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 Polypeptides and Their Applications

related application

This application claims priority to US Provisional Application No. 60/497,988, filed August 26, 2003, which is hereby incorporated by reference in its entirety.

field of invention

The present invention relates to a chimeric protein therapeutic agent useful in the treatment of diseases such as acute and chronic inflammation.

Background of the invention

Inflammation is the body's defense response to injuries such as those caused by mechanical injury, infection or antigenic stimulation. Inflammatory responses can be manifested pathologically when inflammation is induced by an inappropriate stimulus such as a self-antigen that is expressed in an exaggerated manner or persists after removal of injurious agents.

Two important inflammatory response mediators are tumor necrosis factor (TNF) and interleukin-1 (IL-1). TNF neutralizers and IL-1 antagonists have been used to treat inflammation-dependent diseases.

Tumor necrosis factor-α (TNF-α) and tumor necrosis factor-β (TNF-β) are mammalian secreted proteins capable of inducing a wide variety of effects on a large number of cell types. The great similarity in structure and functional properties of these two cytokines has led to their collective description being "TNF".

TNF proteins initiate their biological effects on cells by binding to specific TNF receptor (TNFR) proteins expressed on the cell membrane of TNF-responsive cells. Two different types of TNFR are known to exist: type I TNFR (TNFRI), which has a molecular weight of about 75 kDa, and type II TNFR (TNFRII), which has a molecular weight of about 55 kDa. TNFRI and TNFRII bind to TNF-alpha and TNF-beta, respectively.

TNF antagonists, such as soluble TNFRs and TNF-binding proteins, bind to TNF and prevent TNF from binding to cell membrane-bound TNF receptors. This protein is used to inhibit the biological activity caused by TNF.

The role of TNF in regulating inflammatory diseases is well established. For indications of TNF-dependent diseases such as rheumatoid arthritis and psoriasis, TNFRII has been proven to be clinically safe and effective.

One of the most potent inflammatory cytokines is IL-1. IL-1 is produced by cells of the macrophage/monocyte lineage and can be produced in two forms: IL-1α and IL-1β. IL-1 proteins initiate their biological effects on cells by binding to specific IL-1 receptors (IL-1R), proteins expressed on the cell membrane of an IL-1 responsive cell.

IL-1 receptor antagonist (IL-1ra) is a human protein that acts as a natural inhibitor of IL-1. IL-1ra binds to cell membrane bound IL-1 receptors and prevents IL-1 from binding to the same IL-1 receptors. This protein has been used to inhibit the biological activity caused by IL-1.

Theoretically, simultaneously neutralizing or blocking two important inflammatory mediators (such as TNF and IL-1) should have the best therapeutic value for the treatment of inflammation-dependent diseases. However, a clinical trial of 242 patients and 24 weeks of co-administration of a soluble TNFRII and an aglycosylated IL-1ra, marketed by Immunex and Amgen, did not improve efficacy and was more effective than using a soluble Monotherapy with TNFRII and IL-1ra resulted in higher rates of infection and neutropenia.

Summary of the invention

The present invention relates to a new chimeric polypeptide for treating TNF and IL-1 dependent diseases. The chimeric polypeptide includes: (1) a TNF neutralizer domain, (2) an IL-1 receptor antagonist domain, and (3) a dimerization domain. The three domains are operably linked to each other. The TNF neutralizer domain can include an extracellular domain of human TNFRII: the IL-1 receptor antagonist domain can include IL-1ra; and the dimerization domain can include a human IgG1 Fc fragment or human Immunoglobulin heavy chain constant region. In particular, the IL-1ra is a glycosylated mammalian polypeptide.

In one embodiment, the chimeric polypeptide comprises, from N-terminus to C-terminus, a TNF neutralizer domain, a dimerization domain and an IL-1 receptor antagonist domain. For example, the chimeric polypeptide can include an extracellular domain of human TNFRII, human IgG1 Fc, and IL-1ra (eg, SEQ ID NO: 2).

In another aspect, the invention features a polynucleotide comprising a sequence encoding a chimeric polypeptide of the invention, and a cell producing the polynucleotide. For example, the cell can be a mammalian cell, such as a CHO cell, NSO cell, and SP2/0 cell. Polynucleotides and cells of the invention can be used to produce chimeric polypeptides of the invention.

"Polynucleotide" or "nucleic acid" means a DNA molecule (eg, cDNA or genomic DNA), an RNA molecule (eg, mRNA), or an analog of DNA or RNA. Analogs of DNA or RNA can be synthesized from nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. An "isolated nucleic acid" is a nucleic acid that is structurally distinct from any naturally occurring nucleic acid or any fragment of a naturally occurring genomic nucleic acid. Thus, the term encompasses, for example: (a) a DNA which has a partial sequence of a naturally occurring genomic DNA molecule, but which is flanked by parts other than those located within the genome of the naturally occurring organism. coding sequences flanking part of a molecule; (b) a nucleic acid that is incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in such a way that the resulting molecule is different from any A naturally occurring vector or genomic DNA; (c) an isolated molecule such as cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide A sequence that is part of a hybrid gene (ie, a gene encoding a fusion protein). Specifically excluded from this definition are nucleic acids present in a mixture of (i) DNA molecules, (ii) transfected cells, or (iii) cell clones, e.g., as in DNA libraries (such as cDNA or genomic DNA libraries). The nucleic acids described above can be used to express fusion proteins of the invention. For this purpose, the nucleic acid can be operably linked to appropriate regulatory sequences to produce an expression vector.

The present invention further provides a composition, which comprises: the chimeric polypeptide or polynucleotide of the present invention, and a pharmaceutically acceptable carrier. The composition can be used to treat TNF and IL-1 dependent diseases.

Also within the scope of this invention is a method of treating TNF and IL-1 dependent diseases by administering to an individual in need thereof an effective amount of a composition of this invention. For example, the disease can be an inflammatory disease such as rheumatoid arthritis or psoriasis.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the detailed description below. Other technical features, objects and advantages of the present invention become apparent from the detailed description and claims.

Description of drawings

Figure 1: CHO cell clones of TNFRII-Fc and TNFRII-Fc-IL-1ra chimeras produced at passage 1: expression in 24-well culture plates in serum-free medium; direct Coomasie blue (Coomasie blue) ) protein staining; all recombinant proteins are visible in the range of 0.5 to 1.0 μg; load 10 to 15 μL per lane;

Figure 2: Affinity purification of TNFRII-Fc-IL-1ra chimera: reducing and non-reducing conditions of SDS-PAGE; Coomassie blue protein staining;

Figure 3: An example of our problem-solving ability to reduce a degradation problem for TNFRII-Fc-IL-1ra chimera by modifying the first purification step - using TNFRII-Fc (control group) for complete and partial HPLC analysis of degraded TNFRII-Fc-IL-1ra chimera;

Figure 4: SEC-HPLC analysis of TNFRII-Fc-IL-1ra chimera after formulation and lyophilization;

Figure 5: IL-1 receptor binding analysis shows that TNFRII-Fc-IL-1ra chimera binds human IL-1 receptor with higher affinity than the marketed non-glycosylated IL-1ra (Kineret);

Figure 6: TNFα binding assay showing that TNFRH-Fc (homemade) (+ve control group) specifically binds TNFα when compared to -ve control group Tie2-Fc;

Figure 7: TNFα binding assay shows that, similar to TNFRII-Fc (Figure 6), the TNFRII-Fc-IL-1ra chimera specifically binds TNFα;

Figure 8: Cell-based TNFα neutralization test (neutralization test) shows that, similar to the listed TNFRII-Fc (Enbrel), TNFRII-Fc-IL-1ra chimera neutralizes the killing activity of TNFα for L979 cells ( killing activity); and

Figure 9: Cell-based IL-1 neutralization assays show that both marketed IL-1ra (Kineret) and TNFRII-Fc-IL-1ra chimera neutralize IL-1 biologically against D10 cell proliferation active. As expected, glycosylated IL-1ra had lower in vitro activity than non-glycosylated IL-1ra (Kineret) produced by E. coli.

Detailed description of the invention

The present invention is based on the discovery of a chimeric polypeptide that can be used to treat TNF and IL-1 dependent diseases. The chimeric polypeptide comprises: (1) a TNF neutralizer domain, (2) an IL-1 receptor antagonist domain, and (3) a dimerization domain. The three domains are operably linked to each other. Surprisingly, such chimeric molecules were produced in mammalian hosts at commercial production levels, taking as reference the level of TNFRII-Fc production. It contains intact TNF and IL-1 neutralizing activity after chimerization. In other words, fusion of a large TNFRII-Fc molecule to the N-terminus of IL-1ra does not interfere with IL-1 receptor binding and IL-1 neutralization activity of IL-1ra. Unlike the simultaneous use of TNFRII-Fc and IL-1ra, this chimeric molecule is more widely distributed at the site of inflammation via the systemic route of administration, making it a site-directed TNFRII-Fc. Our results further show that the chimeric molecule produced in mammalian hosts contains glycosylated IL-1ra and has a larger molecular weight than both TNFRII-Fc and IL-1ra. Therefore, it has a longer biological life, and less frequent effective injectable doses. Such a chimeric molecule may have fewer side effects when compared to TNFRII-Fc and when TNFRII-Fc is used with IL-1ra due to its inflammatory site-guiding nature and less effective dose and dose frequency.

"TNF neutralizer domain" means a domain capable of neutralizing TNF, ie, inhibiting the activity of TNF (eg, TNF-α). For example, the TNF neutralizer domain can include the extracellular domain of human TNFRII, the extracellular domain of the IL-6 receptor, an antibody against TNF or a TNF receptor, or an antibody against IL-6 or an IL-6 receptor. Antibody.

TNF neutralizer domains are well known in the art. For example, U.S. Patent No. 5,605,690 discloses that TNF receptors (TNFR) have amino acid sequences that are substantially similar to those of natural mammalian TNF receptors or TNF-binding proteins, and these TNF receptors are able to bind TNF molecules and inhibit TNF binds to TNFR bound to cell membranes.

As mentioned earlier, two different types of TNFR are known to exist: TNFR type I (TNFRI) and TNFR type II (TNFRII). Preferred TNFRs of the invention are soluble forms of TNFRI and TNFRII, and soluble TNF-binding proteins. Soluble TNFR molecules include, for example, analogs or subunits of the native protein having at least 20 amino acids and which exhibit at least some similar biological activity to TNFRI, TNFRII, or a TNF-binding protein. Soluble TNFR constructs lack the transmembrane region (and are secreted from the cell), but retain the ability to bind TNF. Different bioequivalent (bioequivalent) proteins and amino acid analogs have an amino acid sequence corresponding to all of the extracellular regions of a native TNFR (e.g., huTNFRI.DELTA.235, huTNFRI.DELTA.185, and huTNFRI.DELTA.163) or part, or have an amino acid sequence substantially similar to the sequence of amino acid 1-163, amino acid 1-185 or amino acid 1-235 of SEQ ID NO: 1 disclosed in U.S. Patent No. 5,605,690, and when they bind to the TNF ligand is biologically active. Equivalent soluble TNFRs include polypeptides that are altered from these sequences by one or more substitutions, deletions or additions and which retain the ability to bind TNF or inhibit TNF signaling activity via a cell surface bound TNF receptor protein, such as huTNFRI .DELTA.x, wherein x is selected from the group consisting of any one of amino acids 163-235 of SEQ ID NO: 1 disclosed in US Patent No. 5,605,690. Similar deletions can result in muTNFR. Inhibition of TNF signaling activity can be determined by transfecting cells with recombinant TNFR DNAs to obtain recombinant receptor expression. The cells were then exposed to TNF and the resulting metabolic effects were measured. If an effect is due to the action of the ligand, then the recombinant receptor has signaling activity. Exemplary methods for determining whether a polypeptide has signaling activity are disclosed in: 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 J. 5: 2179 (1986) and Chou et. al., J. Biol. Chem. 262: 1842 (1987). Alternatively, primary cells or cell lines expressing endogenous TNF receptors and having a detectable biological response to TNF may also be used.

The nomenclature for "TNFR analogues" as used herein follows the conventional nomenclature by prefixing the protein (e.g., TNFR) with "hu" (for human) or "mu" (for mouse), followed by a DELTA.( To indicate a deletion) and the number of C-terminal amino acids. For example, huTNFR.DELTA.235 means having Asp.sup.235 as the C-terminal amino acid (that is, a polypeptide having the sequence of amino acids 1-235 of SEQ ID NO: 1 disclosed in U.S. Patent No. 5,605,690 ) of human TNFR. In the absence of any human or murine species name, TNFR generally means mammalian TNFR. Likewise, in the absence of any specific designation for deletion mutants, the term TNFR refers to all types of TNFR, including routants and analogs that possess TNFR biological activity.

As used in this specification, a characteristic of TNF receptors, "biological activity" means that a particular molecule shares sufficient amino acid sequence similarity with the embodiments of the invention disclosed herein to be capable of binding measurable amounts of TNF Quantitative, delivering TNF stimulation to cells [eg, as a component of a hybrid receptor construct] or cross-reacting with anti-TNFR antibodies against TNFR of natural origin (ie, non-recombinant). Preferably, biologically active TNF receptors within the scope of the present invention are capable of binding greater than 0.1 nanomoles (nmoles) of TNF per nanomole of receptor, and most preferably, each nanomoles in standard binding assays. Moles of acceptor are greater than 0.5 nmoles of TNF.

In a preferred aspect of the invention, the TNF neutralizing agent is selected from the group consisting of soluble human TNFRI and TNFRII. The pCAV/NOT-TNFR vector containing human TNFRI cDNA clone 1 was used to express and purify soluble human TNFRI. pCAV/NOT-TNFR has been deposited with the American Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.) under the name "pCAV/NOT-TNFR" (deposit number: 68088).

Like most mammalian genes, mammalian TNF receptors are presumably encoded by multi-exon genes. Different mRNA constructs that are due to different mRNA splicing consequences after transcription and share most of the same or similar regions as the cDNA disclosed in US Pat. No. 5,605,690 can also be used.

Other mammalian TNFR cDNAs can be isolated by screening specific mammalian cDNA libraries by cross-species hybridization using an appropriate human TNFR DNA sequence as a probe. Mammalian TNFRs used in the present invention include, for example, primate, human, murine, canine, feline, bovine, ovine, equine, and porcine TNFRs. Mammalian TNFRs can be obtained by isolating TNFR cDNA from mammalian cDNA libraries by cross-species hybridization using a single-stranded cDNA derived from a human TNFR DNA sequence as a hybridization probe.

Functional equivalents of TNFR can be used in the present invention. "Functional equivalent" means a polypeptide derived from a TNFR polypeptide, for example, a fusion protein or a protein with one or more point mutations, insertions, deletions, truncations or combinations thereof. It substantially retains the activity of the TNFR polypeptide, ie, the ability to bind to TNF. The isolated polypeptide may comprise SEQ ID NO: 3 or a fragment of SEQ ID NO: 3, or may be SEQ ID NO: 3 or a fragment of SEQ ID NO: 3.

Primary amino acid structure can be achieved by forming covalent or aggregate conjugates with other chemical moieties such as glycosylation groups, lipids, phosphate, acetyl groups, and the like, or by generating amino acid sequence mutants are modified. Covalent derivatives are prepared by attaching specific functional groups to TNFR amino acid side chains or N- or C-termini. Other TNFR derivatives include covalent or aggregated conjugates of TNFR or fragments thereof plus other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugated peptide can be a signal (or leader) polypeptide sequence in the N-terminal region of the protein, which co-translationally or post-translationally directs the The protein is transferred from its site of synthesis to its functional site inside or outside the cell membrane or cell wall (eg, the yeast alpha-factor leader sequence). TNFR protein fusions may contain peptides (eg, poly-His) added to facilitate purification and identification of TNFR. The amino acid sequence of the TNF receptor can also be linked to the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (Hopp et al., Bio/Technology 6:1204, 1988). The latter sequence is highly antigenic and provides an epitope that is reversibly bound by a specific monoclonal antibody enabling rapid analysis and easy purification of the expressed recombinant protein. This sequence is also specifically cleaved by bovine mucosal enterokinase at residues immediately following Asp-Lys pairing. Fusion proteins capped with this peptide may also be resistant to intracellular degradation in E. coli.

TNFRs associated or unrelated to native forms of glycosylation may also be used. TNFR expressed in yeast or mammalian expression systems (eg, COS-7 cells) may be similar or slightly different from the native molecule in molecular weight and glycosylation pattern depending on the expression system. Expression of TNFR DNAs in bacteria such as E. coli provides aglycosylated molecules. Functional mutant analogs of mammalian TNFR with inactive N-glycosylation sites can be generated by oligonucleotide synthesis and ligation reactions or by site-directed mutagenesis techniques. These analog proteins can be produced in good yields in homogeneous, reducing carbohydrate forms using yeast expression systems. The N-glycosylation sites in eukaryotic proteins are characterized by Amino acid triplet Asn-A ₁ -Z, wherein A ₁ is any amino acid except Pro, and Z is Ser or Thr. In this sequence, Asn provides a side chain amino group for covalent attachment of carbohydrates. This site can be replaced by another amino acid to replace Asn or residue Z, delete Asn or Z, or insert a non-Z amino acid between _A1 and Z, or insert a non-Z amino acid between Asn and _A1 . Amino acids of Asn are removed.

TNFR derivatives can also be obtained by mutation of TNFR or its subunits. As intended herein, a TNFR mutant is a homologous polypeptide to TNFR, but which has an amino acid sequence that differs from native TNFR due to a deletion, insertion or substitution.

Bioequivalent analogs of TNFR proteins can be constructed by, for example, making substitutions of different residues or sequences, or deleting terminal or internal residues or sequences not required for biological activity. For example, cysteine residues can be deleted (eg, Cys ¹⁷⁸ ) or substituted with other amino acids to avoid unwanted or incorrect intramolecular disulfide bond formation due to renaturation. Other mutagenesis methods include modification of adjacent dibasic amino acid residues to enhance expression in yeast systems where KEX2 protease activity is present. In general, substitutions should be made conservatively, ie, the most preferred substituting amino acids are those with similar physiochemical properties to the residue to be substituted. Likewise, when deletion or insertion strategies are employed, the potential impact of deletions or insertions on biological activity should be considered. Substantially similar polypeptide sequences, as defined above, generally comprise the same number of amino acid sequences, although a C-terminal truncation for the purpose of constructing a soluble TNFR will contain fewer amino acid sequences. In order to preserve the biological activity of TNFR, deletions and substitutions will preferably result in homologous or conservative substitution sequences, meaning that a given residue is replaced by a biologically similar residue. Examples of conservative substitutions include substitution of one aliphatic residue for another, as in Ile, Val, Leu, or Ala for each other; or substitution of one polar residue for another, as in Lys with Arg, Glu with Asp , or between Gln and Asn. Other such conservative substitutions, eg, substitutions of entire regions of similar hydrophobic properties, are well known. Furthermore, specific amino acid differences between human, murine and other mammalian TNFRs imply additional conservative substitutions that can be made without altering the basic biological properties of TNFRs.

Subunits of TNFR can be constructed by deleting terminal or internal residues or sequences. Particularly preferred sequences include those in which the transmembrane and intracellular domains of TNFR are deleted or substituted with hydrophilic residues to facilitate secretion of the receptor into the cell culture medium. The resulting protein, called a soluble TNFR molecule, retains the ability to bind TNF. A particularly preferred soluble TNFR construct is TNFRI.DELTA.235 (sequence of amino acids 1-235 of SEQ ID NO: 1 disclosed in U.S. Patent No. 5,605,690), which comprises the entire extracellular domain of TNFRI, terminating in Asp ²³⁵ immediately adjoins the transmembrane region. Additional amino acids can be deleted from the transmembrane region and still retain TNF binding activity. For example, huTNFRI.DELTA.183 comprising the sequence of amino acids 1-183 of SEQ ID NO: 1 disclosed in US Patent No. 5,605,690, and huTNFRI.DELTA.183 comprising SEQ ID NO: 1 disclosed in US Patent No. TNFRI.DELTA.163 of the sequence amino acids 1-163 retains the ability to bind TNF ligands. However, TNFRI.DELTA.142 did not retain the ability to bind TNF ligands. This means that either or both Cys ¹⁵⁷ and Cys ¹⁶³ are required to form an intramolecular disulfide bond for proper TNFRI folding. Cys ¹⁷⁸ , whose deletion does not have any apparent adverse effect on the ability of soluble TNFRI to bind TNF, does not appear to be necessary for proper TNFRI folding. Therefore, any C-terminal deletion to Cys ¹⁶³ would be expected to result in biologically active soluble TNFRI. The present invention contemplates the use of such soluble TNFR constructs corresponding to all or part of the extracellular region of TNFR ending at any amino acid after Cys ¹⁶³ . Other C-terminal deletions (such as TNFRI.DELTA.157) can be made in a suitable manner by cutting the TNFR cDNA with appropriate restriction enzymes and, if necessary, reconstructing the specific sequence with synthetic oligonucleotide linkers Finish. Soluble TNFRs with N-terminal deletions can also be used in the present invention. For example, the N-terminus of TNFRI can be initiated with Leu ¹ , Pro ² , or Ala ³ without significantly affecting the ability of TNFRI to effectively act as a TNF antagonist. The resulting soluble TNFR constructs were then inserted and expressed in appropriate expression vectors and analyzed for the ability to bind TNF.

Mutations in the nucleotide sequence constructed to express the analog TNFR must of course preserve the reading frame of the coding sequence, and preferably will not produce secondary mRNA structures [e.g., loops or hairpins] capable of hybridization ], this secondary mRNA structure will adversely affect the translation of the receptor mRNA. While the location of a mutation can be predetermined, the nature of the mutation itself need not be predetermined. For example, in order to select mutants with optimal properties at a given position, random mutagenesis can be performed at the target codon and the expressed TNFR mutants are screened for the desired activity.

Not all mutations in the nucleotide sequence encoding TNFR will be expressed in the final product, for example, nucleotide substitutions can be made to enhance expression, mainly to avoid secondary structure loops in the transcribed mRNA ( See EPA 75,444A, hereby incorporated by reference), or provide codons that are more readily translated by the chosen host (e.g., known E. coli preferred codons for E. coli expression).

Mutations can be introduced at specific loci by synthesizing oligonucleotides containing mutant sequences flanked by restriction sites capable of ligation to fragments of the native sequence. Following the ligation reaction, the resulting reconstructed sequence encodes an analog with the desired amino acid insertions, substitutions or deletions.

Alternatively, an oligonucleotide-site-specific mutagenesis step can be used to provide an engineered gene with specific codons altered according to desired substitutions, deletions or insertions. Exemplary methods for causing the modifications described above are described by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37: 73, 1985); Craik (Bio Techniques, Jan. 1985, 12-19): Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and US Pat. Nos. 4,518,584 and 4,737,462 disclose suitable techniques and are incorporated herein by reference.

Antibodies against TNF or TNFRs can be used as antibody therapeutics for TNF-dependent diseases. These antibodies contain at their C-terminus an immunoglobulin heavy chain constant region similar to a dimeric IgG Fc fragment and at their N-terminus two TNF or TNFR binding Fab domains.

The activity of these antibodies can be determined using TNF-dependent cells such as L979 cells (ATTC). TNF-dependent cells can be killed by adding effective doses of recombinant TNF[alpha]. This TNF-dependent activity can be neutralized by adding these antibodies to the reaction. The activity of these antibodies can also be determined by using TNF binding assays in 96-well culture plates in vitro.

Soluble IL-6 receptors or antibodies directed against IL-6, or IL-6R with an IgG heavy chain constant region at its C-terminus similar to a dimerized IgG Fc fragment can be used in place of this chimera. Synthesized molecules of TNFRII. The activity of these molecules can be determined by using an IL-6 receptor binding assay in 96-well culture plates in vitro. This activity can also be determined using recombinant IL-6 and IL-6-dependent D10 cells.

"Interleukin-1 receptor antagonist domain" means a domain capable of specifically binding to IL-1 receptor and preventing activation of IL-1 by cellular receptors. Examples of interleukin-1 receptor antagonists include IL-1ra (U.S. Patent No. 6,096,728) and anti-IL-1 receptor monoclonal antibodies (EP 623674), and their functional equivalents (i.e., derived from IL-1 -1ra or anti-IL-1 receptor monoclonal antibody polypeptide), for example, a fusion protein or a protein with one or more point mutations, insertions, deletions, truncations, or combinations thereof. They essentially retain the activity of specifically binding to the IL-1 receptor and prevent activation of the cellular receptor for IL-1. They may comprise SEQ ID NO:5 or a fragment of SEQ ID NO:5. Preferably, the IL-1ra is a glycosylated mammalian polypeptide. The activity of interleukin-1 receptor antagonists can be determined by a cell-based IL-1 neutralization assay using IL-1 dependent D10 cells (see Example 4).

Preferred IL-1ra proteins are disclosed in U.S. Patent No. 5,075,222 (referred to herein as the '222 patent), WO 91/08285, WO 91/17184, AU 9173636, WO 92/16221 and WO 96/22793, among others The disclosure is hereby incorporated by reference into this case. These proteins include glycosylated as well as non-glycosylated IL-1 receptor antagonists.

In particular, three useful IL-1ra types and their variants are disclosed and described in the '222 patent. The first of these, IL-1ra.α, was characterized as a 22-23 kD molecule on SDS-PAGE with an isoelectric point of about 4.8, at about 52 mM NaCl (in Tris buffer, pH 7.6), Eluted from Mono Q FPLC column. The second, IL-1ra.β, characterized as a 22-23 kD protein, eluted from the Mono Q column at 48 mM NaCl. Both IL-1ra.α and IL-1ra.β are glycosylated. The third, IL-1rax, was characterized as a 20kD protein that eluted from the Mono Q column at 48mM NaCl and was non-glycosylated. All three of these inhibitors possess similar functional and immunological activities.

Modified types of IL-1ra are also encompassed by the present invention. As used herein, modified types of IL-1ra include variant polypeptides in which amino acids have been (1) deleted from residues within the amino acid sequence of IL-1ra ("deletion variants"), (2) inserted ( "addition variant"), or (3) is substituted ("substitution variant").

For IL-1ra deletion variants, each polypeptide will typically have a range of from about 1 to 30 residues, more typically about 1 to 10 residues, and most typically about 1 to 5 contiguous residues. range of amino acid sequence deletions. N-terminal, C-terminal and intrasequence deletions are contemplated. Deletions within the IL-1ra amino acid sequence can be made in regions of low homology to sequences of other members of the IL-1 family. Deletions within the IL-1ra amino acid sequence may be made in regions of substantial sequence homology to other members of the IL-1 family and will more likely significantly modify biological activity.

For IL-1ra addition variants, individual polypeptides may include an amino and/or carboxyl terminal fusion ranging in length from 1 residue to 100 or more residues, and single or multiple amino acid residues Intra-sequence insertions within the . Internal additions typically range from about 1 to 10 amino acid residues, more typically about 1 to 5 amino acid residues, and most typically about 1 to 3 amino acid residues.

N-terminal addition variants include the addition of methionine [eg, like an artifact or artifact of direct protein expression in bacterial recombinant cell culture] or additional amino acid residues or sequences. Further examples of amino-terminal insertions include fusion signal sequences and/or other pre-pro sequences to facilitate secretion of proteins from recombinant host cells. Each polypeptide may contain a signal sequence selected for recognition and processing (ie, cleavage by a signal peptidase) by the host cell. For prokaryotic host cells, which do not recognize and process the native IL-1ra signal sequence, each polypeptide may comprise a prokaryotic protein selected from the group consisting of, for example, alkaline phosphatase, penicillinase, or thermostable enterotoxin II leader sequences. Biological signal sequence. In the case of yeast cells, each polypeptide may have a signal sequence selected from the group of, for example, yeast invertase, alpha factor or acid phosphatase leader sequences. For expression in mammalian cells, individual polypeptides may have a native IL-1ra signal sequence, although other mammalian signal sequences may be suitable, eg, sequences derived from other IL-1 family members.

For IL-1ra substitution variants, each such polypeptide may have at least one amino acid residue in IL-1ra that is removed and a different residue inserted in its place. Substitution variants include allelic variants, which are characterized by nucleotide sequence changes that occur naturally in a population of species that may or may not result in amino acid changes. Those skilled in the art can use any known information about the binding or active site of the polypeptide to select possible mutation sites. Exemplary substitution variants are taught in WO 91/17184, WO 92/16221 and WO 96/09323.

One method used to identify amino acid residues or regions for protein mutagenesis is known as "alanine scanning mutagenesis" (Cunningham and Wells (1989), Science, 244:1081-1085, et al. The disclosure is hereby incorporated into this case by reference). In this method, amino acid residues or groups of target residues in proteins (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and neutralized or negatively charged Amino acids (most preferably alanine or polyalanine) are substituted to achieve the interaction of these amino acids with the surrounding aqueous environment inside or outside the cell. Those residues exhibiting functional sensitivity to substitutions are then improved by introducing additional or alternative residues at the substituted positions. Thus, alanine scanning or random mutagenesis can be performed as the positions to introduce amino acid sequence modifications are predetermined, and optimal mutation performance is performed at the given positions, and the resulting variant polypeptides target the optimal all. The combination of desired activity and activity level is screened out.

Positions of greatest interest for substitutional mutagenesis include those found in IL-ra that are substantially different from IL-1ra-like proteins such as IL-1ra in terms of side chain size, charge and/or hydrophobicity. Amino acid positions of different species or other IL-1 family members). Other positions of interest include those where specific IL-1ra residues are identical to these IL-1ra-like proteins. Such positions are generally important for the biological activity of the protein. Initially, these positions were modified via substitutions in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions". If such substitutions result in a change in biological activity, more substantial changes (exemplary substitutions) are introduced and/or other additions/deletions can be made and the resulting polypeptides screened.

Table 1

Conservative modifications to the amino acid sequence of IL-1ra (and corresponding modifications to the encoding nucleic acid sequence) are expected to result in proteins with similar functional and chemical properties. Conversely, substantial modifications in the functional and/or chemical properties of IL-1ra can be accomplished by selecting substitutions that differ significantly in their effectiveness in maintaining: (a) in the substituted region, the structure of the polypeptide backbone (eg, like a folded or helical conformation), (b) the charge and hydrophobicity of the protein at the target position, or (c) the size of the side chain. Naturally occurring residues are divided into the following groups based on common side chain properties:

(1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral and hydrophilic: Cys, Ser, Thr;

(3) acidic: Asp, Glu;

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

(5) Aromatic: Trp, Tyr, Phe; and

(6) Residues affecting chain orientation: Gly, Pro.

Non-conservative substitutions may involve exchanging a member of one of these groups for a member of another. These substituted residues can be introduced into regions of IL-1ra that are homologous or nonhomologous to other IL-1 family members.

Specific mutations in the IL-1ra sequence may involve an unnatural amino acid substitution at the N-terminus, C-terminus, or anywhere in the protein that is modified through the addition of N-linked or O-linked carbohydrates. Such modifications may be of particular utility, such as the addition of amino acids (eg, cysteine), which facilitate attachment of water-soluble polymers to form derivatives. Still further, the sequence of IL-1ra can be modified to add glycosylation sites or to delete N-linked or O-linked glycosylation sites. The asparagine-linked glycosylation recognition site contains a tripeptide sequence that is specifically recognized by the appropriate cellular glycosylase. These triple peptide sequences are Asn-Xaa-Thr or Asn-Xaa-Ser, where Xaa can be any amino acid other than Pro.

In a particular embodiment, the variant is substantially homologous to the amino acid of IL-1ra (SEQ ID NO:5). As used herein, the term "substantially homologous" means a degree of homology which is preferably greater than 70%, more preferably greater than 80%, still more preferably greater than 90%, or most preferably even 95%. As described herein, when 4 gaps can be introduced within 100 amino acids to facilitate alignment, the alignment is based on the amino acid residue in the smaller of the two sequences that is identical to the amino acid residue in the compared sequence Homology percentages are calculated as percentages of homology as described by Dayhoff in Atlas of Protein Sequence and Structure, 5:124 (1972), National Biochemical Research Foundation, Washington, D.C. (the disclosure of which is hereby incorporated by reference in this case) . Those variants of IL-1ra that can be isolated by antibody cross-reactivity to the amino acid sequence of SEQ ID NO: 5, or genes thereof can be identified by hybridizing to the DNA encoding SEQ ID NO: 5 or a fragment of the DNA Variants of IL-1ra that have been isolated are also considered to be substantially homologous.

IL-1ra variants can be prepared by introducing appropriate nucleotide changes into the DNA encoding the IL-1ra variant, or by chemically synthesizing the desired IL-1ra variant in vitro. It will be appreciated by those skilled in the art that many combinations of deletions, insertions and substitutions can be made, provided the final IL-1ra variant is biologically active.

Mutagenesis techniques for substitution, insertion or deletion of one or more selected amino acid residues are well known to those skilled in the art (e.g., U.S. Patent No. 4,518,584, the disclosure of which is incorporated herein by reference) . There are two major variables in constructing each amino acid sequence variant: the location of the mutation and the nature of the mutation. In designing each variant, the location of each mutation and the nature of each mutation will depend on the biochemical property to be modified. Each mutated position can be achieved by, for example, (1) first, substitution with a conserved amino acid species, and then, depending on the result to be achieved, more thorough selection, (2) deletion of the target amino acid residue, or (3) insertion of one or more Amino acid residues adjacent to the identified position are modified individually or sequentially.

"Dimerization domain" means a domain capable of joining two copies of the polypeptide of the present invention into one molecule. For example, a dimerization domain can include a dimerized IgG Fc fragment or human IgG heavy chain constant region. An example of an Fc fragment of this type includes SEQ ID NO:4.

IgG Fc fragments dimerize via the formation of inter-chain disulfide bonds (covalently) by their cysteine residues. Sometimes non-covalent dimerization occurs without involving disulfide bonds. The dimerized IgG Fc fragment in this chimeric molecule can present two functional TNFRII molecules at its N-terminus and two functional IL-1ra molecules at its C-terminus. Such an arrangement increases the chances of receptor/ligand binding in vivo for neutralizing both TNFα and IL-1 receptors.

The activity of covalent dimerization via disulfide bonds can be determined by electrophoresis using reducing and non-reducing SDS-PAGE. When reducing conditions are used, the molecular weight of the protein should be reduced by half. Non-covalent dimerization can be determined by electrophoresis using native and denaturing conditions. When denaturing conditions are used, the molecular weight of the protein should be reduced to half.

In a polypeptide of the invention, the TNF neutralizer domain, dimerization domain and IL-1 receptor antagonist domain are operably linked. As used herein, "operably linked" means that the structural configuration of the polypeptide does not interfere with the activity of each domain, that is, the TNF neutralizer domain retains the ability to neutralize TNF; the interleukin-1 The receptor antagonist domain retains its ability to specifically bind the IL-1 receptor and prevent activation of the cellular receptor for IL-1; and the dimerization domain retains its ability to bind two copies of the polypeptide of the invention The ability to synthesize a molecule capable of presenting two functional TNFRII molecules at its N-terminal and two functional IL-1ra molecules at its C-terminal.

For example, a chimeric polypeptide of the invention may include, from N-terminus to C-terminus, a TNF neutralizer domain, a dimerization domain, and an IL-1 receptor antagonist domain. An example of this class of TNF neutralizer domains includes SEQ ID NO:3. In particular, the chimeric polypeptide comprises an extracellular domain of human TNFRII, human IgG1 Fc, and IL-1ra, an example of such a chimeric polypeptide includes SEQ ID NO:2.

The invention also provides an isolated polynucleotide comprising a DNA sequence encoding a chimeric polypeptide of the invention. Polynucleotides of this type can be constructed using recombinant DNA techniques well known in the art. For example, a polynucleotide of the invention may be a vector comprising a DNA sequence encoding a chimeric polypeptide of the invention. The vector can be used to make the polypeptide.

As used herein, the term "vector" means a polynucleotide capable of transporting another polynucleotide into which it has been ligated. Different types of vectors are well known in the art. See, eg, US Patent No. 6,756,196. One type of vector is a "plasmid," which means a circular double-stranded DNA loop into which additional DNA segments have been ligated. Another type of vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of a host cell after introduction into the host cell and replicate along with the host genome. Furthermore, certain vectors (expression vectors) are capable of directing the expression of genes to which they are operably linked. In recombinant DNA techniques, useful expression vectors generally take the form of plasmids (vectors). However, the invention is intended to include those other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

The recombinant expression vector of the present invention comprises the polynucleotide of the present invention in a form suitable for expressing the polynucleotide in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences (based on the host cell), which is operably linked to the polynucleotide sequence to be expressed. In recombinant expression vectors, "operably linked" is intended to mean that the nucleotide sequence of interest is expressed in a manner that allows expression of the nucleotide sequence (for example, in an in vitro transcription/translation system, or When the vector is introduced into a host cell, in the host cell) is linked to regulatory sequences. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements [eg, polyadenylation signals]. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calf. (1990). Regulatory sequences include those that direct the constitutive expression of a nucleotide sequence in many types of host cells, as well as those that direct the expression of the nucleotide sequence in only certain host cells (e.g., tissue-specific regulation sequence). Those skilled in the art can understand that the design of the expression vector may depend on factors such as the host cell to be transformed, the desired protein expression level and the like. Expression vectors of the invention can be introduced into host cells to produce proteins or polypeptides encoded by polynucleotides as described herein.

The recombinant expression vectors of the present invention can be designed to express the present invention in prokaryotic or eukaryotic cells [for example, bacterial cells (such as E. coli), insect cells (using baculovirus expression vectors), yeast cells or mammalian cells]. A polypeptide invented. Suitable host cells are discussed further in: Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.

In one embodiment, polynucleotides of the invention are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 [Seed (1987) Nature 329:840], pCI (Promega), and pMT2PC [Kaufman et al. (1987) EMBO J. 6:187-195]. When used in mammalian cells, the control functions of the expression vector are usually provided by viral regulatory elements. For example, frequently used promoters are those derived from polyoma, adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17, Sambrook et al.eds., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, a recombinant mammalian expression vector is capable of directing expression of a polynucleotide preferentially in a particular cell type [eg, tissue-specific regulatory elements are used to express the polynucleotide]. Tissue-specific regulatory elements are well known in the art. Non-limiting examples of suitable tissue-specific promoters include: albumin promoter [liver-specific: Pinkert et al. (1987) Genes Dev. 1:268-277]; lymphoid-specific promoter [Calame and Eaton (1988) Adv. Immunol.43:235-275], in particular, T cell receptors [Winoto and Baltimore (1989) EMBO J.8:729-733] and immunoglobulins [Banerji et al. (1983) Promoters of Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748]; neuron-specific promoters [e.g., neurofilament promoter; Byrne and Ruddle (1989) Proc.Natl.Acad .Sci.USA 86:5473-5477]; pancreas-specific promoters [Edlund et al. (1985) Science 230:912-916]; and mammary gland-specific promoters [for example, the milk whey promoter; US Patent No. 4,873,316 and European Application Publication No. 264,166]. Developmentally regulated promoters (developmentally-regulated promoters) are also included, such as the mouse hox promoter [Kesse] and Gruss (1990) Science 249:374-379] and the alpha-fetoprotein promoter [Campes and Tilghman (1989 ) Genes Dev. 3:537-546].

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention or an isolated polynucleotide molecule of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that these terms refer not only to a particular individual cell, but also to the progeny or potential progeny of that cell. Because certain modifications may occur in succeeding generations as a result of mutations or environmental influences, such descended generations may not, in fact, be identical to the parental cells, but are still encompassed by the terms as used herein within the scope of this word.

The host cell can be any prokaryotic or eukaryotic cell. For example, the polypeptide of the present invention can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells [such as Chinese hamster ovary cells (CHO) or NSO cells]. Other suitable host cells are well known to those skilled in the art.

The vector DNA or isolated polynucleotide molecule of the present invention can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various art-recognized techniques for introducing foreign polynucleotides (e.g., DNA) into host cells ), including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (supra) and other laboratory manuals.

With regard to stably transfected mammalian cells, it is known that only a small fraction of cells can integrate foreign DNA into their genome, depending on the expression vector used and the transfection technique. In some cases, the vector DNA is retained by the host cell. In other cases, the host cell does not retain the vector DNA, but only the isolated polynucleotide molecule of the invention brought into it by the vector. In some cases, isolated polynucleotide molecules of the invention are used to transform cells without the use of vectors.

To identify and select these integrants, a gene encoding a selectable marker (eg, resistance to antibiotics) is usually introduced into the host cell along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate. A polynucleotide encoding a selectable marker can be introduced into the host cell in the same vector as encoding the polypeptide of the present invention, or can be introduced in a separate vector. Cells stably transfected with the introduced polynucleotide can be identified by drug selection (eg, cells that have incorporated a selectable marker gene will survive while other cells die).

A host cell of the invention (such as a prokaryotic or eukaryotic host cell in culture) can be used to produce (ie, express) a polypeptide of the invention. Accordingly, the invention further provides methods of using the host cells of the invention to produce polypeptides of the invention. In one embodiment, the method comprises culturing the host cell of the present invention (into which a recombinant expression vector or an isolated polynucleotide molecule encoding a polypeptide of the present invention has been introduced) in a suitable medium, whereby the polypeptide is generated. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.

The polypeptides of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the polypeptide and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and (isotonic) and absorption delaying agents (delaying agents), and the like. The use of such media and agents as pharmaceutically active substances is well known in the art. Its use in the compositions is contemplated in addition to any conventional media or agents incompatible with the active compounds. Additional active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (eg, intravenous, intradermal, subcutaneous), oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous administration may contain the following ingredients: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, Glycerin, propylene glycol, or other synthetic solvents; antimicrobials, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers , such as acetate, citrate, or phosphate; and agents for adjusting tonicity, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be presented in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (which are water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline solution, bacteriostatic water, Cremophor EL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In any event, the composition must be sterile and should be fluid so as to allow easy syringability. It must be stable under the conditions of manufacture and storage and should be protected against the contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size (in the case of dispersants), and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (eg, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). In many cases it will be preferable to include isotonic agents in the compositions, for example, sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption [eg, aluminum monostearate and gelatin].

Sterile injectable solutions can be prepared by incorporating the polypeptide in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, if desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the polypeptide into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder having the active ingredient plus any additional desired powder from a previously sterile-filtered solution thereof. Ingredient powder.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the polypeptides may be incorporated with excipients and administered in the form of tablets, troches or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, wherein the compound in the liquid carrier is used orally and swished and expectorated or swallowed. Pharmaceutically suitable binding agents and/or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth ( gum tragacanth) or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel or cornstarch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal bismuth silicon oxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring. For administration via inhalation, the compound is presented as an aerosol spray from a pressurized container or dispenser containing a suitable propellant (e.g., a gas such as carbon dioxide)] or a nebulizer. form is delivered.

Systemic administration can also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally well known in the art and include, for example, for the purposes of transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are generally formulated into ointments, salves, gels, or creams as well known in the art.

The compositions may also be prepared for rectal delivery in the form of suppositories (eg, containing traditional suppository bases such as cocoa butter and other glycerides) or retention enemas.

In one embodiment, the polypeptide is prepared in a carrier that will protect the polypeptide against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters ), and PLA can be used. Methods for preparation of such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals Corporation. Liposomal suspensions (comprising liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods well known to those skilled in the art (eg, as described in US Patent No. 4,522,811).

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, "dosage unit form" means physically discrete units suitable as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of a pharmaceutical carrier calculated together with the required pharmaceutical carrier. An active compound that produces the desired therapeutic effect. Specifications for the dosage unit forms of the invention are dictated by and are directly dependent upon the unique properties of the polypeptide and the particular therapeutic utility to be achieved, as well as the inherent limitations of the art of compounding such polypeptides for the treatment of individuals.

The polypeptides of the present invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be administered, for example, by intravenous injection, topical administration (US Patent No. 5,328,470), or stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 : 3054-3057) and are transported into a body. Pharmaceutical preparations of gene therapy vectors may comprise the gene therapy vector in an acceptable diluent, or may comprise a sustained release matrix embedded with the gene delivery vector. Alternatively, where complete gene delivery vectors (eg, retroviral vectors) can be produced entirely from recombinant cells, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

The pharmaceutical composition can be contained in a container, pack or dispenser together with instructions for administration.

The present invention further provides a method for treating TNF and IL-1 dependent diseases, which comprises administering an effective amount of the composition of the present invention to an individual in need. "TNF and IL-1 dependent disease" means a disease associated with abnormal gene expression level or activity of TNF or IL-1. Examples of such diseases include, but are not limited to, acute and chronic inflammation [e.g., inflammatory conditions of the joints, such as osteoarthritis, psoriatic arthritis, and/or rheumatoid arthritis]; psoriasis; acute Hepatitis; cardiovascular disease; brain damage from trauma; epilepsy; bleeding or stroke; and graft resistance disease.

An individual to be treated can be identified as being in need of treatment for a TNF and IL-1 dependent disease. Identification of an individual in need of such treatment can be based on the opinion of the individual or a health care professional, and can be subjective (eg, opinion) or objective (eg, measurable by a test or diagnostic method). The word "treat" is defined as curing, alleviating, relieving, remedying, preventing or ameliorating a disease, its symptoms, secondary to the disease A substance is administered to an individual for the purpose of a disease state, or a predisposition to the disease. An "effective amount" is an amount of substance capable of producing a medically desirable result as described herein in the individual being treated. The medically desired result can be objective (ie, measurable by some test or marker) or subjective (ie, the individual gives an indication or perception of an effect).

The effective dose of the composition of the present invention is 1 to 4 times per two weeks, between 2.5 and 300 mg per person. The effective amount can be any specific amount falling within the above range. The effective amount can be applied to monotherapy or combination therapy to treat TNF and IL-1 dependent diseases. Those skilled in the art will appreciate that lower or higher dosages than those recited above may be required. The effective amount and treatment regimen for any one unique individual [e.g., a mammal such as a human] will depend on a variety of factors, including the activity of the particular extract or components used therein; age; body weight; General health; sex; diet; time of administration; excretion rate; drug combination; severity and development of disease, condition or symptom; individual's management of disease, condition or symptom;

The following examples are for illustration only and do not limit the rest of the disclosure in any way. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.

Example

Example 1

Construction and expression of TNFRII-Fc-IL-1ra and control group TNFRII-Fc

The constructs described in SEQ ID NO: 1, 6 have been successfully constructed, sequenced and expressed in mammalian cell lines. Natural signal-human TNFRII extracellular domain-human IgG1 Fc gene-human IL-1ra (SEQ ID NO: 1) and natural signal-human TNFRII extracellular domain-human IgG1 Fc gene (SEQ ID NO: 6) at 24 The expression titers in the serum-free medium of the -well culture plates ranged from 50 mg to 100 mg/L each (Fig. 1). Higher expression of TNFRII-Fc-IL-1ra than TNFRII-Fc in CHOK1 cells [assessed by direct Coomassie blue protein staining of conditioned media] has been identified in our recent production clone screening experiments. Discover.

sequence listing

(1) General information:

(i) Applicant: Hui Mizhou (Hui, Mizhou)

(ii) Title of the invention: a novel method for treating TNF and IL-1 dependent application.

(iii) Number of sequences: 7

(iv) Mailing address:

(a) Amprotien Corporation

(b) Street: 355N Lantana Street#220

(c) City: Camarillo

(d) State: California

(e) Country: United States

(f) ZIP: 93010

(2) Information on SEQ ID NO: 1:

(i) Sequence features: (A) length: 1923bp; (B) type: nucleic acid; (C) strand (Strandedness); single strand; (D) topology: linear.

(ii) Molecular Type: cDNA

(iii) Antisense: No

(iv) Original source: Humans (homo sapiens)

(v) Immediate source: synthetic

(vi) Features: full-length coding sequence

(vii) Features: signal peptide 1-66

(viii) sequence description: SEQ ID NO: 1 (the full-length nucleotide of coding TNFRII-Fc-IL-Lra sequence without stop codon)

atggcgcccgtcgccgtctgggccgcgctggccgtcggactggagctctgggctgcggcgcacgccttgcccgcc

caggtggcatttacaccctacgccccggagcccgggagcacatgccggctcagagaatactatgaccagacagctc

agatgtgctgcagcaaatgctcgccgggccaacatgcaaaagtcttctgtaccaagacctcggacaccgtgtgtgact

cctgtgaggacagcacataacacccagctctggaactgggttcccgagtgcttgagctgtggctcccgctgtagctctg

accaggtggaaactcaagcctgcactcgggaacagaaccgcatctgcacctgcaggcccggctggtactgcgcgc

tgagcaagcaggaggggtgccggctgtgcgcgccgctgcgcaagtgccgcccgggcttcggcgtggccagacca

ggaactgaaacatcagacgtggtgtgcaagccctgtgccccggggacgttctccaacacgacttcatccacggatatt

tgcaggccccaccagatctgtaacgtggtggccatccctgggaatgcaagcatggatgcagtctgcacgtccacgtc

ccccacccggagtatggccccaggggcagtacacttaccccagccagtgtccaacacgatcccaacacacgcagcc

aactccagaacccagcactgctccaagcacctccttcctgctcccaatgggccccagccccccagctgaagggagc

actggcgacgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggac

cgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtgg

tggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagac

aaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggct

gaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagcc

aaagggcagccccgagaaccacaggtgtacacccctgcccccatcccgggatgagctgaccaagaaccaggtcag

cctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaa

caactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaaga

gcaggtggcagcagggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagag

cctctccctgtctccgggtaaacgaccctctgggagaaaatccagcaagatgcaagccttcagaatctgggatgttaa

ccagaagaccttctatctgaggaacaaccaactagttgctggatacttgcaaggaccaaatgtcaatttaaaagaaaag

atagatgtggtacccattgagcctcatgctctgttcttgggaatccatggagggaagatgtgcctgtcctgtgtcaagtct

ggtgatgagaccagactccagctggaggcagttaacatcactgacctgagcgagaacagaaagcaggacaagcgc

ttcgccttcatccgctcagacagtggccccaccaccagttttgagtctgccgcctgccccggttggttcctctgcacag

cgatggaagctgaccagcccgtcagcctcaccaatatgcctgacgaaggcgtcatggtcaccaaattctacttccag

gaggacgag

(2) Information on SEQ ID NO: 2:

(i) Sequence features: (A) length: 619 amino acids; (B) type: amino acids; (C) topology: linear

(ii) Molecule type: protein

(iii) sequence description: SEQ ID NO: 2 (translated mature TNFRII-Fc-IL-1ra amino acid sequence)

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTV

CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYC

ALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDIC

RPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPE

PSTAPSTSFLLPMGGPSPPAEGSTGDEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDLAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKRPSGRK

SSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLKEKIDVVPIEPHALFL

GIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSF

ESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE

(2) Information of SEQ ID NO: 3: (amino acid sequence of mature TNFRII extracellular domain)

(i) Sequence features: (A) length: 235 amino acids; (B) type: amino acids; (C) topology: linear

(ii) Molecule type: protein

(iii) sequence description: SEQ ID NO: 3 (amino acid sequence of mature TNFRII extracellular domain)

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTV

CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYC

ALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDIC

RPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPE

PSTAPSTSFLLPMGGPSPPAEGSTGD

(2) Information of SEQ ID NO: 4: (amino acid sequence of human immunoglobulin Fc fragment)

(i) Sequence features: (A) length: 232 amino acids; (B) type: amino acids; (C) topology: linear

(ii) Molecule type: protein

(iii) Sequence description: SEQ ID NO: 4 (amino acid sequence of human immunoglobulin Fc fragment)

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE

DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPG

(2) Information of SEQ ID NO: 5: (mature IL-1ra amino acid sequence)

(i) Sequence features: (A) length: 152 amino acids; (B) type: amino acids; (C) topology: linear

(ii) Molecule type: protein

(iii) Sequence description: SEQ ID NO: 5 (amino acid sequence of mature IL-1ra)

RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLKEKIDVVPIE

PHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDS

GPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE

(2) Information on SEQ ID NO: 6:

(i) Sequence features: (A) length: 1467bp; (B) type: nucleic acid; (C) strand (Strandedness): single strand; (D) topology: linear

(ii) Molecular type: cDNA

(iii) Antisense: No

(iv) Original source: Humans (homo sapiens)

(v) Immediate source: synthetic

(vi) Features: full-length coding sequence

(vii) Features: signal peptide 1-66

(viii) Sequence description: SEQ ID NO: 6 (full-length nucleotide encoding TNFRII-Fc sequence without stop codon)

atggcgcccgtcgccgtctgggccgcgctggccgtcggactggagctctgggctgcggcgcacgccttgcccgcccag

gtggcatttacaccctacgccccggagcccgggagcacatgccggctcagagaatactatgaccagacagctcagatgtg

ctgcagcaaatgctcgccgggccaacatgcaaaagtcttctgtaccaagacctcggacaccgtgtgtgactcctgtgagga

cagcacatacaccccagctctggaactgggttcccgagtgcttgagctgtggctcccgctgtagctctgaccaggtggaaac

tcaagcctgcactcgggaacagaaccgcatctgcacctgcaggcccggctggtactgcgcgctgagcaagcaggaggg

gtgccggctgtgcgcgccgctgcgcaagtgccgcccgggcttcggcgtggccagaccaggaactgaaacatcagacgt

ggtgtgcaagccctgtgccccggggacgttctccaacacgacttcatccacggatatttgcaggccccaccagatctgtaa

cgtggtggccatccctgggaatgcaagcatggatgcagtctgcacgtccacgtcccccacccggagtatggccccaggg

gcagtacacttaccccagccagtgtccaacacgatcccaacacacgcagccaactccagaacccagcactgctccaagca

cctccttcctgctcccaatgggccccagccccccagctgaagggagcactggcgacgagcccaaatcttgtgacaaaact

cacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacac

cctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaact

ggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtg

gtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctccc

agcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacacccctgcccccatcccg

ggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtggg

agagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagc

aagctcaccgtggacaagagcaggtggcagcagggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaacca

ctacacgcagaagagcctctccctgtctccgggtaaa

(2) Information on SEQ ID NO: 7:

(i) Sequence features: (A) length: 467 amino acids; (B) type: amino acids; (C) topology: linear

(ii) Molecule type: protein

(iii) sequence description: SEQ ID NO: 7 (translated mature TNFRII-Fc amino acid sequence)

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTV

CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYC

ALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDIC

RPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPE

PSTAPSTSFLLPMGGPSPPAEGSTGDEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 2

Scale up and purification of TNFRII-Fc-IL-1ra and control group TNFRII-Fc

Cell lines were serum-free suspensions in CHO-CD4 medium (Irvine Scientific) and in-house feed medium, and were grown in 3-liter bioreactors (Eplikon) and roller bottles (roller bottles) are enlarged. Both TNFRII-Fc-IL-1ra (SEQ ID NO: 2) and the control group TNFRII-Fc (SEQ ID NO: 7) can be produced at commercial levels (at least 180 mg/L in a 3 liter bioreactor) . These proteins were purified by protein-A direct capture followed by ion exchange and hydrophobic chromatography (Fig. 2, 3). A large amount of purified protein was formulated, lyophilized and analyzed by SEC-HPLC (Figure 4).

Example 3

The lyophilized protein was then used in TNFα binding and IL-1 receptor binding assays

For IL-1 receptor binding assays, recombinant human IL-1 receptor extracellular domains were first produced in house using mammalian hosts. TNFRII-Fc-IL-1ra, negative control TNFRII-Fc and positive control IL-1ra (Kineret) have been coated in 96-well culture plates. Next, the IL-1 receptor was incubated at 37°C for binding. The binding was detected using a rabbit anti-human IL-1 receptor extracellular domain antibody followed by goat anti-rabbit IgG conjugated to HRP. Figure 5 shows that both TNFRII-Fc-IL-1ra and IL-1ra (Kineret) bind to the IL-1 receptor, whereas TNFRII-Fc (Enbrel) does not.

For TNFα binding assays, recombinant TNFα was coated in 96-well culture plates. TNFRII-Fc-IL-1ra, positive control TNFRII-Fc (Enbrel) and negative control Tie2 (ANG-1 receptor extracellular domain)-Fc were then incubated at 37°C for binding. The binding was detected by an anti-human IgG Fc antibody conjugated to HRP. Figures 6 and 7 show that both the TNFRII-Fc-IL-1ra chimera and TNFRII-Fc bind to TNFα, while the negative control Tie2-Fc does not bind to TNFα.

Example 4

Bioanalysis and functional testing of TNFRII-Fc-IL-1ra, control TNFRII-Fc (Enbrel), and control IL-1ra (Kineret)

For cell-based IL-1 neutralization assays, IL-1-dependent D10 cells (ATCC) were used to test IL-1ra (Kineret) and TNFRII-Fc-IL-1ra chimera against recombinant human IL-1 for Blocking activity of proliferation stimulating activity of D10 cells.

For cell-based TNF neutralization assays, L929 cells (ATCC) were used to test the blocking activity of TNFRII against TNFα (Biosource International).

The results of the cell-based analysis are shown in Figures 8-9. Meanwhile, functional TNFRII-Fc-IL-1ra chimeras have been successfully generated. It maintains TNFα and IL-1 neutralizing activity. It has a longer biological lifetime than TNFRII-Fc (Enbrel) due to its mammalian nature of glycosylation and large size fusion molecule.

Example 5

125-I labeling and animal testing of TNFRII-Fc-IL-1ra chimera and control TNFRII-Fc

125-I-labeled TNFRII-Fc-IL-1ra was produced using the lodogen method and purified by size exclusion chromatography (M Hui et al., 1989). IL-1 receptor binding assays have been established using homemade fusions of recombinant mammalian IL-receptor extracellular domains (see Example 3). IL-1 receptor binding to 125-I labeled TNFRI-Fc-IL-1ra was compared simultaneously with non-radiolabeled TNFRII-Fc (Enbrel) and negative control TNFRII-Fc.

Our data show that 125-I labeled TNFRII-Fc-IL-1ra is functional in terms of IL-1 receptor binding (Table 2).

¹²⁵ -I-labeled TNFRII-Fc-IL-1ra was injected together with ¹²⁵ -I-labeled TNFRII-Fc (Enbrel) into a skin inflammation mouse model (see below). Surprisingly, our results showed a greater distribution than TNFRII-Fc at sites of inflammation (Table 3). This is most likely due to its IL-1 receptor binding affinity. Meanwhile, TNFRII-Fc was also more distributed in inflammatory sites, but to a lower extent than TNFRII-Fc-IL-1ra. This is explained by its TNFILα binding affinity and the high concentration of TNFα at the site of inflammation.

Mice treated with 6 nmol TPA consistently developed skin inflammation with 200 ul of acetone through the ears for 2 to 3 days. TNFRII-Fc (5 μg and 10 μg) and TNFRII-Fc-IL-1ra (2.5 μg, 5 μg and 10 μg) were administered throughout the development of skin inflammation. Eight mice per group were injected with TNFRII-Fc and TNFRII-Fc-IL-1ra chimera daily from days 0 to 3. Systemic (ip) administration of TNFRII-Fc was shown to be effective in suppressing skin inflammation symptoms in mice (Table 3). Surprisingly, the TNFRII-Fc-IL-1ra chimera was shown to be more effective than TNFRII-Fc, although the effective dose of TNFRII-Fc-IL-1ra was demonstrated to be significantly lower than that of TNFRII-Fc (Table 4).

Animal experiments have been used to evaluate the properties of TNFRII-Fc-IL-1ra chimeras in collagen-induced arthritis models. Mice previously immunized with porcine type II collagen (CII) in complete Freund adjuvant consistently developed collagen-induced arthritis (CIA )]. Clinical symptoms of arthritis began to appear in the mice approximately 14 to 17 days after immunization, with 90 to 100% of the mice exhibiting severe arthritis by day 28. Mice were injected intraperitoneally with TNFRII-Fc, TNFRII-Fc-IL-1ra and negative control Fc fragments to test the effect on CIA, effective dose and effective dosing frequency. Mice were assessed for arthritis symptoms 12 weeks after immunization.

TNFRII-Fc (5 ug and 10 ug) and TNFRII-Fc-IL-1ra (2.5 ug, 5 ug and 10 ug) were administered throughout the period of CIA development. From day 0 to day 35, 8 mice in each group were injected with TNFRII-Fc and TNFRII-Fc-IL-1ra every other day and once every 4 days. Systemic (ip) administration of TNFRII-Fc was shown to be effective in suppressing symptoms of CIA in mice (Table 5). Surprisingly, TNFRII-Fc-IL-1ra was shown to be more efficacious than TNFRII-Fc, with a significantly reduced effective dose and frequency of effective dosing (Table 5).

Table 2: IL-1 receptor binding to 125-I labeled and unlabeled TNFRII-Fc-IL-1ra (n=3).

name Binding OD(X±SD) TNFRII-Fc-IL-1ra 125-I labeling 1.5±0.3 TNFRII-Fc-IL-1ra 1.5±0.2 TNFRII-Fc (Fnbrel) 0.4±0.3

Table 3: Distribution of 125-I labeled TNFRII-Fc-IL-1ra and TNFRII-Fc (Enbrel) in inflamed and non-inflamed skin tissues 4 hours after injection. The distribution is expressed as % of injected dose per gram of tissue (n=6).

deal with organize Injections per gram of tissue TNFRII-Fc-IL-1ra 125-I inflamed skin 3.8±0.2 TNFRII-Fc-IL-1ra 125-I normal skin 1.5±0.1 TNFRII-Fc(Enbrel)125-I inflamed skin 2.8±0.2 TNFRII-Fc(Enbrel)125-I normal skin 1.4±0.2

Table 4: Effects of systemic administration of TNFRII-Fc-IL-1ra, TNFRII-Fc (Enbrel), simultaneous use of TNFRII-Fc and IL-1ra, and negative control Fc fragments during the induction phase of skin inflammation. Skin inflammation was expressed as ear swelling thickness x 10-2 mm (X±SD)/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: Effect of systemic administration of TNFRII-Fc-IL-1ra, TNFRII-Fc (Enbrel), simultaneous use of TNFRII-Fc and IL-1ra, and negative control Fc fragments during the induction phase of the onset of arthritis. Onset of arthritis is expressed as day of onset (X±SD)/incidence (% positive in total number of animals) (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 features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by other features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each disclosed feature is one example only of a generic series of equivalent or similar features.

From the above description, those skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from its spirit and scope, various changes and modifications of the present invention can be made, so that It is adapted to various uses and conditions. Accordingly, other implementations are within the scope of the 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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