JP2008515970A - Chimeric protein - Google Patents

Abstract

Provided is a chimeric protein therapeutic useful for the treatment of various diseases such as inflammation, asthma and cancer.
A first portion that is located at the amino terminus of a fusion protein and specifically binds to and neutralizes a first cytokine or growth factor, and is located at the carboxyl terminus of the fusion protein, specifically a disease. A fusion protein comprising a second portion that binds to a second cytokine receptor that is often abundant at a site (eg, an inflammatory site that is rich in IL-1 receptor). Furthermore, the second part is usually a receptor antagonist (eg an IL-1 receptor antagonist) and functionally equivalent analogues thereof. Also disclosed are nucleic acids encoding fusion proteins, vectors and host cells having the nucleic acids, and related compositions and methods targeting inflammatory diseases and indications that coexist with inflammation.
[Selection figure] None

Description

(Related application)
This patent application includes US Provisional Application No. 60 / 618,476 (filed October 12, 2004), US Provisional Application No. 60 / 628,994 (filed November 17, 2004), and Claims priority of US provisional application filed Feb. 1, 2005 (Title of Invention “IL-1ra as a fusion partner to target angiogenesis”), the disclosure of which is incorporated herein by reference. .

(Technical field)
The present invention relates to chimeric protein therapeutics useful for the treatment of various diseases such as inflammation, asthma and cancer.

  Inflammation is a biodefense response to damage caused by physical injury, infection or antigenic stimulation. Pathologically, the inflammatory response is triggered by an inappropriate stimulus such as a self-antigen and may develop in an abnormal manner or persist for a long time after removal of harmful substances. Inflammation often appears with asthma and angiogenesis-related indications. A number of therapeutic proteins have been developed to inhibit inflammatory responses, treat inflammation-related asthma, and reduce pathological angiogenesis.

  However, many of them are unsatisfactory due to low efficacy, risk of side effects or instability.

  The present invention relates to the use of an IL-1 receptor antagonist (IL-1ra) or a functional equivalent thereof as a fusion partner for a bioactive protein or a therapeutic protein. Examples of bioactive proteins or therapeutic proteins include, but are not limited to, tumor necrosis factor (TNF) neutralizing substance, IL-18 neutralizing substance, IL-4 / IL-13 neutralizing substance, VGEF neutralizing substance, angiopoietin Neutralizing substances and other substances useful for the treatment of inflammation, asthma and angiogenesis-related indications.

  One aspect of the invention features a fusion protein, the fusion protein located at the N-terminus of the fusion protein, a first portion that specifically binds and neutralizes a first cytokine or growth factor, and It is located at the C-terminus of the fusion protein and specifically comprises a second portion that binds to a second cytokine or growth factor receptor (eg, an IL-1 receptor that is abundant at the site of inflammation). These domains are functionally linked, and the first or second cytokine is present in large numbers at the site of inflammation.

  The fusion proteins described herein can be glycosylated. In addition, a linker moiety that joins the first and second moieties can be included. The linker moiety can be dimerized. In one example, the linker moiety includes an Fc fragment of an immunoglobulin or a functional equivalent thereof. Preferably, the immunoglobulin is IgA, IgE, IgD, IgG or IgM. More preferably, the immunoglobulin is IgG or an Fc fragment thereof (eg, SEQ ID NO: 2). The immunoglobulin chain comprises SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 23 or SEQ ID NO: 24, or functional equivalents thereof.

  In the fusion protein described above, the first portion can bind to and neutralize VEGF, angiopoietin, TNF, IL-18, IL-4 or IL-6, or functional equivalents thereof. For example, the first portion specifically comprises an immunoglobulin sequence that binds to and neutralizes VEGF, angiopoietin, TNF, IL-8, IL-4, IL-13 or IgE, or functional equivalents thereof. Including. The first part also contains the sequence of the receptor for VEGF, Angiopoietin, TNF, IL-18, IL-4, IL-13 or IgE (eg SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 15 or SEQ ID NO 19). Can be included.

  In the fusion protein described above, the second portion specifically binds to the IL-1 receptor. The second portion can be an antagonist of IL-1 (eg, a portion comprising the sequence of IL-1ra (SEQ ID NO: 1)) or an analog of a functional equivalent thereof. Accordingly, the fusion proteins include those of SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24 or SEQ ID NO: 25. included. Another aspect of the invention features an isolated nucleic acid comprising a sequence encoding the fusion protein. It can comprise a sequence encoding one of SEQ ID NO: 1 to SEQ ID NO: 25.

  Compositions comprising (i) the fusion protein or nucleic acid encoding it and (ii) a pharmaceutically acceptable carrier are within the scope of the present invention. Also included within the scope of the present invention are methods for modulating the immune response of a patient. The method diagnoses a patient having or at risk for symptoms characterized by excessive inflammatory, immune and angiogenic responses and encodes the patient with an effective amount of the fusion protein or fusion protein Administering a nucleic acid. The patient can be a patient who has undergone or will undergo allogeneic or xenotransplantation. Examples of symptoms include inflammatory diseases, autoimmune diseases, allergic diseases or cancer. Where the symptom is angiogenesis-dependent cancer, a fusion protein comprising SEQ ID NO: 24 is preferred.

  In another aspect, the invention features a method for extending the half-life of a recombinant protein in a patient. The method includes conjugating the recombinant protein to a portion comprising SEQ ID NO: 1 or a functional equivalent thereof to form a fusion protein chimera, which affects the half-life of the fusion protein in the patient's body. The recombinant protein binds to a cytokine or growth factor.

  The invention also features a method for enhancing the efficacy of a recombinant protein in a patient. The method includes linking the recombinant protein to a portion containing SEQ ID NO: 1 or a functional equivalent thereof to form a fusion protein chimera, which affects the efficacy of the fusion protein in a patient. In one embodiment, the fusion protein chimera binds to IL-1 receptor and cytokines or growth factors and simultaneously disables at the patient's inflammatory site or disease site rich in IL-1 receptor. In other embodiments, the fusion protein chimera neutralizes or competitively inhibits the activity of IL-1 and cytokines or growth factors at the site of inflammation in the patient or at a disease site rich in IL-1 receptors. In another aspect, the invention features a method of transporting a therapeutic protein to a target site in a patient, the method comprising linking the therapeutic protein to a portion comprising SEQ ID NO: 1 or a functional equivalent thereof to a fusion protein A chimera and administering the fusion protein chimera to a patient in need thereof. Therapeutic proteins target inflammatory sites that are rich in IL-1 receptors. In one embodiment, the portion comprising SEQ ID NO: 1 or a functional equivalent thereof binds to the IL-1 receptor and the recombinant protein is a therapeutic protein that binds to cytokines or growth factors and is neutralized.

  Isolated polypeptide refers to a polypeptide that is substantially free of naturally occurring related molecules, which is at least 75% pure by dry weight (ie, any value greater than or equal to 75% and less than or equal to 100%). Purity can be measured by any appropriate standard method such as column chromatography, polyacrylamide gel electrophoresis or HPLC. The isolated polypeptide of the present invention may be purified from a natural source (wild type polypeptide) or may be produced by recombinant DNA techniques or chemical synthesis methods.

A nucleic acid refers to 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 preferably is double-stranded DNA. An “isolated nucleic acid” refers to a nucleic acid having a structure that is not identical to any naturally occurring nucleic acid or fragment of any naturally occurring genomic nucleic acid. Thus, the terms include, for example:
(A) DNA having a portion of the sequence of a naturally occurring genomic DNA molecule (provided that none of the coding sequences adjacent to that portion in the genome molecule of a naturally occurring organism is adjacent to it);
(B) a nucleic acid bound to a vector or a prokaryotic or eukaryotic genomic DNA, provided that the resulting molecule is not identical to any naturally occurring vector or genomic DNA;
(C) cDNA, genomic fragments, fragments obtained by polymerase chain reaction (PCR), or isolated molecules such as restriction enzyme degradation products; and (d) part of a hybrid gene (ie, a gene encoding a fusion protein). A recombinant nucleotide sequence comprising
The above nucleic acid can be used for expression of the polypeptide of the present invention. For this purpose, an expression vector can be constructed by operably linking nucleic acids to appropriate control sequences.

  A vector refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. The vector can autonomously replicate or can be integrated into the host DNA. Examples of vectors include plasmids, cosmids or viral vectors. The vector contains the nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably, the vector includes one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed. “Control sequences” include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Control sequences include not only tissue-specific regulatory and / or inducible sequences but also those that induce constitutive expression of nucleotide sequences. The design of the expression vector may depend on factors such as the choice of the host cell to be transformed and the required expression level of the protein or RNA. Expression vectors can be introduced into host cells to produce the polypeptides of the present invention. In addition, host cells containing the above nucleic acids are within the scope of the present invention. Examples include E. coli cells, insect cells (eg, using baculovirus expression vectors), yeast cells or mammalian cells. See, eg, Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA. checking ... For production of the polypeptide of the present invention, host cells can be cultured in a medium under conditions that allow expression of the polypeptide encoded by the nucleic acid of the present invention, and the polypeptide can be purified from the cultured cells or cell culture medium. . Alternatively, the nucleic acid of the present invention can be used for transcription and translation in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

  By “functional equivalent” of a proteinaceous element is meant a polypeptide derivative of the protein, eg, a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins or combinations thereof. It substantially retains the activity of the factor (eg, the ability to bind to a cytokine, growth factor or its receptor). The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

  The present invention is based, at least in part, on IL-Ira or its functional equivalent as a fusion partner to demonstrate the in vivo lifetime and efficacy of many bioactive proteins (eg, anti-inflammatory proteins, anti-asthma proteins and anti-angiogenic proteins). Based on the discovery to extend. Examples of these proteins include tumor necrosis factor (TNF) neutralizing substance, IL-18 neutralizing substance, IL-4 / IL-13 neutralizing substance, VEGF neutralizing substance, and angiopoietin neutralizing substance.

  Protein fusion to the N-terminus of a bioactive protein (especially for large protein fusion partners) often leads to a complete loss of activity. For example, proenzymes and prohormones have no activity by fusing with propeptides at the N-terminus. These pre-digested proenzymes and prohormones become biologically active only after their propeptides are cleaved. Furthermore, fusion with large proteins often leads to a decrease in expression yield. Unexpectedly, IL-1ra fusion proteins can be produced at an industrial level in mammalian derived host cells. Fusion also does not interfere with the binding and neutralizing activity of IL-1ra with the IL-1 receptor, nor does it interfere with the binding and neutralizing activity of the bioactive protein to which it is fused. Unexpectedly, IL-1ra (eg, a glycosylated product derived from a mammal) or a functional equivalent thereof not only prolongs the in vivo life of a bioactive protein, but also causes IL-1 receptor It leads to many inflammatory sites.

IL-1ra
IL-1 is a cytokine produced by macrophage / monocyte lineage cells. It is produced in two forms, IL-1α and IL-1β. IL-1 protein produces its biological effect on cells by specifically binding to the IL-1 receptor (IL-1R). IL-1R is usually expressed on the cell membrane of IL-1 sensitive cells.

  An IL-1 receptor antagonist (IL-1ra) is a human protein that acts as a natural IL-1 inhibitor. IL-1ra is used to suppress the physiological activity produced by IL-1. It binds to the cell membrane bound IL-1 receptor and prevents its binding to IL-1. The majority of IL-1 receptors are expressed at sites of inflammation (Deleuran et al, 1992; Laken VD et al, 1997) and lymphocytes (Dower SK et al, 1990). Therefore, IL-1ra can lead a therapeutic protein fused thereto (for example, a substance neutralizing TNF described later) to an inflammatory site rich in IL-1 receptor. This targeting effect requires a reduction in the effective amount of the therapeutic protein, thus reducing side effects or enhancing efficacy. Furthermore, the synergistic effect of IL-1ra and the fusion partner leads to a higher therapeutic effect than each of the two proteins, alone or in combination, due to (at least some) fusion proteins going to the same site.

  IL-1ra and its functional equivalents can be used in the practice of the present invention. The functional equivalent of IL-1ra refers to a polypeptide derivative of IL-1ra (SEQ ID NO: 1) as described in [Problems to be Solved by the Invention]. It has substantially IL-1ra activity (eg, binding activity to the IL-1 receptor and activity that prevents binding of the IL-1 receptor to IL-1). IL-1ra and its functional equivalents contain at least one interleukin 1 receptor antagonist domain, which can specifically bind to the IL-1 receptor family, and activation of cell receptors by IL-1 and its family Refers to a domain that can interfere with The IL-1 receptor family includes several receptor members. Thus, there are several different IL-1 family agonists and antagonists. These IL-1 antagonists may not necessarily bind to members of the same IL-1 receptor family. Here, IL-1ra is used to denote all IL-1 antagonists that bind to the IL-1 receptor family and / or neutralize the activity of members of the IL-1 family.

  The IL-1ra functional equivalent includes an interleukin 1 receptor antagonist domain. This domain refers to a domain that can specifically bind to the IL-1 receptor family and inhibit activation of cellular receptors for IL-1 and its family. Examples of interleukin 1 receptor antagonists include IL-1ra (US Pat. No. 6,096,728), IL-1 HYl or IL-1 family member 5 (US Pat. No. 6,541,623), IL −1 Hy2 or IL-1 family member 10 (US Pat. No. 6,365,726), IL-1raβ (US Pat. No. 6,399,573), other IL-1 antagonist members and their functional equivalents ( And polypeptides derived from IL-1ra (eg, proteins having one or more point mutations, insertions, deletions, truncations, or combinations thereof). They retain substantially the activity of specifically binding to the IL-1 receptor and inhibiting the activation of cellular receptors for IL-1. They can comprise SEQ ID NO: 1 or fragments of SEQ ID NO: 1. Preferably, IL-1ra is a glycosylated mammalian polypeptide. Interleukin 1 receptor antagonist activity can be measured by cell-based IL-1 neutralization assays using IL-1 dependent D10 cells (see Example 3) and other IL-1 family neutralization assays .

  Preferably, IL-1ra or a functional equivalent thereof is a glycosylated polypeptide. Natural IL-1ra is glycosylated by two N-linked glycosylation sites (US Pat. No. 6,096,728). These two N-linked glycosylation sites are important for the in vivo activity of IL-1ra, in particular its in vivo lifetime, and its binding to serum proteins. Kineret (IL-1ra produced by E. coli) is not post-translationally modified. As a result, it has a significant tendency to bind to human serum proteins and has low efficacy in vivo.

  Antagonist activity of IL-1ra or its functional equivalent is due to cell-based IL-1 neutralization assay using IL-1 dependent D10 cells (see Example 3) and other standard IL-1 families It can be measured by neutralization assay. Fusion of IL-1ra to any proteinaceous substance results in an increase in molecular weight and an in vivo lifetime. Fusion of IL-1ra to other molecules via immunoglobin Fc (eg IgG1 Fc) can further increase the molecular weight. Since immunoglobin Fc has dimerization ability, its presence can double the concentration of fusion protein at the target site.

TNF
Tumor necrosis factor α (TNFα) and tumor necrosis factor β (TNFβ) are mammalian secreted proteins that can induce diverse effects on a variety of cells. These are summarized as “TNF” due to the remarkable similarities in the structure and function of these two cytokines.

  Specific binding of TNF to the TNF receptor (TNFR) expressed on the cell membrane of a TNF-responsive cell produces its physiological effect on the cell. Two different types of TNFRs are known: type I TNFR (TNFRI) (molecular weight of about 55 kilodaltons (kd)), type II TNFR (TNFRII) (molecular weight of about 75 kd). TNFRI and TNFRII bind to TNFα and TNFβ, respectively.

  The role of TNF in inflammatory diseases has been widely elucidated. TNFRII (trade name “Enbrel”) fused to a human IgG1 Fc fragment has been used to treat certain TNF-dependent diseases such as rheumatoid arthritis and psoriasis. Soluble TNFRI (Onecept, Serono) is in clinical trials for the treatment of psoriasis.

  Antagonists of TNF have been identified. These antagonists (eg, soluble TNFRII and TNFRI) bind to TNF and prevent TNF binding to the TNF receptor. Such proteins can be used to suppress biological activity caused by TNF. A protein-based TNF neutralizing material can be fused to IL-1ra or a functional equivalent thereof. Like IL-1, TNF is an important regulator of inflammatory responses. Examples of the TNF neutralizing substance include TNF and functional equivalents thereof. Each of them contains one or more TNF neutralizer domains, which can neutralize TNF, ie inhibit the activity of TNF. The TNF neutralizer domain may include an extracellular domain of human TNFRII, an extracellular domain of TNFRI, or a variable domain of an anti-TNF antibody. For example, the extracellular domain of type II TNF receptor (TNFRII), TNF binding protein 1 (rhTBP-1) or type I TNF receptor type (TNFRI), humanized anti-TNF antibody (eg, Humira®, Abbott Laboratories), and chimeric anti-TNF antibodies (e.g., Remicade of Johnsons & Johnson).

  Because TNFα and IL-1 play a major role in inflammatory diseases, fusions or chimeras of TNF antagonists with IL-1ra or functional equivalents should be used to block the TNFα and IL-1 pathways Therefore, each can be used individually and effectively to treat diseases associated with acute and chronic inflammation. The TNF neutralization activity of the chimeric protein can be measured using TNF-dependent cells (eg, L979 cells (ATTC)). Specifically, TNF-dependent cells can be killed with an effective amount of recombinant TNFα. This TNF-dependent activity can be neutralized by adding these TNF neutralizing substances to the reaction system. The activity of these TNF neutralizing substances can be measured by an in vitro binding test using TNF. A combination of IL-1ra and type I TNF receptors (not type II) has been proposed for the treatment of diseases mediated by TNFα and IL-1. However, the results of a 24-week clinical trial with 242 patients in 2003 were published by Immunex Inc and Amgen Inc, which concluded that the combination of Enbrel and Kineret (individual dose (biweekly Do not increase Enbrel by 25 mg and Kineret by 10 mg daily at a molar ratio of 1:12)), but do not increase efficacy compared to treatment with Enbrel or Kineret single administration, rather than infection and favorable It was to increase the incidence of neutropenia.

IL-18 and IL-4
The IL-1ra or functional equivalent thereof can also be fused with other anti-inflammatory, anti-asthma or anti-angiogenic proteins. For example, the following are mentioned.
(I) IL-18 neutralizing substances such as IL-18 binding protein (IL-18bp), IL-18 receptor (IL-18R) extracellular domain and humanized anti-IL-18 antibody (ii) IL-4 IL-4R extracellular domain (trade name “Nuvance”, Immunex), and IL-4 neutralizing substance (iii) anti-VEGF antibody such as humanized anti-IL-4 antibody (Protein Design Labs) and Soluble Tie2 extracellular domain of erythropoietin neutralizer.
As mentioned above, by binding of IL-1ra to the C-terminus of these proteins,
(1) its molecular weight increases,
(2) when produced in a mammalian-derived host, two additional glycosylation sites are added;
(3) direct them to transport targeting inflammatory sites rich in IL-1 receptors;
(4) Simultaneously block IL-18, IL-4, VBEGF, or angiopoietin and IL-1 at a 1: 1 molar ratio.

  A clinical trial (Serono) has been undertaken for the treatment of psoriasis for skin inflammatory indications with recombinant IL-18bp. The good safety of this IL-18 bp is shown. Fusion of IL-1ra to the C-terminus can significantly increase its in vivo lifetime. Inflammatory site targeting via IL-1ra fusion can significantly increase its efficacy. Dual neutralization of IL-18 and IL-1 by IL-1ra fusion also exerts a synergistic effect in the treatment of inflammation-dependent diseases such as psoriasis (Yudoh K et al. (2004)). Interestingly, IL-18 and IL-1 use the same IL-1 receptor family and almost the same cellular signaling pathway. The double block of IL-1 and IL-18 almost completely blocks the inflammatory mechanism mediated by the entire IL-1 receptor family. The double block of IL-1 and IL-18 by the chimeric protein of the present invention is the most effective anti-inflammatory therapeutic agent.

  The functional equivalent of IL-18bp can also be used for the practice of the present invention. IL-18bp or a functional equivalent thereof comprises an IL-18 neutralization domain, which can neutralize IL-18, ie inhibit the activity of IL-18. For example, the IL-18 neutralizing agent domain includes an extracellular domain of human IL-18 receptor (US Pat. No. 6,589,764), IL-18bp, anti-IL-18 antibody, or IL-18 mutation. An antagonist protein is mentioned.

  The IL-18 neutralizing activity of the chimeric protein of the present invention can be measured using IL-18-dependent KG-1 cells. For example, human IL-18 induces IFN-g secretion from KG-1 cells in a dose-dependent manner (when TNFa is present). This IL-18-dependent IFN-g secretion can be inhibited by an effective amount of IL-18 neutralizer. These IL-18 neutralization activities can also be measured in the IL-18 / IL-18 receptor binding test.

  Clinical trials for the treatment of asthma have been conducted on soluble recombinant IL-4 receptor. A high safety profile is shown. However, its efficacy is not sufficient. Interestingly, IL-1 has been reported to be required for allergen-specific Th2 cell activation and the development of hypersensitivity reactions due to airborne infection (Iwakura Y et al., 2003). Furthermore, the coexistence or interdependence of asthma and chronic inflammation, and their interactions are known in the clinic. The blocking of IL-1 provides a clear therapeutic effect on asthma, at least in model animals. The simultaneous blocking of IL-4 and IL-1 with a 1: 1 molar ratio fusion of IL-1ra and soluble IL-4 receptor can significantly enhance the therapeutic effect of severe asthma. Inflammatory site targeting with IL-1ra can enhance the therapeutic value of soluble IL-4 receptor in the treatment of severe asthma exacerbated by inflammation. Furthermore, IL-1ra fusion can significantly increase the in vivo lifetime of soluble IL-4 receptor.

  A soluble IL-4 receptor or functional equivalent thereof can be fused to IL-1ra. The IL-4 receptor or functional equivalent thereof comprises an IL-4 neutralization domain, which can neutralize IL-4, ie inhibit the activity of IL-4. For example, IL-4 neutralizer domains include extracellular domains of human IL-4 receptors, anti-IL-4 antibodies, or IL-4 mutant protein antagonists with double mutation R121D / Y124D (Schnarr et al., 1997). Interestingly, this IL-4R subunit not only binds to IL-4, but also binds to the IL-13 receptor due to the subunit nature common to IL-4 and IL-13. The IL-4 neutralizing activity of the chimeric protein of the present invention can be measured by an IL-4-dependent TF-1 cell-based assay. For example, IL-4-dependent proliferation of human TF-1 cells can be inhibited by the addition of an effective amount of IL-4 neutralizing agent. The activity of the IL-4 neutralizing substance can be measured by the IL-4 / IL-4 receptor binding test.

VEGF and Angiopoietin The above method can also be applied to antagonists of VEGF and Angiopoietin, and functional equivalents thereof. VEGF is important for angiogenesis. Anti-VEGF antibodies (trade name Avastin, Genentech) are used for the treatment of cancer indications. Similarly, soluble VEGF receptor extracellular domain fused with IgG1 Fc has also been used to neutralize VEGF in angiogenesis-related indications. A functional equivalent of VEGF contains a VEGF neutralization domain, which can neutralize VEGF, ie inhibit the activity of VEGF. For example, the VEGF neutralizer domain may comprise the extracellular domain of human VEGF and the variable domain of an anti-VEGF antibody.

  The VEGF neutralization activity of the chimeric protein of the present invention can be measured using VEGF-dependent HUVEC cells. For example, human VEGF induces proliferation of HUVEC cells. This VEGF-dependent growth of HUVEC cells can be inhibited by an effective amount of VEGF neutralizing agent. The activity of the VEGF neutralizing agent can be measured using the VEGF / VEGF receptor binding test.

  The angiopoietin soluble receptor Tie2 has also been proposed as an anti-angiogenic therapeutic for cancer or angiogenesis-related rheumatoid arthritis. The coexistence and interdependence of angiogenesis and inflammation has been observed so far in the clinical field. The most common example is rheumatoid arthritis, where angiogenesis and inflammation coexist. The angiopoietin soluble receptor Tie2 or functional equivalent thereof comprises an angiopoietin neutralization domain, which can neutralize angiopoietin, ie inhibit the activity of angiopoietin 1. For example, an angiopoietin neutralizing substance domain includes an extracellular domain of human Tie2 and an anti-Tie2 or anti-angiopoietin antibody.

  The Tie-2 neutralizing activity of the chimeric protein of the present invention can be measured by Tie-2-dependent HUVEC cells. For example, human angiopoietin 1 induces intracellular phosphorylation of HUVEC cells. This Tie-2 dependent phosphorylation of HUVEC cells can be inhibited by an effective amount of Tie-2 neutralizing substance. The activity of the Tie-2 neutralizing agent can be measured using the Tie-2 / Angiopoietin 2 binding assay.

  It is known that IL-1 is an important pathological angiogenic stimulator. Disabling IL-1 with IL-1ra or its functional equivalent inhibits angiogenesis and tumor growth in model animals, suggesting that inflammation enhances angiogenesis. For example, the most malignant breast cancer is inflammatory breast cancer. Use of fusions of IL-1ra and angiogenic substances (eg anti-VEGF antibodies, soluble VEGF receptor extracellular domain or soluble Tie2 extracellular domain) in the treatment of cancer or rheumatoid arthritis related indications It is preferred that a marked effect is obtained over the anti-angiogenic substance alone.

In addition to the above therapeutic agents, other suitable protein therapeutics that can be fused to IL-1ra or functional equivalents thereof are listed below.
1. E25 (olizumab). E25 is a humanized anti-IgE antibody (Novatis) and is used to treat allergic asthma (seasonal allergic rhinitis).
2. H5G1.1. H5G1.1 is a humanized anti-C5 antibody (Alexion Pharmaceuticals) and can be used to treat psoriasis and autoimmune diseases.
3. TP10. TP10 is a soluble complement receptor 1 (sCR1) used for the treatment of acute respiratory failure syndrome and organ transplantation (AVANT immunotherapy).
4). ABX-IL8. ABX-IL8 is an anti-IL-8 monoclonal antibody (Abgenix) and can be used to treat psoriasis.
5. CTLA4Ig. CTLA4Ig is a soluble recombinant receptor (Bristol-Myers Squibb) and can be used for immunosuppression.

  In the fusion of the above substance with IL-1ra / its functional equivalent partner, the two fusion partners are active synergistically or complementary to each other. IL-1ra binds to the IL-1 receptor and leads to a therapeutic agent fused to an inflammatory site rich in IL-1 receptor. It also disables IL-1 activity. Fusions of IL-lra with any of these proteins can be used to treat inflammation, asthma and angiogenesis related diseases, or endothelial cell proliferation related diseases.

  Angiogenesis-related diseases refer to any disorder that requires angiogenesis or exhibits abnormal angiogenesis. For example, but not limited to, cancer, solid cancer, tumor metastasis, benign tumors (eg hemangiomas), acoustic neuromas, neurofibromas, trachoma and pyogenic granulomas, rheumatoid arthritis, psoriasis, diseases of ocular blood vessels (eg Diabetic retinopathy), retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, post-lens fibroproliferation and rubeosis, Osier-Webber syndrome, myocardial angiogenesis, plaque angiogenesis, peripheral vasodilatation, Hemophilia arthropathy, angiofibromas and damaged granulation. In the present invention, endothelial cell proliferation-related diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma and hypertrophic scars. The fusion proteins described herein can also be used to treat the diseases listed above by inhibiting angiogenesis necessary for embryo transfer.

  Preferably, the fusion protein of the invention comprises a dimerization domain. “Dimerization domain” refers to a domain capable of binding two polypeptides. For example, the dimerization domain includes an IgG Fc fragment (eg, a human IgG heavy chain constant region). Such an Fc fragment comprises, for example, SEQ ID NO: 2. IgG Fc fragments dimerize by the formation of disulfide bonds (covalent bonds) by cysteine residues between chains. In addition, dimerization by a non-covalent bond that does not contain a disulfide bond may occur. The dimerized IgG Fc fragment is, for example, a bifunctional TNFRII or a soluble IL-4R or IL-18bp or soluble Tie-2 molecule at the N-terminus and a bifunctional IL-1ra molecule at the C-terminus. Can be shown. This configuration increases the possibility of receptor / ligand binding in vivo to neutralize any of TNFα or IL-4 or IL-18 or angiopoietin, and the IL-1 receptor.

  The activity of covalent dimerization via a disulfide bond can be measured using SDS-PAGE under reducing and non-reducing conditions. The molecular weight of the protein must be reduced by half under reducing conditions. Non-covalent dimerization can be measured using electrophoresis under non-denaturing and denaturing conditions. The molecular weight of the protein must be reduced by half under denaturing conditions.

  In the polypeptides of the present invention, (NF neutralization domain or IL-4 / IL-13 neutralization domain or IL-18 neutralization domain or VEGF neutralization domain or angiopoietin neutralization domain, dimerization domain, and IL- 1 receptor antagonist domains are operably linked The term “operably linked” in the present invention refers to the structure of the polypeptide being constructed in a manner that does not interfere with the activity of each domain. For example, the IL-4 neutralization domain retains its ability to neutralize IL-4 and the interleukin 1 receptor antagonist domain specifically binds to its IL-1 receptor and binds to the cellular receptor for IL-1 The dimerization domain binds two polypeptides of the invention, eg, has two functions. The IL-4 receptor extracellular domain to the N-terminus that retains the ability to demonstrate IL-1ra molecules having 2 functionality to the C-terminus.

By fusion of IL-1ra to the C-terminus of the above TNF neutralizing substance, IL-18 neutralizing substance, IL-4 neutralizing substance, VEGF neutralizing substance or angiopoietin neutralizing substance,
(1) its molecular weight increases,
(2) when produced in a mammal-derived host, two additional glycosylation sites are added to IL-1ra;
(3) directing the neutralizing substance to transport targeting inflammatory sites rich in IL-1 receptors;
(4) Simultaneously block IL-1 and TNF, IL-18, IL-4, IL-13, IgE, VEGF and angiopoietin in a 1: 1 molar ratio.
The resulting double block enhances the efficacy of treatment of inflammatory diseases and provides a more complete block against inflammatory disease processes. The simultaneous double block of IL-4 / IL-13 / VEGF / Angiopoietin and IL-1 has a therapeutic effect on diseases in which the coexistence and interdependence of inflammation and asthma or angiogenesis plays an important role in the disease process, Improved and complete.

  The polypeptide of the present invention can be obtained as a synthetic or recombinant polypeptide. To prepare a recombinant polypeptide, the nucleic acid encoding it is linked to another nucleic acid encoding a fusion partner (eg, glutathione-S-transferase (GST), 6 × His epitope tag, or M13 gene 3 protein). May be. The resulting fusion nucleic acid can be isolated by known methods by expressing the fusion protein in a suitable host cell. A variety of host-expression vector systems can be used. They include, but are not limited to, microorganisms (eg, bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors), yeast transformed with recombinant yeast expression vectors, recombinant viruses or plasmid expression vectors. Examples include transfected human cell lines. Isolation and purification of the recombinant polypeptide or fragment thereof can be performed by conventional means such as chromatographic preparation involving monoclonal or polyclonal antibodies and immunological separation methods. The isolated fusion protein may be further treated with an enzyme, for example, and the fusion partner can be removed to obtain the recombinant polypeptide of the present invention.

Compositions and Methods of Treatment A method of treating a disease characterized by an excessive immune response or angiogenesis-related disease by administering an effective amount of a fusion protein of the invention to a patient in need thereof is also provided by the invention. Is within the range. Patients undergoing treatment (eg, patients suffering from autoimmune disease, transplant rejection, allergic disease or cancer derived from immune cells) have or are at risk of developing symptoms characterized by excessive or unnecessary immune responses It can be diagnosed as having sex. This method can be performed alone or in combination with other drugs or other therapies.

  The term “treat” refers to the administration of a composition to a patient for the purpose of curing, reducing, alleviating, treating, preventing or ameliorating a disease, symptoms of the disease, disease states or disease trends derived from the disease. Point to. “Effective amount” refers to an amount of the composition that can provide a medically desirable result for the patient to be treated. Medically desirable results may be objective (ie, measurable by testing or labeling) or subjective (ie, the patient shows or feels the effect). Typical diseases to be treated include acute and chronic inflammation, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis and psoriatic arthritis), multiple sclerosis, cerebral spinal cord Inflammation, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and dermatitis with eczema), psoriasis, Sjogren's syndrome, Crohn's disease, aphthous ulcer, iritis , Conjunctivitis, keratoconjunctivitis, type I diabetes, inflammatory bowel disease, ulcerative colitis, asthma, allergic asthma, lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruption, leprosy reversal reaction, Erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensory nerve hearing loss, aplastic anemia, true erythrocytic anemia, idiopathic Thrombocytopenia , Polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, posterior uveitis, mediated Pulmonary fibrosis, graft-versus-host disease, bone marrow transplantation, liver transplantation, etc. (including transplantation using allogeneic or heterogeneous tissues) or any organ or tissue transplantation, allergy such as atopic allergy, AIDS , T cell tumors (eg leukemia or lymphoma), acute hepatitis, diseases related to angiogenesis (eg rheumatoid arthritis and cancer), and cardiovascular diseases.

  A patient undergoing treatment may be diagnosed as in need of treatment for one or more of the disorders described above. Diagnosis of a patient in need of such treatment is made at the discretion of the patient or health professional and may be subjective (eg, opinion) or objective (eg, measurable by testing or diagnostic methods) .

  In an in vivo embodiment, a therapeutic composition (eg, a composition comprising a fusion protein of the invention) is administered to a patient. Usually, the protein is suspended in a pharmaceutically acceptable carrier (eg, physiological saline) and administered by oral administration or intravenous injection, or subcutaneous, intramuscular, intrathecal, intraperitoneal, intrarectal, It is injected or transplanted into the vagina, nasal cavity, stomach, trachea or lung.

  The required dosage will depend on the choice of route of administration, the nature of the formulation, the nature of the patient's disease, the patient's height, weight, surface area, age and sex, the other drugs being administered, and the diagnosis of the attending physician. A suitable dose is in the range of 0.01 mg / kg to 100.0 mg / kg. Necessary dosage changes are expected to take into account the type of composition available and the efficiency differences in the various routes of administration. For example, oral administration may require a higher dose than administration by intravenous injection. Changes in these dosage levels can be adjusted using standard routine procedures for empirically obtained optimization well known in the art. Encapsulation of the composition in a suitable transport carrier (eg, polymeric microparticles or implantable means) can improve transport efficiency, particularly in oral transport.

  Also included within the scope of the invention are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of a fusion protein of the invention. The pharmaceutical composition can be used for the treatment of the above-mentioned diseases. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents.

  The pharmaceutical compositions of the invention can be formulated into dosage forms for different routes of administration utilizing conventional methods. For example, it can be formulated as a capsule for oral administration, a gel embedding or a tablet. Capsules can contain any standard pharmaceutically acceptable material (eg gelatin or cellulose). According to conventional methods, it can be formulated as a tablet by compressing a mixture of the composition together with a solid carrier and a lubricating oil. Examples of solid carriers include starch and sugar bentonite. The composition can also be administered in the form of hard coated tablets or capsules containing a binder (eg lactose or mannitol, conventional fillers and tableting). The pharmaceutical composition can be administered via a parenteral route. Examples of dosage forms for parenteral administration include solutions in which the active agent is dissolved in water, isotonic saline or 5% glucose solution, or other well-known pharmaceutically acceptable additives Is mentioned. Cyclodextrins, or other solubilizers known to those skilled in the art, can be utilized as pharmaceutical excipients for the delivery of therapeutic compounds.

  The efficacy of the composition of the present invention can be evaluated in vitro and in vivo. See the examples below. Briefly, the composition can be tested for its ability to suppress the immune response in vitro. In an in vivo test, the composition can be injected into an animal (eg, a model mouse), and its therapeutic effect can be observed. Based on the results, an appropriate dosage range and administration route can be determined.

  The following examples are disclosed for illustrative purposes only, and should not be construed as limiting the content of any part other than this disclosure in any way. Those skilled in the art will be able to complete the practice of the invention based on the description in the present specification without further refining. All publications cited herein are hereby incorporated by reference in their entirety.

  Our results also show that IL-1ra fusion molecules produced in mammalian hosts contain glycosylated IL-1ra and have a higher molecular weight than non-IL-1ra fusion molecules. They have a longer in vivo life and provide an effective infusion volume that does not require frequent administration. Due to its inflammatory site-specific nature, and low effective dose and lower dosing frequency, IL-1ra fusion molecules are non-IL-1ra fusion molecules or a combination of non-IL-1ra fusion molecules and IL-1ra There are fewer side effects than.

Various expression vectors were constructed. Each vector encodes the following protein:
A) TNFRII-Fc-IL-1ra (SEQ ID NO: 5), TNFRI-Fc-IL-1ra (SEQ ID NO: 8), and control TNFRII-Fc (SEQ ID NO: 4) or TNFRI-Fc (SEQ ID NO: 7),
B) Humira® (D2E7) -IL-1ra (SEQ ID NO: 10 and 11), Remicade® (cA2) -IL-1ra (SEQ ID NO: 13 and 14), and control dimerized Humira (Registered trademark) (D2E7) (SEQ ID NO: 9 and SEQ ID NO: 11) and Remicade (registered trademark) (cA2) (SEQ ID NO: 12 and SEQ ID NO: 14),
C) IL-18bp (SEQ ID NO: 15), dimerized IL-18bp-Fc (SEQ ID NO: 16) and dimerized IL-18bp-Fc-IL-1ra (SEQ ID NO: 17),
D) The extracellular domain of soluble IL-4R (SEQ ID NO: 19), IL-4R-Fc (SEQ ID NO: 19) and IL-4R-Fc-IL-1ra (SEQ ID NO: 21);
E) VEGFR1-Fc-IL-1ra and light chain (SEQ ID NO: 24 and SEQ ID NO: 23) and anti-VEGF heavy chain-IL-1ra and light chain (SEQ ID NO: 25 and SEQ ID NO: 23).

  Most constructs encoding the protein (SEQ ID NO: 4 to SEQ ID NO: 25) were sequenced and expressed in mammalian cell lines. SEQ ID NO: 4 to SEQ ID NO: 25 were expressed in natural suspensions adapted for mammalian hosts using natural or optimized codons as well as artificial or natural secretory signal sequences. The dimerized antibody product was detected by non-heated SDS-PAGE and Western blot.

  The expression titers of TNFRII-Fc (SEQ ID NO: 4) and TNFRII-Fc-IL-1ra (SEQ ID NO: 5) in serum-free medium in 24-well plates were 50 mg / l to 100 mg / l, respectively (FIG. 1). there were. Compared with TNFRII-Fc, high expression of TNFRII-Fc-IL-1ra was observed in the CHOK1 cell suspension (estimated by direct protein staining of conditioned medium with Coomassie Blue). This result shows that IL-1ra fusion chimeric protein can be produced on an industrial scale using a host derived from a mammal.

  Scale-up and purification of TNFRII-Fc-IL-1ra, IL-4R-ECD-Fc-IL-1ra and IL-18bp-Fc-IL-1ra were performed. The cell line was cultured in serum-free suspension prepared with CHO-CD4 medium (Irvine Scientific) and a self-made feed medium and scaled up in a 3 L bioreactor (Eplikon). TNFRII-Fc-IL-1ra (SEQ ID NO: 5), IL-4R-ECD-Fc-IL-1ra (SEQ ID NO: 20) and IL-18bp-Fc-IL-1ra (SEQ ID NO: 17) on an industrial scale Manufactured. These proteins were directly captured and purified by protein A, followed by ion exchange and hydrophobic chromatography (FIGS. 2, 3 and 4). Bulk purified protein was formulated, lyophilized and analyzed by SEC-HPLC.

  The activities of TNFRII-Fc-IL-1ra, IL-4R-Fc-IL-1ra, IL-18bp-Fc-IL-1ra and VEGFR1-Fc-IL-1ra were tested by bioassay.

  For cell-based IL-1 neutralization assay, IL-1 dependent D10 cells (ATCC) were used and IL-1ra (Kineret), TNFRII for proliferation of recombinant human IL-1-dependent D10 cells. -The blocking activity of Fc-IL-1ra, IL-4R-Fc-IL-lra and IL-18bp-Fc-IL-1ra was tested.

In short:
Human IL-1α induces D10 cell proliferation in a dose-dependent manner,
The concentration at which IL-1a induces 50% of total cell proliferation (ie, EC50) was measured.
The EC50 range of hIL-1a for D10 cells is usually 1 pg / ml to 5 pg / ml. When cells were incubated with an effective amount of IL-1 receptor antagonist, IL-1ra inhibited cell proliferation by blocking cell surface IL-1 receptor. This blocking effect was also dose dependent. When the concentration of receptor antagonist was low, it did not block cell surface receptors. IL-1 then induced cell growth recovery. The concentration of receptor antagonist at which 50% of IL-1 activity was blocked was taken as the EC50 of the antagonist.

  Recombinant proteins (TNFRII-Fc-IL-1ra, IL-4R-Fc-ILlra, IL-18bp-Fc-IL-1ra or VEGFR1-Fc-IL-1ra) are similar to IL-1 receptor antagonists, Functions such as soluble TNFRII, IL-18, IL-4 or VEGF neutralizers were performed. Cell-based bioassays confirmed the biological activity of these chimeric molecules (FIGS. 6, 8, 10, and 11).

  In a cell-based TNF neutralization assay, L929 cells (mouse passage cell line, ATCC) were used to test the blocking activity of TNFRII against TNFα. Briefly, TNFα was used to induce dose-dependent rapid cell death. The EC50 of TNFα (the concentration at which TNFα induces 50% of total cell death) was less than 50 pg / ml. When preincubated with TNFα molecules and a high concentration of soluble TNF receptor (sTNFR), the soluble receptor bound to TNFα and inhibited its binding to cell surface receptors. This blocked TNFα activity that induced cell death. This blocking effect was also dose dependent. When sTNFR was diluted to a constant concentration, no block of TNFα activity was observed and the degree of cell death decreased. From the above, the EC50 of sTNFR (that is, the concentration that blocks 50% of TNFα activity) was determined.

Human TNFα (BioSource) was seeded at a total volume of 150 μl / well with a fixed number of L929 cells (in DMEM medium supplemented with 10% horse serum, L-glutamine and 1 μg / ml actinomycin D) in a 2-fold dilution series. To the 96-well assay plate. Control wells containing cells in medium alone were also prepared. The assay plate was incubated for 1 day in a humid chamber of 37 ° C., 5% CO ₂ incubator. The cells in each well were then fixed with 10% paraformaldehyde and stained with 1% crystal violet solution. The dye was solubilized with 30% acetic acid. The optical density (OD) (in direct proportion to the total number of cells) of each well of the assay plate was then measured using a plate reader at a wavelength of 540 nm. O. D. The relationship of TNFα concentration vs. TNFα concentration was plotted as a cytotoxicity curve. TNFRII-Fc (Enbrel) and TNFRII-Fc-IL-1ra were diluted in a 2-fold dilution series in a DMEM medium supplemented with a constant concentration of human TNF-α (10% horse serum, L-glutamine and 1 μg / ml actinomycin D). ) In a 96-well assay plate previously seeded at a total volume of 150 μl / well. The assay plate was preincubated for 1 hour at 37 ° C. The mixture in each well of the assay plate was transferred to another 96-well plate previously seeded with a fixed number of L929 cells. The final concentration of human TNFα per well was 500 pg / ml with a total volume of 150 μl / well. The assay plate was incubated for 1 day in a humid chamber of 37 ° C., 5% CO ₂ incubator. The cells in each well were then fixed with 10% paraformaldehyde and stained with 1% crystal violet solution. The dye was solubilized with 30% acetic acid. The optical density (OD) of each well of the assay plate was then measured using a plate reader at a wavelength of 540 nm. O. D. The relationship between TNFRII-Fc and TNFRII-Fc-IL-1ra concentration was plotted as a neutralization curve.

  The test results show that human TNFα induced L929 cell death in a dose-dependent manner. Background O.D. containing only actinomycin D and cells. D was 0.5. The dose curve for human TNFα decreased from a base level of 0.5 to a minimum level of 0.1. Furthermore, at a human TNFα concentration of 100 pg / ml or higher, O.D. D. Did not decrease, indicating a saturation stage of human TNFα. All tests were performed twice and the CV% at each position was <9%. The EC50 of human TNFα under these conditions was 8 pg / ml.

  TNFRII-Fc (Enbrel) and TNFRII-Fc-IL-1ra were found to inhibit human TNFα activity in L929 cells in a dose-dependent manner. O. of base level (for cells in the presence of human TNFα (500 pg / ml) and actinomycin D). D was 0.1. In the presence of different concentrations of TNFRII-Fc-IL-1ra, O.D. D. Increased to a base level of 0.1-0.5 and complete neutralization was observed. TNFRII-Fc and TNFRII-Fc-IL-1ra completely neutralized human TNFα activity at a concentration of 50 ng / ml. All dilution tests were performed twice and the CV% at each position was <10%. The EC50s of TNFRII-Fc (Enbrel) and TNFRII-Fc-IL-1ra under these conditions were 3 ng / ml to 4 ng / ml and 10 ng / ml.

A cell-based IL-4 neutralization assay was performed using human IL-4 induced TF-1 cell proliferation. TF-1 cells were incubated in media containing different concentrations of human IL-4, and 96-well plates were cultured in an incubator for 3 days at 37 ° C. and 5% CO ₂ . MTS was added to the cultured tissue and incubated for 5 hours. The optical density (OD) of the plate was measured at 490 nm using a plate reader. Cell growth curves (OD vs. human IL-4 concentration) were plotted. In neutralization, IL-4R-Fc and IL-4R-Fc-IL-1ra prepared by dilution series were added to the medium with a constant concentration of human IL-4 (2 ng / ml) and 1 in a 96-well plate. Pre-incubation was carried out at 37 ° C for a period of time. The same number of TF-1 cells were added to each well of a 96-well plate after the end of the culture. The plate was incubated for 3 days in an incubator at 37 ° C. and 5% CO ₂ . MTS was added and incubated for 5 hours. The OD of the plate was measured at 490 nm using a plate reader. The relationship of OD versus IL-4R-Fc and IL-4R-Fc-IL-1ra concentration was plotted as a cell growth inhibition curve.

  Combined with the results of the IL-1 neutralization assay (FIG. 8), the results in FIG. 7 show that IL-4R-Fc-IL-1ra is functional and has IL-4R and IL-1 neutralization activity. Was shown.

  In a cell-based IL-18 neutralization assay, dose-dependent human IL-18-induced IFN-g secretion from KG-1 cells (if TNFα was present) was analyzed. The EC50 of human IL-18 (the concentration that induces 50% of the maximum amount of IFNg secretion from KG-1 cells) is usually 20 ng / ml / ml to 40 ng / ml. When human IL-18 binding protein (IL-18bp) was preincubated with human IL-18 prior to addition to cultured cells, IL-18bp bound IL-18 and blocked its activity. This blocking effect was dose dependent. The concentration of binding protein at which 50% of maximum IFNg secretion was prevented was taken as its EC50.

Two samples of IL-18bp-Fc-IL-1ra prepared by serial dilution and IL-18bp-Fc as a control were added to the medium together with a constant concentration of human IL-18 (R & D System, 50 ng / ml), 96 wells The plate was preincubated for 1 hour at 37 ° C. As a positive control, two samples each containing only human IL-18 prepared by serial dilution were prepared. The same number of KG-1 cells (ATCC, CCL246) together with a certain amount of human TNFa (BioSource) was added to each well of a 96-well assay plate after the end of the culture. The assay plate was incubated for an additional 24 hours in an incubator at 37 ° C., 5% CO ₂ . From each well of the assay plate, 50 μl / well of culture was transferred to the ELISA plate. Using human IFNg ELISA (Biosource), the test was performed according to the instructions of the kit. The optical density (OD) of the plate was measured at 450 nm using a plate reader. The relationship between OD versus human IL-18 concentration was plotted as the IFNg secretion curve induced by human IL-18. The relationship between the concentration of OD vs. IL-18bp-Fc-IL-1ra and OD vs. IL-18bp-Fc (control) was plotted as an IL-18bp neutralization curve.

  The results of the cell-based assay are shown in FIG. Combined with the results of the IL-1 neutralization assay (FIG. 10), it was shown that a functional IL-18bp-Fc-IL-1ra chimera was efficiently obtained. The neutralizing activity of both IL-18 and IL-1 was maintained. Human VEGF (vascular endothelial growth factor) induces the proliferation of HUVE (human umbilical vein endothelial cell) cells in a dose-dependent manner. The concentration that induces 50% of the maximum proliferation of EC50HUVE cells of human VEGF is usually between 2 ng / ml and 6 ng / ml. When soluble human VEGF receptor-1 and human VEGF were preincubated prior to addition to cultured cells, the soluble human VEGF receptor-1 bound to human VEGF and interfered with its activity in the cells. This soluble receptor blocking effect was also dose dependent. The concentration of soluble receptor at which 50% of maximum cell growth is blocked was taken as its EC50. A recombinant protein VEGFR1-Fc-IL-1ra with a soluble VEGF receptor and an IL-1 receptor antagonist on the same molecule was made. Thus, it can act as an IL-1 receptor antagonist and likewise as a soluble VEGFR1.

Two samples each of VEGFR1-Fc-IL-1ra prepared by serial dilution were added to the medium together with a constant concentration of VEGF (BioSource, 10 ng / ml), and incubated in a 96-well assay plate for 1 hour at 37 ° C. Pre-incubated. As a positive control, two samples each containing only human VEGF prepared by serial dilution were prepared. The same number of HUVE cells (Cambrex, CC-2517) was added to each well of the 96-well assay plate after the end of the culture. The assay plate was further incubated for 96 hours in an incubator at 37 ° C., 5% CO ₂ . MTS (Promega) was added to each well of the assay plate 4 hours before the end of the culture. Thereafter, the optical density (OD) of the plate was measured at a wavelength of 490 nm using a plate reader. The relationship between OD vs. VEGF concentration was plotted as a cell growth curve with VEGF. The relationship of OD versus VEGFR1-Fc-IL-1ra concentration was plotted as a VEGF-R neutralization curve.

  Human VEGF stimulated the proliferation of HUVE cells in a dose-dependent manner. The ED50 was 3 ng / ml. Preincubation of 10 ng / ml VEGF with VEGFR1-Fc-IL-1ra prepared by serial dilution prior to administration to cells inhibited VEGF-dependent cell growth in a dose-dependent manner. The EC50-Fc-IL-1ra of VEGFR1 was 15 ng / ml (FIG. 12). When combined with the results of the IL-1 neutralization assay (FIG. 11), it was shown that a functional VEGFR1-Fc-IL-1ra chimera was efficiently produced. The neutralizing activity of VEGF and IL-1 was maintained.

  An animal experiment of IL-4R-Fc-IL-1ra using asthma model mice was performed. Female BALB / c mice (6 to 8 weeks old) were used. Briefly, these mice received an intraperitoneal emulsion on day 0 and 14 with 2.25 mg of aluminum hydroxide (Pierce, Rockford, IL) containing 40 μg of OVA (Sigma). Injected.

  Mice were divided into 8 hour and 48 hour groups. The 8 hour group consisted of saline control-8 hours, OVA-8 hours, IL-4R-Fc / OVA-8 hours and IL-4R-Fc-IL-1ra / OVA-8 hours, while 48 The time group consists of saline control-48 hours, OVA-48 hours, IL-4R-Fc / OVA-48 hours and IL-4R-Fc-IL-1ra / OVA-48 hours.

  On the 28th day, 100 μg OVA dissolved in 0.05 ml of normal saline was administered by intranasal route to all divided groups except the saline control group. For the saline control group, normal saline containing aluminum was administered from the intraperitoneal route on days 0 and 14, and 0.05 ml of normal saline was administered from the intranasal route on day 28. .

  On day 29, 100 μg OVA dissolved in 0.05 ml of normal saline was further administered by intranasal route to the 48-hour divided group excluding the saline control group. The physiological saline control group was further administered 0.05 ml of normal physiological saline by the intranasal route on the 29th day.

Administration of IL-4R-Fc and IL-4R-Fc-IL-1ra IL-4R-Fc / OVA-8 hours, IL-4R-Fc-IL-1ra / OVA-8 hours, IL-4R-Fc / OVA The group of -48 hours and IL-4R-Fc-IL-1ra / OVA-48 hours was administered at day 28 at 200 μg / mouse / day. On day 28 they were administered 60 minutes prior to intraperitoneal injection of OVA. The IL-4R-Fc / OVA-48 hour and IL-4R-Fc-IL-1ra / OVA-48 hour groups received an additional 200 μg / mouse / day on day 29.

Measurement of bronchoalveolar lavage (BLA) cell count In 8 hour subgroups, mice were sacrificed 8 hours after a single dose of OVA into the nasal cavity on day 28, and burs were prepared for histological analysis. went. In the 48 hour split group, mice were sacrificed 48 hours after the second nasal administration of OVA on days 28 and 29.

  After the left lung was bound with the mainstem bronchus, the right lung was washed with 1.0 ml of normal physiological saline using a tracheal cannula. The total number of white blood cells was measured using a hemocytometer. The difference in the number of cells was measured by staining a preparation obtained by centrifuging the cells with leukostat (Fisher Diagnostics, Pittsburgh, PA). Cells were identified as macrophages, eosinophils, neutrophils and lymphocytes by standard hematology methods, and at least 200 cells were counted by × 400 magnification.

Lung tissue Trachea and left lung (upper and lower lobes) were collected and fixed with car noise solution at 20 ° C. for 15 hours. After embedding in paraffin, tissue sections were prepared every 5 μm. For each mouse, 10 airways randomly distributed throughout the left lung were used to analyze the severity of cellular inflammatory reaction and mucus obstruction. The degree of cellular infiltration around the pulmonary blood vessels and airways was evaluated on a semi-quantitative scale from 0 to 4+.

Result 1. Inhibition of early pneumonia by treatment with IL-4R-Fc-IL-1ra.
Table 1 shows the differential of cells in BAL fluid 8 hours after a single dose of OVA intranasally. Differential cell numbers were determined for saline control-8 hours, OVA-8 hours, IL-4R-Fc / OVA-8 hours and IL-4R-Fc-IL-1ra / OVA-8 hours groups (each Group n = 5; mean value ± expressed in SEM).

2. Inhibition of late-stage pneumonia by treatment with IL-4R-Fc-IL-1ra.
Table 2: shows the difference in the number of cells in BAL fluid 48 hours after twice intranasal OVA administration. Differential cell numbers were determined for saline control-48 hours, OVA-48 hours, IL-4R-Fc / OVA-48 hours and IL-4R-Fc-IL-1ra / OVA-48 hours groups (each Evaluation in group: n = 5). Shown as mean ± SEM. P <0.01.

Lung Histological Analysis Significant cell infiltration around the pulmonary blood vessels and airways was observed in the OVA-8 hour and OVA-48 hour groups. Compared to the IL-4R-Fc / OVA-8 hour and IL-4R-Fc / OVA-48 hour groups, the IL-4R-Fc-IL-lra-8 hour and IL-4R-Fc-IL-1ra- A marked phenomenon of cell infiltration around the pulmonary blood vessels and airways was observed in the 48 hour group. This result suggests that IL-4R-Fc-ILlra best treated asthma in this model animal.

An animal experiment of IL-18bp-IgG1Fc-IL-1ra using a mouse CIA model was performed. According to the recently published standard protocol (Banada et al., 2002), DBA / IJ mice aged 8-10 weeks were injected intradermally with bovine type II collagen (CII) to induce CIA. Each mouse was injected on days 0 and 21 with 100 μl of IFA containing 200 μg CII and 200 μg inactivated Mycobacterium tuberculosis (Difco, Detroit, MI). Mice (n = 5) were injected intraperitoneally once every 2 days between days 21 and 36 (control PBS, 3 mg / kg IL-18bp-Fc and 3 mg / kg). IL-18bp-Fc-IL-1ra) was treated with therapeutic intervention. Mice were sacrificed on day 36 by cervical dislocation. At the same time, three normal DBA / 1J mice (control) were sacrificed. Clinical CIA disease activity was assessed blindly by two observers between days 21 and 36, every other day, 3 points per limb:
0 = normal joint;
1 = mild inflammation and redness;
2 = severe erythema and swelling enough to affect all limbs (with concomitant use);
3 = Deformation of limbs or joints (joint ankylosis, loss of joint stiffness and function)
All scores for clinical disease activity were based on all four limbs in each animal (maximum score: 12) (Banda et al., 2002).

  The forelimb and right hind limb are surgically removed from all mice on day 36 and fixed with 10% buffered formalin and subjected to tissue sample preparation and tissue analysis as described above (Bendele et al., 2000). did. Histological findings of limb joints and knees were recorded by blinded and experienced observers. Data were expressed as mean and total scores for inflammation, pannus, cartilage injury and bone injury using 5 joints with a score of 0 to 5 per animal as described above (Bendele et al., 2000).

Results Clinical disease activity and joint histological analysis with IL-18bp-Fc-IL-1ra In all groups, the incidence of arthritis was 100%. Clinical disease in mice treated with either 3 mg / kg IL-18bp-Fc and 3 mg / kg IL-18bp-Fc-IL-lra from day 21 to day 36 compared to control PBS alone A decrease in activity scores was shown (Table 1). According to the histological analysis of the joint, the joint injury was observed when treated with either 3 mg / kg IL-18bp-Fc or 3 mg / kg IL-18bp-Fc-IL-1ra as compared with the PBS group. Suppressed. Significant differences were observed between 3 mg / kg IL-18bp-Fc and 3 mg / kg IL-18bp-Fc-IL-1ra for either clinical disease activity scores or histological scores. IL-18bp-Fc-IL-1ra showed significantly better results than IL-18bp-Fc (Table 1). Table 3: Clinical disease activity of CIA mice treated with IL-18bp-Fc-IL-1ra.

On days 0 and 21, DBA / 1J mice were immunized with IFA containing 200 μg M. tuberculosis and 200 μg CII. Mice were injected intraperitoneally every 3 days for 3 weeks, day 21 and day 36, and injected intraperitoneally once out of two times (control PBS, 3 mg / kg IL-18bp-Fc and 3 mg / kg IL- 18 bp-Fc-IL-1ra.) Was treated with therapeutic intervention. Clinical CIA disease activity was assessed every other day, 3 points per limb, with 2 observers, blinded to treatment and between observers. Data were expressed as clinical disease activity score (mean ± SEM) by each treatment group versus days after initial collagen infusion.

  In vivo testing of IL-18bp-Fc-IL-1ra using a contact hypersensitivity (CHS) mouse model was performed. This was done by induction of CHS and treatment of C57BL / 6 mice (8 and 14 weeks of age) with IL-18bp chimera. DNFB, acetone, Evans blue, formamide, BSA, PMA, ionomycin, brefeldin A and LPS (derived from E. coli 026: B6) were purchased from Sigma-Aldrich (St. Louis, Mo). DNFB was diluted with acetone / olive oil (4/1) immediately before use. Mice that had been sensitized by applying 25 μl of 0.5% DNFB solution to the shaved back skin or that had not been treated (control) were prepared. Five days later, 10 μl (non-stimulated dose) of 0.2% DNFB was administered to the right ear and the same amount of solvent to the left ear. From day 5 onwards, ear thickness was monitored daily using a caliper splint before dosing. Ear swelling was calculated by ((Tn-T5) right ear- (Tn-T5) left ear) (Tn and T5 values represent ear thickness before administration on day n and day 5 of the test, respectively). ). A non-sensitized but exposed control group was included in each study to demonstrate that the observed swelling was due to DNFB-specific inflammation rather than non-specific stimulation. From day 5 60 min prior to exposure, 250 μg of IL-18bp-Fc or IL-18bp-Fc-IL-1ra per animal was injected intraperitoneally daily to neutralize IL-18 and / or IL-1. . Control animals received saline alone as a carrier. Treatment during the first re-exposure period was stopped on day 7.

Results The therapeutic treatment with IL-18bp-Fc-IL-1ra provides a CHS protective effect. To experimentally induce CHS, hapten DNFB was sensitized on the shaved back of mice. Five days later, DNFB was applied to the ear to induce CHS. Inflammation was recorded as the swelling of the DNFB-exposed ear relative to the swelling of the control ear that received solvent alone.

  Administration of IL-18BP-Fc and IL-18bp-Fc-IL-1ra during the induction period of 5-7 days significantly reduced DNFB-exposed ear swelling during the entire period of response (Table 1). A significant difference was observed between IL-18bp-Fc and IL-18bp-Fc-IL-1ra (Table 1), a double block of IL-1 and IL-18 at the same location, or IL-18bp-Fc-IL It was suggested that the specific properties of the -1ra receptor-rich site play an important role in the IL-1 effect. IL-18bp-Fc-IL-1ra was significantly better than IL-18bp-Fc. Table 4: CHS protection effect was obtained by treatment with IL-18BP during the induction period.

C57BL / 6 mice were sensitized with DNFB on day 0 and exposed to the ear after 5 days. Ear swelling was measured daily and expressed as an increase in DNFB-sensitized ear swelling relative to the swelling of the control-coated control ear. Animals were treated daily with IL-18bp chimera or carrier alone. Data show the average of 5 mice per group.

  An IL-1 receptor binding test was performed.

  Briefly, the extracellular domain of recombinant human IL-1 receptor was first expressed and independently purified using mammalian CHO cells. Using TNFRII-Fc-IL-1ra, negative control TNFRII-Fc and positive control IL-1ra (Kineret) dissolved in 100 μl of coating buffer (Sigma), a 96-well plate was coated at a concentration of 1 μg / well. did. Purified IL-1 receptor (0.1 μg / well) was then incubated for 45 minutes at 37 ° C. in PBS. Receptor / ligand binding was detected using a rabbit anti-human IL-1 receptor extracellular domain antibody (R & D Systems) followed by a goat anti-rabbit IgG-HRP complex (Pierce). After washing with PBS-T, it was mixed with TMB (Sigma, T8665) to cause a color reaction. Using an EL800 universal microplate reader (Bio-Tek), the optical density (OD) of the plate at 650 nm was measured. The OD value against the dilution time was plotted. FIG. 13 shows that TNFRII-Fc-IL-1ra and IL-1ra (Kineret) bound to the IL-1 receptor and TNFRII-Fc (Enbrel) did not. Interestingly, TNFRII-Fc-IL-1ra (prepared from mammals) bound significantly more to the IL-1 receptor than IL-1ra (Kineret) prepared from E. coli. Furthermore, IL-1ra obtained from mammals contains two N-linked glycosylation sites, thereby binding to less serum proteins than IL-1ra (Kineret) obtained from E. coli, and consistently different in Has in vitro binding properties.

125-I labeling and animal experiments of TNFRII-Fc-IL-1ra, IL-4R-Fc-IL-1ra and IL-18bp-Fc-IL-1ra, and their non-IL-1ra fusion controls were performed.

  125-I labeled TNFRII-Fc-IL-1ra, IL-4R-Fc-IL-1ra and IL-18bp-Fc-IL-1ra were prepared by the method of Iodogen and subjected to molecular sieve chromatography (M Hui et al., 1989). An IL-1 receptor binding test was established using a fusion of recombinant IL-receptor extracellular domain from a self-made mammal (see Example 4 above). Binding of IL-1 receptor to 125-I labeled TNFRI-Fc-IL-1ra was compared to the results of TNFRII-Fc-IL-1ra, which did not discriminate with radioisotopes performed in parallel. As a result, it is shown that 125-I-labeled TNFRII-Fc-IL-1ra has a function with respect to IL-1 receptor binding.

  Mice treated with 6 nmol of TPA in 200 μl acetone applied to the ear consistently developed dermatitis for 2-3 days. 125-I labeled TNFRII-Fc-IL-1ra was injected into dermatitis model mice (see below) along with 125-I labeled TNFRII-Fc (Enbrel). As a result, it was surprisingly shown that 125-I-labeled TNFRII-Fc is more distributed at the inflammatory site than TNFRII-Fc (Table 1). This is thought to be due to the binding affinity of the IL-1 receptor. In addition, 125-I-labeled IL-4R-Fc-IL-1ra and IL-18bp-Fc-IL-1ra were injected into dermatitis model mice together with 125-I-labeled IL-4R-Fc and IL-18bp-Fc. . Similar results were obtained (Tables 2 and 3). Table 5: Distribution of 125-I labeled TNFRII-Fc-IL-1ra and TNFRII-Fc (Enbrel) in inflamed and non-inflamed skin tissue 4 hours after injection.

Distribution is expressed as a percentage of the dose injected per gram of tissue (n = 6).

Table 6: Distribution of 125-I labeled IL-4R-Fc-IL-1ra and IL-4R-Fc in inflamed and non-inflamed skin tissue 4 hours after injection.

Distribution is expressed as a percentage of the dose injected per gram of tissue (n = 6).
Table 7: Distribution of 125-I labeled IL-18bp-Fc-IL-1ra and ILK-18bp-Fc in inflamed and non-inflamed skin tissue 4 hours after injection.

Distribution is expressed as a percentage of the dose injected per gram of tissue (n = 6).

  The immunogenicity of IL-4R-Fc-IL-1ra was estimated using two cynomolgus monkeys. 10 mg of IL-4R-Fc-IL-1ra was injected subcutaneously every week for 8 weeks. Serum samples were collected before and after injection (Day 1 and Day 56). The samples were analyzed by a neutralization assay to confirm the presence of anti-chimeric IL-4R-Fc-IL-1 antibodies that neutralize the bioactivity of IL-4 and IL-1 on the chimeric protein. Serum samples were affinity purified with protein A and anti-human IgM antibody to detect even lower concentrations of neutralizing antibody. Antibodies that neutralize IL-4 and IL-1 bioactivity on the chimeric protein were not detected using untreated serum of purified monkeys and purified IgG and IgM. The above results suggested that the chimeric IL-4R-Fc-IL-1ra has no immunogenicity to monkeys and humans.

The production of TNFRII-Fc and TNFRII-Fc-IL-Ira chimeras by the first generation of CHO cell clones is shown. The protein was expressed in a serum-free medium in a 24-well plate, the protein was directly stained with Coomassie blue, and all recombinant proteins were visually observed in the range of 0.5 μg to 1.0 μg. 10 μl to 15 μl was added per lane. FIG. 6 shows affinity purification of TNFRII-Fc-IL-Ira chimera. SDS-PAGE under reducing and non-reducing conditions; protein staining with Coomassie Blue. Here is an example of troubleshooting ability. The modification of the first purification step reduced the degradation problem of the TNFRII-Fc-IL-Ira chimera. With TNFRII-Fc as a control, intact and partially degraded TNFRII-Fc-IL-Ira chimeras were analyzed by HPLC. Affinity purification of IL-4R-Fc, IL-4R-Fc-IL-1ra and IL-18bp-Fc-IL-lra is shown. FIG. 9 shows a cell-based TNFα neutralization test showing that TNFRII-Fc-IL-Ira chimera neutralizes TNFα killing activity of L979 cells, similar to commercially available TNFRII-Fc (Enbrel). FIG. 5 shows a cell-based IL-1 neutralization test showing that commercially available IL-Ira (Kineret) and TNFRII-Fc-IL-Ira chimeras neutralize the biological activity of IL-1 on D10 cell proliferation. Figure 3 shows human IL-4 neutralization assay with IL-4R-Fc-IL-1ra and control IL-4R-Fc. Figure 3 shows a human IL-1 neutralization assay with IL-4R-Fc-IL-1ra. IL-18 neutralization activity by IL-18bp-Fc-IL-1ra is shown. The IL-1 neutralization activity by IL-18bp-Fc-IL-1ra is shown. FIG. 6 shows IL-1 neutralization activity by VEGFR1-Fc-IL-1ra in D10 cells. FIG. 6 shows VEGF neutralization activity by VEGFR1-Fc-IL-Ira in HUVE cells. IL-1 receptor binding assay.

Claims (32)

A fusion protein,
A first portion located at the amino terminus of the fusion protein and specifically binding and neutralizing with a first cytokine or growth factor; and
Located at the carboxyl terminus of the fusion protein, specifically binds to a receptor for a second cytokine or growth factor, has a functionally linked domain, and the receptor for the second cytokine is located at an inflammatory site or disease site A fusion protein containing a second part, which is contained in large quantities.   The fusion protein of claim 1, further comprising a linker moiety that joins the first part and the second part, wherein the linker part has the ability to dimerize.   The fusion protein of claim 2, wherein the linker moiety comprises an Fc fragment of an immunoglobulin or a functional equivalent thereof.   The fusion protein according to claim 3, wherein the immunoglobulin is IgA, IgE, IgD, IgG or IgM.   The fusion protein of claim 4, wherein the immunoglobulin is IgG.   6. The fusion protein of claim 5, wherein the Fc fragment comprises SEQ ID NO: 2.   2. The fusion protein of claim 1, wherein the first portion binds and neutralizes VEGF, angiopoietin, TNF, IL-18, IL4 or IL-13, or a functional equivalent thereof.   The first portion comprises an immunoglobulin chain sequence that specifically binds and neutralizes VEGF, angiopoietin, TNF, IL-18, IL-4, IL-13 or IgE, or a functional equivalent thereof. Item 8. The fusion protein according to Item 7.   9. The fusion protein of claim 8, wherein the immunoglobulin chain comprises SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 23 or SEQ ID NO: 24, or functional equivalents thereof.   8. The fusion protein of claim 7, wherein the first portion comprises a receptor or binding protein sequence for VEGF, angiopoietin, TNF, IL-18, IL-4 and IL-13.   11. The fusion protein of claim 10, wherein the first portion comprises SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 15 or SEQ ID NO: 19.   The fusion protein of claim 1, wherein the fusion protein is glycosylated.   The fusion protein of claim 1, wherein the second cytokine is IL-1.   14. The fusion protein of claim 13, wherein the second portion is an IL-1 antagonist.   15. The fusion protein of claim 14, wherein the second portion comprises a sequence of IL-1ra (SEQ ID NO: 1) or a functionally equivalent analog thereof.   15. The fusion protein comprises SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24 or SEQ ID NO: 25. The fusion protein described.   An isolated nucleic acid comprising a sequence encoding the fusion protein of claim 1.   The nucleic acid according to claim 17, wherein the nucleic acid comprises a sequence encoding any one of SEQ ID NO: 1 to SEQ ID NO: 25.   A vector comprising the nucleic acid according to claim 17.   A host cell comprising the nucleic acid of claim 17.   A method for producing a polypeptide, comprising culturing the host cell of claim 20 in a medium under conditions capable of expressing the polypeptide encoded by the nucleic acid, and purifying the cultured cell or the cell medium. A method for producing the polypeptide, comprising:   A composition comprising the fusion protein of claim 1 or a nucleic acid encoding the fusion protein and a pharmaceutically acceptable carrier.   A method for modulating a patient's immune response, wherein a patient having or at risk of having a condition characterized by an excessive immune response is diagnosed, and the nucleic acid encoding the fusion protein of claim 1 or a nucleic acid encoding the fusion protein. A method of modulating an immune response comprising administering an effective amount of.   24. The method of modulating an immune response according to claim 23, wherein the patient has received or will undergo allogeneic or xenotransplantation.   24. The method for regulating an immune response according to claim 23, wherein the symptom is an inflammatory disease, an autoimmune disease, an allergic disease or an angiogenesis-dependent cancer.   26. The method of modulating an immune response according to claim 25, wherein the symptom is cancer and the fusion protein comprises SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 25. A method for extending the half-life of a recombinant protein in a patient comprising:
Linking the recombinant protein to a portion comprising SEQ ID NO: 1 or a functional equivalent thereof to form a fusion protein chimera;
A method for extending the half-life of a recombinant protein in a patient, comprising: affecting the half-life of the fusion protein in the patient, wherein the recombinant protein binds and neutralizes a cytokine or growth factor. A method for enhancing the efficacy of a recombinant protein in a patient comprising:
A method for enhancing the efficacy of a recombinant protein in a patient, comprising binding the recombinant protein to a portion containing SEQ ID NO: 1 or a functional equivalent thereof to form a fusion protein chimera, and affecting the efficacy of the fusion protein in the patient. A method for transporting therapeutic proteins to a target site of a patient, comprising:
Binding the therapeutic protein to a portion containing SEQ ID NO: 1 or a functional equivalent thereof to form a fusion protein chimera;
Administering the fusion protein chimera to a patient in need thereof, wherein the therapeutic protein targets an inflammatory site rich in IL-1 receptors, to the target site of the patient for the therapeutic protein Transportation method.   29. In a patient according to claim 28, wherein the portion comprising SEQ ID NO: 1 or a functional equivalent thereof binds to the IL-1 receptor and the recombinant protein is a therapeutic protein that binds to and neutralizes cytokines or growth factors. A method for enhancing the efficacy of a recombinant protein.   31. A set in a patient according to claim 30, wherein said fusion protein chimera binds and disables IL-1 receptor and cytokine or growth factor simultaneously at the patient's inflammatory site or disease site rich in receptors at IL-1. A method for enhancing the efficacy of a replacement protein. 31. Recombinant protein in a patient according to claim 30, wherein the fusion protein chimera neutralizes or antagonizes IL-1 and cytokine or growth factor activity at the patient's inflammatory site or disease site rich in IL-1 receptor. How to increase the efficacy of