence of translation)

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTV

CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYC

ALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDIC

RPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPE

PSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Embodiment 2

The amplification of TNFRII-Fc-IL-1ra and control group TNFRII-Fc (Scale up) and purifying

Cell line is the suspension that does not contain serum in CHO-CD4 culture medium (Irvine Scientific) and homemade culture medium (in-house feed medium), and is exaggerated with rotating in the bottle (roller bottles) at 3 liters bioreactor (Eplikon). The two can be produced at business level (in 3 liters bioreactor at least 180mg/L) TNFRII-Fc-IL-1ra (SEQ ID NO:2) and control group TNFRII-Fc (SEQ ID NO:7). These protein are by protein-A Direct Acquisition method, then are purified out with ion-exchange and hydrophobic chromatography (the 2nd, 3 figure). The protein of large-scale purification is formulated, freeze drying and SEC-HPLC analyze (the 4th figure).

Embodiment 3

Cryodesiccated protein then is applied to TNF α combination and IL-1 receptor binding assay

For the IL-1 receptor binding assay, recombinant human IL-1 receptor extracellular structural domain at first uses mammalian hosts in inside (in house) and is generated.TNFRII-Fc-IL-1ra, negative contrast TNFRII-Fc and over against being coated in the 96 hole culture plates according to IL-1ra (Kineret).Then, the IL-1 acceptor by at 37 ℃ of incubations to carry out combination.This is used in combination the anti-people IL-1 of rabbit receptor extracellular domain antibodies, is then detected out with the goat anti-rabbit igg that is conjugated with HRP.The 5th figure demonstrates TNFRII-Fc-IL-1ra and IL-1ra (Kineret), and the two is bonded to the IL-1 acceptor, and TNFRII-Fc (Enbrel) does not have combination.

For TNF α binding analysis, reorganization TNF α is coated in the 96 hole culture plates.TNFRII-Fc-IL-1ra is over against following at 37 ℃ of incubations to carry out combination according to TNFRII-Fc (Enbrel) and negative contrast Tie2 (ANG-1 receptor extracellular structural domain)-Fc.This combination is detected out by the anti-human IgG Fc antibody that is conjugated with HRP.6th, 7 figure demonstrate TNFRII-Fc-IL-1ra mosaic and TNFRII-Fc the two are bonded to TNF α, and negative contrast Tie2-Fc is not bonded to TNF α.

Embodiment 4

TNFRII-Fc-IL-1ra, contrast TNFRII-Fc (Enbrel), and bioanalysis and the function test of contrast IL-1ra (Kineret)

For the IL-1 neutralization analysis based on cell, IL-1 dependent form D10 cell (ATCC) is used to test I L-1ra (Kineret) and the blocking-up activity of TNFRII-Fc-IL-1ra mosaic antagonism recombinant human IL-1 for the multiplication-stimulating activity of D10 cell.

For the TNF neutralization analysis based on cell, L929 cell (ATCC) is used to test the blocking-up activity of TNFRII to anti-TNF alpha (Biosource International).

Analytical results based on cell is displayed among the 8th to 9 figure.Simultaneously, functional TNFRII-Fc-IL-1ra mosaic is successfully generated.It keeps among TNF α and the IL-1 and activity.Because the essence that its Mammals produced has glycosylation and large-sized fusion molecule, it has the biological existence phase that also will grow than TNFRII-Fc (Enbrel).

Embodiment 5

TNFRII-Fc-IL-1ra mosaic and the 125-I mark and the animal testing that contrast TNFRII-Fc

The Iodogen method is manufactured comes out the TNFRII-Fc-IL-1ra of 125-I mark by using, and is purified out via size exclusion chromatography method (M Hui et al., 1989).The IL-1 receptor binding assay is established out (referring to embodiment 3) by the Mammals reorganization IL-receptor extracellular structural domain that uses homemade fusion.The IL-1 receptors bind is synchronously made comparisons with nonradioactive labeling's TNFRII-Fc (Enbrel) and negative contrast TNFRII-Fc to the TNFRI-Fc-IL-1ra of 125-I mark.

The TNFRII-Fc-IL-1ra that our data presentation goes out the 125-I mark is (table 2) that function is arranged with regard to the IL-1 receptors bind.

¹²⁵The TNFRII-Fc-IL-1ra of-I mark with ¹²⁵The TNFRII-Fc of-I mark (Enbrel) is injected in the mouse model (skin inflammation mice model) of skin inflammation (referring to the below) together.Surprisingly, our result demonstrates it has more distribution (table 3) at inflammation part than TNFRII-Fc.This most possibly is because its IL-1 receptor binding affinity.Simultaneously, TNFRII-Fc also has more distribution at inflammation part, but its degree is lower than TNFRII-Fc-IL-1ra.This can be illustrated by its TNFIL α binding affinity and at the TNF α of the high density of inflammation part.

Processed mouse with 6nmol TPA is coated with the acetone of 200ul consistence ground by ear and develops skin inflammation and last 2 to 3 days.TNFRII-Fc (5 μ g and 10 μ g) and TNFRII-Fc-IL-1ra (2.5 μ g, 5 μ g and 10 μ g) are applied and go through whole skin inflammation between evolution period.Every group of 8 mouse are injected with TNFRII-Fc and TNFRII-Fc-IL-1ra mosaic from 0 to 3 day every day.TNFRII-Fc general ground (intraperitoneal ground) is used and demonstrated in mouse is effective (table 3) on the inhibition skin inflammation symptom.Surprisingly, the TNFRII-Fc-IL-1ra mosaic demonstrates more more effective than TNFRII-Fc, though the effective dose of TNFRII-Fc-IL-1ra is proved significantly also lower (table 4) than TNFRII-Fc.

Animal experiment has been used to assess the characteristic of TNFRII-Fc-IL-1ra mosaic in the arthritis model that collagen protein brought out.Before the mouse consistence ground that gives immunization with the II collagen type (CII) that is assigned in the pig in the Lun Shi adjuvant (completeFreund adjuvant) fully not developed and bringing out property of collagen protein sacroiliitis [collagen-inducedarthritis, (CIA)].Big after immunization 14 to 17 days, the clinical arthritis symptom begins to occur in mouse, has 90 to 100% mouse to the to show serious sacroiliitis in 28 days.Mouse is detected effectiveness to CIA, effective dose and effective dosage frequency (effectivedosing frequency) by intraperitoneal ground injection with TNFRII-Fc, TNFRII-Fc-IL-1ra and negative contrast Fc fragment.Mouse 12 all pins after immunization are assessed arthritic symptom.

TNFRII-Fc (5ug and 10ug) and TNFRII-Fc-IL-1ra (2.5ug, 5ug and 10ug) are applied and go through whole C IA between evolution period.Every group of 8 mouse are from the 0th day to the 35th day, every other day and per 4 days once, injected with TNFRII-Fc and TNFRII-Fc-IL-1ra.TNFRII-Fc general ground (intraperitoneal ground) is used and demonstrated on the symptom that suppresses CIA in mouse is effective (table 5).Surprisingly, TNFRII-Fc-IL-1ra demonstrates more effective than TNFRII-Fc, and has the effective dose and effective dosage frequency (table 5) of remarkable minimizing.

Table 2:IL-1 receptors bind to the 125-I mark with cold TNFRII-Fc-IL-1ra (n=3).

Title	In conjunction with OD (X ± SD)
		TNFRII-Fc-IL-1ra 125-I mark	1.5±0.3
TNFRII-Fc-IL-1ra	1.5±0.2
		TNFRII-Fc(Fnbrel)	0.4±0.3

Table 3: in injection back 4 hours, the TNFRII-Fc-IL-1ra of 125-I mark and TNFRII-Fc (Enbrel) inflammation with uninflamed skin histology in distribution.This distribution is represented as the % (n=6) of the injected dose in every gram tissue.

Handle	Tissue	Injection in every gram tissue
			TNFRII-Fc-IL-1ra 125-I	The skin of inflammation	3.8±0.2
TNFRII-Fc-IL-1ra 125-I	Normal skin	1.5±0.1
			TNFRII-Fc(Enbrel)125-I	The skin of inflammation	2.8±0.2
TNFRII-Fc(Enbrel)125-I	Normal skin	1.4±0.2

Table 4: bringing out the stage of skin inflammation, TNFRII-Fc-IL-1ra, TNFRII-Fc (Enbrel) uses TNFRII-Fc and IL-1ra simultaneously, and bears the effect that the segmental general of contrast Fc is used.Skin inflammation is represented as ear swelling thickness x10-2mm, and (X ± SD)/sickness rate (incidence) (%onset of total animal # at day 3) (n=10).

	5ug	10ug	20ug
				TNFRII-Fc-IL-1ra	4±0.3/100％	3±0.2/100％	3±0.2/100％
TNFRII-Fc(Enbrel)	8±0.2/100％	8±0.2/100％	7±0.2/100％
				TNFRII-Fc+IL-1ra	8±0.2/100％	7±0.2/100％	7±0.2/100％
Fc fragment (control group)	16±0.2/100％	15±0.2/100％	16±0.3/100％

Table 5: bringing out the stage that sacroiliitis begins, TNFRII-Fc-IL-1ra, TNFRII-Fc (Enbrel) uses TNFRII-Fc and IL-1ra simultaneously, and bears the effect that the segmental general of contrast Fc is used.Arthriticly initially be represented as that initial day (X ± SD)/sickness rate (% of whole animal number middle-jiao yang, function of the spleen and stomach) (n=10).

	2.5ug	5ug	10ug
				TNFRII-Fc-IL-1ra	27±0.3/100％	28±0.2/100％	32±0.2/100％
TNFRII-Fc(Enbrel)	24±0.2/100％	24±0.2/100％	24±0.2/100％
				TNFRII-Fc+IL-1ra	24±0.2/100％	25±0.2/100％	25±0.2/100％
Fc fragment (control group)	18±0.2/100％	19±0.2/100％	18±0.3/100％

All disclosed features in this specification sheets can be bonded in the middle of any combination.Each disclosed feature in this specification sheets can be applied identical, of equal value or the similar purpose further feature replaces.Therefore, unless otherwise indicated, each disclosed feature only is the Equivalent of broad sense series (generic series) or an example of similar features.

It seems from top description, those skilled in the art can easily know essential feature of the present invention, and do not departing under its spirit and the scope, can make various variation of the present invention and modification, so that it is adapted to various use and condition.Therefore, other embodiment also drops in the scope of following claims.

Claims (26)

1. chimeric polyeptides, it comprises:

(1) TNF neutralizing agent structural domain;

(2) IL-1 receptor antagonist structural domains; And

(3) dimerization structural domains,

Wherein these three structural domains are operably connected.

2. chimeric polyeptides as claimed in claim 1, wherein this TNF neutralizing agent structural domain comprises the structural domain in conjunction with Mammals TNF or IL-6.

3. chimeric polyeptides as claimed in claim 2, wherein this TNF neutralizing agent structural domain comprises Mammals TNFR's or Mammals IL-6 acceptor extracellular domain or its function equivalent.

4. chimeric polyeptides as claimed in claim 3, wherein this Mammals TNFR is TNFRII or TNFRI.

5. chimeric polyeptides as claimed in claim 3, wherein this Mammals TNFR is human TNF RII.

6. chimeric polyeptides as claimed in claim 1, wherein this IL-1 receptor antagonist structural domain comprises IL-1ra or its function equivalent.

7. chimeric polyeptides as claimed in claim 6, wherein this IL-1ra is by glycosylated Mammals polypeptide.

8. chimeric polyeptides as claimed in claim 1, wherein this dimerization structural domain comprises human Ig Fc fragment.

9. chimeric polyeptides as claimed in claim 8 should mankind Ig Fc fragment be an IgG1 Fc fragment wherein.

10. chimeric polyeptides as claimed in claim 1, wherein this chimeric polyeptides is held to C-end from N-and is comprised: a TNF neutralizing agent structural domain, a dimerization structural domain and an IL-1 receptor antagonist structural domain; Or their function equivalent.

11. as the chimeric polyeptides of claim 10, wherein this chimeric polyeptides comprises: the extracellular domain of a human TNFRII, IgG 1 Fc and IL-1ra; Or their function equivalent.

12. as the chimeric polyeptides of claim 11, wherein this chimeric polyeptides comprises SEQ ID NO:2.

13. polynucleotide, it comprises the sequence of the chimeric polyeptides of the claim 1 of encoding.

14. a cell, it comprises the polynucleotide as claim 13.

15. as the cell of claim 14, wherein this cell is mammalian cell, bacterial cell, yeast cell, insect cell or vegetable cell.

16. as the cell of claim 15, wherein this cell is Chinese hamster ovary celI or NSO cell or SP/2/0 cell.

17. a composition, it comprises the chimeric polyeptides and the pharmaceutically acceptable carrier of claim 1.

18. a composition, it comprises the polynucleotide and the pharmaceutically acceptable carrier of claim 13.

19. the method in order to treatment TNF and IL-1 dependent form disease, it comprises needs the individuality of this treatment to use the composition of the claim 17 of significant quantity to one.

20. as the method for claim 19, wherein this disease is a diseases associated with inflammation.

21. as the method for claim 20, wherein this diseases associated with inflammation is rheumatoid arthritis or psoriasis.

22. the method in order to treatment TNF and IL-1 dependent form disease, it comprises needs the individuality of this treatment to use the composition of the claim 18 of significant quantity to one.

23. as the method for claim 22, wherein this disease is a diseases associated with inflammation.

24. as the method for claim 23, wherein this diseases associated with inflammation is rheumatoid arthritis or psoriasis.

25. a carrier, it comprises the polynucleotide of claim 13.

26. one kind in order to produce the method for polypeptide, it is included under the condition of the expression that allows the polynucleotide encoded polypeptide, with the cell cultures of claim 14 in substratum, and from the institute's cultured cells or the substratum of this cell this polypeptide of purifying.

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