CN117327198A

CN117327198A - Interleukin-1 receptor antagonist fusion protein and application thereof

Info

Publication number: CN117327198A
Application number: CN202311264159.XA
Authority: CN
Inventors: 邓磊修; 纪王双; 曲莉莉
Original assignee: Shandong Baimaiji Biotechnology Co ltd
Current assignee: Shandong Baimaiji Biotechnology Co ltd
Priority date: 2023-09-27
Filing date: 2023-09-27
Publication date: 2024-01-02

Abstract

The invention provides an interleukin-1 receptor antagonist fusion protein and application thereof, wherein the fusion protein consists of three parts of interleukin-1 receptor antagonist, connecting peptide and nano antibody of albumin. The interleukin-1 receptor antagonist fusion protein and the application thereof solve the technical problems of short half-life period of the recombinant interleukin-1 receptor antagonist in the related technology and frequent administration of patients.

Description

Interleukin-1 receptor antagonist fusion protein and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an interleukin-1 receptor antagonist fusion protein and application thereof.

Background

Interlukin-1 (IL-1) is an important class of pro-inflammatory cytokines, and when an organism is wounded or inflamed, mononuclear phagocytes at the site of inflammation produce large amounts of IL-1, bind to receptors located on the cell surface such as fibroblasts and T cells, and stimulate signaling and cell activation. This interaction results in a multicellular response, such as promotion of bone marrow release of neutrophils, induction of chemotactic infiltration of monocytes and polynuclear granulocytes into the local inflammation, and localized release of lysosomal enzymes. IL-1 also causes degranulation of basophils and mast cells, releasing inflammatory mediators.

The interleukin-1 family mainly includes IL-1α and IL-1β and IL-1 receptor antagonists (IL-1Ra), which bind to the IL-1R1 receptor but do not stimulate signaling and do not cause activation of downstream pathways, and are endogenous anti-inflammatory cytokines, which are natural specific antagonists of the pro-inflammatory cytokines IL-1α and IL-1β. IL-1ra blocks the pro-inflammatory effects of IL-1, including prostaglandin E2 and IL-6 release from endothelial cells, fever, thrombocytosis, production of acute phase proteins in the liver, and neutrophil expansion and infiltration.

Anakina, a genetically recombinant IL-1Ra produced by America Ind (Amgen), has been approved by the United states national drug and food administration (FDA) for improving symptoms and signs in patients with moderate and severe active rheumatoid arthritis, at 9 and 14 days 2001, slowing the progression of arthritic lesions. The medicine is suitable for patients with rheumatoid arthritis with age of more than or equal to 18 years and ineffective treatment by one or more anti-rheumatoid medicines. The most important difference between Anakinra and natural IL-1Ra is: (1) it is a non-glycosylated protein; (2) Its nitrogen terminal is one methionine more than the natural IL-1 Ra. Because of its small molecular weight (about 17 KD), it is easily cleared by the kidneys, has a short half-life, needs to be injected once a day, and sometimes accompanies an injection site reaction.

Accordingly, the prior art is subject to further development.

Disclosure of Invention

The invention aims to overcome the technical defects, and provides an interleukin-1 receptor antagonist fusion protein and application thereof, so as to solve the technical problems of short half-life period of recombinant interleukin-1 receptor antagonist drugs and frequent administration of patients in the related technology.

In order to achieve the technical purpose, the invention adopts the following technical scheme: an interleukin-1 receptor antagonist fusion protein is provided, which is formed by coupling an interleukin-1 receptor antagonist and a nano antibody of albumin through a connecting peptide. The interleukin-1 receptor antagonist is shown in SEQ ID NO.3, and has one more methionine at the nitrogen terminus than the native IL-1 Ra. The nanometer antibody (anti-HAS Nanobody) capable of being combined with human serum albumin is obtained after alpaca immunity, phage technology screening and humanized transformation optimization, and the single-domain antibody can be combined with human serum albumin with high affinity.

Further, the linker peptide is of the general formula [ GlyGlyGlySer ] n, wherein n is an integer from 1 to 4. The linker peptide is a flexible linker peptide consisting of glycine and serine, and mainly plays a role in maximally regulating the activity of proteins at both ends.

Preferably, the connecting peptide is GlyGlyGlySer.

According to the invention, through constructing IL-1Ra-Linker-anti-HSA Nanobody or anti-HSA Nanobody-Linker-IL-1Ra fusion proteins, the affinity and activity of two functional groups of IL-1Ra-Linker-anti-HSA Nanobody are verified through different coupling strategies, and the preferred sequences are respectively shown as SEQ ID No.1 and SEQ ID No. 2. Wherein SEQ ID NO.1 consists of: IL1Ra-GGGGS-VHH15, SEQ ID NO.2 consists of: VHH15-GGGGS-IL1Ra, wherein VHH15 is a humanized nano antibody which can be specifically combined with human serum albumin and is obtained through a series of screening optimization.

The nucleotide for encoding the interleukin-1 receptor antagonist fusion protein is obtained by adopting an artificial synthesis method, and codons preferred by a host can be selected during the artificial synthesis, so that the expression of a product can be often improved. The nucleic acid encoding the fusion protein may be inserted into the host chromosome or may exist as an episome.

The invention also provides a carrier carrying the interleukin-1 receptor antagonist fusion protein nucleotide sequence. The nucleic acid of the target protein sequence was cloned into various expression vectors using standard molecular cloning procedures described in J.Sam Brookfield et al, second edition, science Press, 1995.

The invention also provides host cells, including but not limited to mammalian, plant, insect, fungal and bacterial cells, bearing interleukin-1 receptor antagonist fusion protein nucleotide sequences. Bacterial cells include cells of gram-positive bacteria such as bacillus, streptomyces and staphylococcus species, cells of gram-negative bacteria such as escherichia and pseudomonas. Fungal cells include yeast cells such as Saccharomyces, pichia pastoris and Hansenula polymorpha (Hansenula polymorpha). Insect cells include Drosophila cells and Sf9 cells. Plant cells include cells from crop plants such as cereals, medicinal and ornamental plants or bulbs, and the like. Mammalian cells suitable for use in the present invention include epithelial cell lines (porcine etc.), osteosarcoma cell lines (human etc.), neuroblastoma cell lines (human etc.), epithelial cancers (human etc.), glial cells (murine etc.), hepatocyte lines (monkey etc.), CHO cells (chinese hamster ovary), COS cells, BHK cells, heLa cells, 911, AT1080, a549, 293 or per.c6, human ECC NTERA-2 cells, D3 cells of mESC cell lines, human embryonic stem cells such as HS293 and BGV01, SHEF1, SHEF2 and HS181, NIH3T3 cells, 293T, REH and MCF-7 and hMSC cells. The fusion protein may be present in the host cell or may be secreted from the host. Transformation of the desired nucleic acid into a host cell can be accomplished by conventional methods such as: electroporation, preparation of competent cells, and the like. Successfully transformed cells, i.e.cells containing the DNA construct of the invention, can be identified by well known techniques, such as by collecting and lysing the cells, extracting DNA, and then identifying by PCR methods. Alternatively, the proteins in the cell culture medium or in the cell disruption solution can be detected with anti-VHH or anti-IL-1 Ra antibodies.

The pharmaceutical composition of the invention comprises the interleukin-1 receptor antagonist fusion protein, a carrier, a host cell and a pharmaceutically acceptable carrier or additive. The fusion proteins and derivatives thereof of the present invention may be used alone, preferably in combination with one or more pharmaceutically acceptable carriers to form pharmaceutical formulations. Drug carriers should generally be compatible with the fusion protein and not deleterious to the recipient itself, typically water, saline, sugars, alcohols or amino acids, which need to be sterile and pyrogen free. The pharmaceutical formulations may be presented in unit dosage form and may be prepared by any method known in the art. These methods include the step of mixing the fusion protein with one or more accessory ingredients. Preferred pharmaceutical formulations include aqueous liquid formulations and lyophilized formulations having a water content of less than 5% or no water. These formulations may contain buffers, salts, small molecule sugars, etc., to provide the drug with a permeability equal to or similar to that of the recipient's blood. The pharmaceutical formulation may be present in a unit dose or multi-dose container, such as a sealed ampoule, a penicillin bottle or a prefilled syringe. The lyophilized preparation is prepared by lyophilizing liquid preparation, and adding sterile, pyrogen-free liquid carrier such as water for injection before use.

The invention provides application of the interleukin-1 receptor antagonist fusion protein, a vector, a host cell and a pharmaceutical composition in preparing medicines for treating diseases caused by the increase of interleukin 1.

The fusion proteins of the invention are useful in diseases caused directly or indirectly by or synergistically involved in the elevation of interleukin 1 including, but not limited to, the following: arthritis, colitis, rheumatoid arthritis, granulomatous lung disease, crohn's disease, periodic fever syndrome, gout acute episodes, IL-1 receptor antagonist deficiency, recurrent pericarditis, still's disease, autoimmune disease, tumor adjuvant therapy, and the like.

The beneficial effects are that:

1. the interleukin-1 receptor antagonist fusion protein of the invention has the long-life characteristic of albumin, can prolong the residence time of the medicine in the body and reduce the requirement of frequent administration.

2. The interleukin-1 receptor antagonist fusion protein provided by the invention combines the targeting characteristic of albumin nano-antibodies, can accurately convey medicines to inflammation sites, improves treatment effects and reduces side effects.

3. The interleukin-1 receptor antagonist fusion protein can be combined with the stability of albumin nano-antibodies, and can maintain the activity and structural integrity of the medicine.

Drawings

FIG. 1 is an electrophoresis diagram of a fusion protein employed in an embodiment of the present invention;

FIG. 2 is a graph of the biological activity of fusion proteins employed in an embodiment of the present invention;

FIG. 3 is a graph showing affinity of fusion proteins with human albumin for use in an embodiment of the invention;

FIG. 4 is a graph of the in vivo half-life of fusion proteins employed in an embodiment of the present invention.

Detailed Description

In order to make the present application solution better understood by those skilled in the art, the following description will be made in detail and with reference to the accompanying drawings in the embodiments of the present application, it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.

The present invention is further illustrated by the following examples, which are provided for illustrative purposes only and are not to be construed as limiting the scope or spirit of the invention. Those skilled in the art will appreciate that other variations or alternatives may be used for the purposes of the present invention. But rather the objects of the invention are defined only by the present description and the appended claims.

The instruments, reagents, and biological materials used in the examples are commercially available and are conventional in the art by experimental means unless otherwise specified.

Example 1 screening of nanobodies (anti-HSA nanobodies) that bind human serum albumin

1. Alpaca immunity

Commercially available Human Serum Albumin (HSA) and immune adjuvantGold Adjuvant, sigma company) to immunize alpaca. 200 microliters of protein plus adjuvant is immunized with 0.3mg each time, two points are injected subcutaneously, two points are injected intramuscularly, immunization is performed 5 times, the first 4 times are two weeks apart, the adjuvant is added each time, the last time is not added, and blood is taken after one week of the last immunization is completed.

2. Antibody library construction

Blood was taken one week after the last immunization of alpaca to isolate PBMC, RNA was extracted using Purelink RNA Micro kit, primeScript was used ^TM II 1st Strand cDNA Synthesis Kit reverse transcription to prepare cDNA, and two rounds of PCR amplification to obtain single-chain VHH antibody fragments (VHH is a nanobody, is an antibody with a single variable domain on a heavy chain, has a VHH domain and lacks a VL domain), connecting the single-chain VHH fragments with Pcomb3XSS phage display plasmids, electrically transforming TG1 competent cells, and establishing a phage antibody library with a library capacity of more than 5X 108.

3. Antibody screening

(1) Coating antigen: commercially available human HSA antigen was diluted with coating buffer, 2ug/ml, mixed well and added to the immune tube for 2.5ml total, 4 degree coating overnight.

(2) Recombinant phage collection: the above overnight culture broth was centrifuged at 600 g×10min and the supernatant was filtered through a 0.45um filter head. 2ml of PEG/NaCl was added, the mixture was placed on ice for 30-60min, centrifuged at 10000 g.times.20 min, the supernatant was removed, and the phage library was dissolved in 5ml of PBS.

(3) Closing: the immunization tube was washed twice with PBS, and blocking solution was added thereto at room temperature for 1h. In addition, 1ml of phage library was mixed with an equal volume of blocking solution and blocked at room temperature for 10-15min.

(4) Incubating the phage library: the immunization tube was washed with PBS 2 times, and the blocked phage library was added to the incubator at 37℃for 2-3 hours.

(5) Eluting: the immune tube was washed 8 times with PBST, 2 times with PBS, 2.5ML of eluent (0.2M glycine,pH 2.5) was added, left to stand for 10min, the eluent was transferred to a 15ML centrifuge tube, and 500ul of 1M Tris-HCl buffer (pH 8.0) was added for neutralization.

(6) Infection by adding 500ul of the above eluent into 10ml of TG1 bacteria solution, standing at 37deg.C for 30min. The bacterial solutions were diluted 10-fold, 100-fold and 1000-fold, 100ul of each of the above diluted bacterial solutions was plated, and 5ul of amp,37℃and 220rpm were added to each of the remaining solutions for 1 hour.

(7) 50ul of the helper phase is added into the bacterial liquid, and the temperature is 37 ℃,220rpm and 2 hours. The supernatant was centrifuged at 2500rpm X5min, and the pellet was suspended by 10ML 2 XYT-AK, and cultured overnight at 37℃and 220 rpm.

(8) Lower-round Panning: repeating the steps (1) - (7) and carrying out 3 rounds of Panning.

4. Antibody specificity, affinity identification, humanization

And (3) carrying out specificity and affinity identification on the antibody obtained in the step (3), carrying out humanization treatment on the antibody, and finally screening to obtain the antibody shown in SEQ ID NO.4, wherein the code of the antibody is VHH-15, and the single-domain antibody has specific high-affinity binding on human albumin.

Example 2 construction and expression of fusion proteins

The coding genes of the fusion proteins shown in SEQ ID No.1 and SEQ ID No.2 are artificially synthesized, an optimized host is Pichia pastoris, the synthesized fusion protein coding gene is connected with a pPIC9 plasmid (Invitrogen), and the synthesized fusion protein gene is inserted between XhoI and EcoRI sites at the downstream of a promoter of a pPIC9 vector AoX. Dissolving 5-20 mug of constructed linearization DNA in 5-10 mu l of TE solution, uniformly mixing with 80 mu l of pre-treated competent cells, transferring into a 0.2cm pre-cooled electric conversion cup, converting by adopting an electric shock method, immediately adding 1ml of 1M pre-cooled sorbitol solution after electric conversion to uniformly mix thalli, transferring into a 1.5ml EP tube, placing in a 30-DEG shaking table for low-speed culture for 1h, coating the electric conversion thalli suspension on an MD plate, and coating one plate every 200-600 mu l; plates were incubated at 30℃until single colonies appeared.

Several single colonies were picked from the MD plate into 5ml BMGY medium (while the corresponding single colony spots were kept on YPD plate for subsequent strain preservation) overnight for the next day of centrifugation, 3000rpm,5min, supernatant was discarded, resuspended in 5ml BMMY medium, induction was started, and induction time was 48-72 hours. After induction, SDS-PAGE electrophoresis detects protein expression, and colonies with high expression are taken and stored. And extracting the DNA of the high-expression strain, and sequencing to determine whether the high-expression strain contains the complete target fusion protein gene.

Inoculating the high-expression strain into shake flask for induction expression, inducing with methanol, centrifuging to collect supernatant, and adopting MonoRab ^TM Affinity chromatography of Anti-Humanized VHH Affinity Resin FF packing, equilibration and washing of Tris buffer salt solution: TBS 50mM Tris-HCl,150mM NaCl,pH 7.4; elution is an acidic elution buffer solution: 0.1M Glycine HCl,pH 2.5; neutralization buffer solution: 1M Tris-HCl, pH 9.0. The electrophoresis of the purified protein is shown in figure 1, wherein the 1st lane is SEQ ID NO.1 protein, and the protein number ILV01; lane 2 is the protein SEQ ID NO.2, protein number VLI02, the molecular weight of which is substantially consistent with the theoretical molecular weight.

EXAMPLE 3 identification of fusion protein IL-1Ra Activity

A375S 2 cell is a cell sensitive to IL-1β, to which 500pg/ml IL-1β is cytotoxic. IL-1ra addition competitively inhibited IL-1 beta cytotoxicity, and the biological activities of IL-1ra and fusion proteins were examined by measuring the cell viability.

A375.s2 cells were cultured in RPM1640/DMEM (1:1) medium containing 10% fetal bovine serum at 37 ℃,5% co2 and saturated humidity, passaged when grown as monolayers, and the cells were harvested by digestion to make a cell suspension of 8.0×104 cells/ml, and plated in 96-well plates at 100 μl per well. The final concentration of 100. Mu.l of the sample was set to a value of 5 groups such as IL-1β control group, 5ng/ml IL-1β+30nM IL-1Ra (R & D company), 5ng/ml IL-1β+30nM ILV01, 5ng/ml IL-1β+30nM VLI02, etc. After the addition of the drug, the mixture was incubated at 37℃for 72 hours with 5% CO2, and then CCK-8 dye was added thereto to measure the OD at a wavelength of 450 nm. See fig. 2 for results, wherein a: normal cell group without IL-1 β, B: adding 5ng/ml IL-1 beta to kill the control cell group; c: IL-1β+30nM IL-1Ra (R & D company) 5 ng/ml; d: IL-1β+30nM ILV01 cell group at 5 ng/ml; e:5ng/ml IL-1β+30nM VLI02 cell group. As can be seen from the figure, the activities of the fusion proteins ILV01 (SEQ ID NO. 1) and VLI02 (SEQ ID NO. 2) are basically consistent with the activities of natural IL-1Ra, and the fusion proteins have the function of competing and inhibiting the biological activity of IL-1 beta.

Example 4 determination of affinity of fusion proteins to human albumin

(1) Human serum albumin HSA protein was diluted with coating solution and added to 96-well elisa plates at 100ng HSA per well overnight at 4 ℃ and a corresponding negative control was set.

(2) Plates were washed with 300. Mu.L of 0.1% PBST, allowed to stand for 3min, and each well was further filled with 300. Mu.L of 5% skimmed milk powder and blocked at 37℃for 2h.

(3) The plate was washed with 300. Mu.L of 0.1% PBST, once, left to stand for 3min, and the purified VHH15 protein, fusion protein ILV01 and fusion protein VLI02 were diluted 8 concentration gradients at an initial concentration of 10nM, at a 1:2 column-fold ratio, added to the ELISA plate at 100. Mu.L/well, and incubated for 1h at 37 ℃.

(4) Plates were washed three times with 300 μl of 0.1% pbst solution added to each well, allowed to stand for 3min each time, unbound antibody removed, and 100 μl1 was added to each well: 5000 dilution of Mouse anti-Human-IL-1Ra mAb (R & D company), incubation at 37℃for 1h.

(5) Plates were washed three times with 300 μl of 0.1% pbst solution added to each well, allowed to stand for 3min each time, unbound antibody removed, and 100 μl1 was added to each well: 5000 dilution of HRP-Goat Anti-Mouse IgG (H+L) (R & D company), incubation at 37℃for 1H.

(6) 300. Mu.L of 0.1% PBST solution is added to each well, the plate is washed five times, each time the plate is kept stand for 3min, 100. Mu.L of TMB solution is added to the ELISA plate, and the plate is incubated at 37 ℃ in a dark place for 10min.

(7) And adding 100 mu L of 2.29% sulfuric acid into each hole of the ELISA plate to terminate the reaction, measuring OD values at 450nm and 630nm by using an ELISA plate, and calculating the difference value of OD 450-OD 630 to obtain the OD value.

Results:

the results are shown in FIG. 3, wherein curve A is the VHH15 control, curve B is the fusion protein ILV01 (SEQ ID NO. 1), and curve C is the fusion protein VLI02 (SEQ ID NO. 2). From the results, it can be seen that the affinity of the fusion proteins ILV01, VLI02 to human serum albumin is substantially consistent with the anti-HSA nanobody VHH 15.

Example 5 half-life assay of fusion proteins

1. Administration and blood sampling method rat subcutaneous injection administration was performed by respectively: IL1ra (1 mg/kg), ILV01 (2 mg/kg) and VLI02 (2 mg/kg), blood samples of animals are collected at different time points, centrifuged for 30min, serum is separated, and the samples are stored at-20 ℃ for testing. The blood sampling parameters were as follows: ILV01 (2 mg/kg), VLI02 (2 mg/kg), blood sampling time: 0,0.5,1,2,4,8, 12, 24, 48, 72, 96, 120h; IL1ra (1 mg/kg) blood sampling time: 0,5, 15, 30min,1,2,4,8, 12, 24h.

2. The samples were tested by the double antibody sandwich ELISA method using the Human IL1ra ELISA kit (R & D).

1) The capture antibody was diluted to working concentration (PBS dilution), the diluted capture antibody was plated at 100 ul/well into 96-well plates, and the plates were incubated overnight at room temperature.

2) The plate washer washes the plate three times, each with 300ul of wash.

3) 300ul of dilution reagent was added to each well and incubated for 1h at room temperature.

4) And (5) repeating the step 2.

5) 100ul of sample or standard (in diluted reagent) was added to each well, sealed and incubated for 2h at room temperature.

6) And (5) repeating the step 2.

7) 100ul of detection antibody (diluted) was added to each well, sealed and incubated for 2h at room temperature.

8) And (5) repeating the step 2.

9) 100ul of secondary antibody is added, sealed and protected from light, and incubated for 20min at room temperature.

10 Step 2) is repeated.

11 100ul of substrate solution was added to each well, incubated at room temperature for 20min, protected from light.

12 Another 50ul of stop solution was added to each well and the plate was gently tapped to ensure adequate mixing.

13 Optical density was measured with a microplate reader at 450 nm.

3. Drawing a pharmaceutical generation curve by adopting Excel software; the results are shown in FIG. 4, wherein A is IL1ra (1 mg/kg) administration group, B is ILV01 (2 mg/kg) administration group, and C is VLI02 (2 mg/kg) administration group, and the half-life of the fusion proteins ILV01 and VLI02 is obviously prolonged.

It should be noted that the terms "first," "second," and the like in the description and claims of the present application and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that embodiments of the present application described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

Alternatively, specific examples in this embodiment may refer to examples described in the foregoing embodiments, and this embodiment is not described herein.

The foregoing embodiment numbers of the present application are merely for describing, and do not represent advantages or disadvantages of the embodiments.

In the foregoing embodiments of the present application, the descriptions of the embodiments are emphasized, and for a portion of this disclosure that is not described in detail in this embodiment, reference is made to the related descriptions of other embodiments.

The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application and are intended to be comprehended within the scope of the present application.

Claims

1. An interleukin-1 receptor antagonist fusion protein, characterized in that the fusion protein consists of three parts of an interleukin-1 receptor antagonist, a connecting peptide and a nano antibody of albumin.

2. The interleukin-1 receptor antagonist fusion protein of claim 1, wherein and the linker peptide is of the general formula [ GlyGlyGlyGlySer ] n, wherein n is an integer from 1 to 4.

3. The interleukin-1 receptor antagonist fusion protein of claim 2, wherein the linker peptide is glyglyglyser.

4. The interleukin-1 receptor antagonist fusion protein of claim 3, wherein said fusion protein is: interleukin-1 receptor antagonist-connective peptide-albumin nanobody and albumin nanobody-connective peptide-interleukin-1 receptor antagonist, the sequences of which are shown in SEQ ID No.1 and SEQ ID No.2 respectively.

5. A nucleic acid encoding the interleukin-1 receptor antagonist fusion protein of claim 1.

6. A vector carrying a vector capable of expressing the nucleic acid of claim 5.

7. A host cell harboring the vector of claim 6.

8. A pharmaceutical composition comprising the interleukin-1 receptor antagonist fusion protein of any one of claims 1-4, the vector of claim 6, the host cell of claim 7, and a pharmaceutically acceptable carrier or additive.

9. Use of an interleukin-1 receptor antagonist fusion protein according to any one of claims 1-4, a vector according to claim 6, a host cell according to claim 7 and a pharmaceutical composition according to claim 8 for the preparation of a medicament for the treatment of a disease caused by elevated interleukin 1.

10. The use according to claim 9, wherein the disease caused by elevated interleukin 1 comprises: rheumatoid arthritis, colitis, rheumatoid arthritis, granulomatous lung disease, crohn's disease, periodic fever syndrome, gout acute episodes, IL-1 receptor antagonist deficiency, recurrent pericarditis, steve's disease, and autoimmune disease.