WO2012002562A1

WO2012002562A1 - Modified protein therapeutics

Info

Publication number: WO2012002562A1
Application number: PCT/JP2011/065407
Authority: WO
Inventors: Yasuhiko Masuho; Hiroaki Nagashima; Kaname Kaneko; Ayaka Yamanoi
Original assignee: Tokyo University of Science
Current assignee: Tokyo University of Science
Priority date: 2010-06-30
Filing date: 2011-06-24
Publication date: 2012-01-05
Anticipated expiration: 2012-12-30

Abstract

The invention relates to a fusion protein including a first region including an extracellular domain of a cytokine receptor, and a second region fused with the first region, and including immunoglobulin Fc domains that are tandemly repeated, or single-chain immunoglobulin Fc domains that are tandemly repeated.

Description

DESCRIPTION

MODIFIED PROTEIN THERAPEUTICS

Technical Field

[0001] The present invention relates to a fusion protein, a therapeutic agent and a therapeutic method.

Background Art

[0002] Tumor necrosis factors (hereinafter sometimes abbreviated to "TNFs") include TNFa, and TNFP (also referred to as "lymphotoxin a"). Tumor necrosis factor receptor 1 (also referred to as, for example, "tumor necrosis factor receptor 1 A isoform") and tumor necrosis factor receptor 2 (also referred to as, for example, "tumor necrosis factor receptor IB") are receptors of these tumor necrosis factors. Binding of TNF to these receptors causes cellular responses of immunity or inflammation.

[0003] Biological drugs of TNF-a antagonists include infliximab, adalimumab, etanercept, certolizumab pegol and golimumab.

[0004] From among these biological drugs, etanercept is a kind of Fc fusion protein consisting of two extracellular domains of the tumor necrosis factor receptor 2 (TNFRII) linked to the Fc region of IgGl (see, for example, J Immunol (1993) 151 :1548-1561).

Etanercept is able to bind to a soluble form of TNF-a (sTNF-a) and a transmembrane TNF-a (tmTNF-a), and exerts a potent clinical effect on rheumatoid arthritis (RA) (see, for example, N Engl J Med (2000) 343:1586-1593).

[0005] Further, infliximab, adalimumab, certolizumab pegol and golimumab are anti-TNFa antibodies.

[0006] When etanercept, which is a fusion protein, is compared with anti-TNFa antibodies, etanercept differs from other antagonists in that etanercept lacks efficacy in Crohn disease (see, for example, Gastroenterology (2001) 121:1088-1094) and is less efficacious in psoriasis (see, for example, N Engl J Med (2003) 349:2014-2022). As to cytotoxicity, several studies showed that infliximab and adalimumab induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in tmTNF-a-transfected cells, whereas etanercept induced no or low levels of cytotoxicities (see, for example, Cytokine (2009) 45:124-131, ARTHRITIS & RHEUMATISM (2008) 58, 1248-1257 and Cancer Res (2008) 68:3863-3872).

[0007] These differences may result from their biological characteristics. Etanercept can bind to TNF-a in 1 : 1 molar ratio, whereas 3 molecules of other monoclonal antibody (mAb) agents bind to one molecule of TNF-a, and the dissociation rate of etanercept is more rapid than those of the others (see, for example, J Pharmacol Exp Ther (2002) 301 :418-426).

Further, etanercept is reported to have a half-life of 70 hours to 100 hours in human blood (see, for example, J Pharm Pharmacol. (2005) ,57 (11), 1407-13), while infliximab and

adalimumab, which are anti-TNFa antibodies, have retention time of about 3 weeks. As demonstrated above, the half-lives of infliximab and adalimumab in blood are known to be longer than that of etanercept.

[0008] Anti-CD20 mAbs having Fc domains connected in tandem bind to Fey receptors, FcyRIA, FcyRIIA, FcyRIIB and FcyRIIIA with higher avidities than the parental mAb, resulting in enhanced ADCC (see, for example, Mol Immunol (2008) 45:2752-2763 and International Publication WO2007/100083 pamphlet).

[0009] Further, production of an antibody having two Fc domains tandemly connected to a monoclonal antibody CAMPATH-IH against CDw52 antigen have been reported (see, for example, Therapeutic Immunol, (1994) 1, 247-255).

SUMMARY OF THE INVENTION

Technical Problem

[0010] Among biological drugs, fusion proteins, typified by etanercept, are desired to have enhanced ADCC activities and enhanced CDC activities, and an elongated half-lives in blood, so as to broaden the applications thereof to treatment of a wide range of diseases.

[0011] Anti-CD20 mAbs to which Fc domains are tandemly connected have been confirmed to have high affinity for Fey receptors compared to ordinary anti-CD20 mAbs, and have an increased ADCC activity. It has also been confirmed that CDC activity is not affected even in the case of a fusion protein of a Fc multimer and a variable region of an anti-CD20 mAb. Further, it has been confirmed that an antibody obtained by tandemly connecting two

CAMPATH-IH antibodies has a lower CDC activity than that of a single CAMPATH-IH antibody. That is, contribution to CDC activity varies depending on the type of antibody and the type of target site. Therefore, tandemly connected Fc regions do not necessarily increases ADCC activity and CDC activity, and further technical development has been requested with a view to enhancing ADCC activity and CDC activity.

[0012] An object of the present invention is provision of a fusion protein which has an enhanced ADCC activity and an enhanced CDC activity, an elongated half-life in blood, and which is capable of exerting high therapeutic effects against various diseases.

Solution to Problem

[0013] Aspects of the present invention include the following.

< 1 > A fusion protein comprising : a first region comprising an extracellular domain of a cytokine receptor; and a second region comprising plural immunoglobulin Fc domains or plural single-chain immunoglobulin Fc domains, and fused with the first region,

wherein the plural immunoglobulin Fc domains or plural single-chain

immunoglobulin Fc domains comprise tandemly repeated immunoglobulin Fc domains or tandemly repeated single-chain immunoglobulin Fc domains.

<2> The fusion protein according to <1>, wherein the cytokine receptor is a TNFa receptor.

<3> The fusion protein according to <2>, wherein the TNFa receptor is a TNFa receptor II.

<4> The fusion protein according to any one of <1> to <3>, wherein the immunoglobulin is IgG.

<5> The fusion protein according to <4>, wherein the IgG is human IgGl .

<6> The fusion protein according to any one of <1> to <5>, wherein adjacent domains in the tandemly repeated immunoglobulin Fc domains or the tandemly repeated single-chain immunoglobulin Fc domains are linked to each other via a linker peptide.

<7> The fusion protein according to <1> to <6>, wherein the linker peptide is a trimer of glycine-glycine-glycine-glycine-serine.

<8> The fusion protein according to any one of <1> to <7>, wherein the tandemly repeated immunoglobulin Fc domains or the tandemly repeated single-chain immunoglobulin Fc domains are composed of two or three immunoglobulin Fc domains that are connected in series, or of two or three single-chain immunoglobulin Fc domains that are connected in series.

<9> A therapeutic agent for treating an inflammation-related disease, the therapeutic agent comprising the fusion protein of any one of <1> to <8>.

<10> The therapeutic agent according to <9>, wherein the inflammation-related disease is Crohn disease.

<11> A method for treating an inflammation-related disease, comprising administering the fusion protein of any one of <1> to <8> to a patient in need thereof.

<12> The method for treating an inflammation-related disease according to <11>, wherein the inflammation-related disease is Crohn disease.

Advantageous Effects of Invention

[0014] According to the invention, a fusion protein which has an enhanced ADCC activity and an enhanced CDC activity, an elongated half-life in blood, and which is capable of exerting high therapeutic effects against various diseases, can be provided. BRIEF DESCRIPTION OF DRAWINGS

[0015] Fig. 1 A shows cDN A constructs of TNFRII-Fc and TNFRII-Fc-Fc.

[0016] Fig. 1 B shows schematic illustrations of TNFRII-Fc, TNFRII-Fc-Fc and

TNFRII-Fc-Fc (single).

[0017] Figs. 2A, 2B, 2C and 2D show gel-filtration HPLC. Figs. 2E and 2F show

SDS-PAGE analyses.

[0018] Fig. 3 A shows binding activities of Fc fusion proteins to sTNF-a.

[0019] Fig. 3B shows neutralizing activities of Fc fusion proteins against sTNF-ot

cytotoxicity.

[0020] Figs. 4A and 4B show binding activities of Fc fusion proteins to tmTNF-a on CHO-DG44.

[0021] Figs. 5 A and 5B show CDC activities of Fc fusion proteins.

[0022] Figs. 6 A, 6B, 6C, 6D and 6E show binding activities of Fc fusion proteins to Fey receptors.

[0023] Figs. 7A, 7B and 7C show ADCC activities of Fc fusion proteins.

[0024] Fig. 8 shows the effect of fusion proteins on the body weight of BALB/c mice with

DSS-induced colitis.

DESCRIPTION OF EMBODIMENTS

[0025] The present invention is described in detail below. Although the below descriptions of the constituent elements sometimes refer to representative embodiments of the invention, the invention is by no means limited to the embodiments.

[0026] The fusion protein of the invention is a fusion protein including a first region including an extracellular domain of a cytokine receptor and a second region which is fused to the first region, and which includes tandemly repeated immunoglobulin Fc domains or tandemly repeated single-chain immunoglobulin Fc domains.

[0027] It is presumed that the fusion protein of the invention exhibits increased frequency and strength of binding to Clq, which is a component of a complement, and exhibits an enhanced ADCC activity and an enhanced CDC activity, due to fusion of the first region including an extracellular domain of a specific cytokine receptor and a second region having a specific structure including tandemly repeated immunoglobulin Fc domains or tandemly repeated single-chain immunoglobulin Fc domains. Further, it is presumed that an elongated half-life of the fusion protein of the invention in blood results from the fusion of the first region and the second region. Therefore, it is presumed that these are the reasons why the fusion protein of the invention produces high therapeutic effects against a wide range of inflammation-related diseases, to which conventional fusion proteins are ineffective.

[0028] <First Region>

[0029] In the fusion protein of the invention, the first region includes an extracellular domain of a cytokine receptor.

[0030] Examples of the cytokine receptor include: interleukin receptors such as TNFa receptor, interleukine-1 receptor, IL-6 receptor and IL-17 receptor; G-CSF receptor; GM-CSF receptor; receptors for vascular endothelial growth factors (VEGF), epidermal growth factors (EGF), thrombopoetin, erythropoietin, CC chemokines, C chemokines, CXC chemokines and CX₃C chemokines; cell adhesion molecules such as integrins; erythropoietin receptor, thrombopoietin receptor, epidermal growth factor receptor, platelet-derived growth factor receptor, vascular endothelial growth factors (VEGF) receptor and insulin receptor.

Preferable examples of the cytokine receptor include TNFa receptor, EGF receptor and VEGF receptor. The cytokine receptor is particularly preferably TNFa receptor, from the viewpoint of suppressing inflammation.

[0031 ] It has been found that both of TNFa receptor I and TNFa receptor II are involved in many TNF-a-related inflammations (Crit. Rev. Eukaryot. Gene. Expr. (2010) 20, 87-103). From this viewpoint, examples of TNFa receptors include TNFa receptor I and TNFa receptor II. Further, TNFa receptor II is most preferable from the viewpoint of the strength of the affinity for TNFa.

[0032] The cytokine receptor is preferably a cytokine receptor derived from human.

However, the cytokine receptor is not limited to a human-derived cytokine receptor, and known cytokine receptors having activities against the binding site comparable to the activities of human-derived cytokine receptors may be used.

[0033] The extracellular domain is not limited as long as the extracellular domain is an extracellular domain of the cytokine receptor described above. The extracellular domain of TNFa receptor II is hereinafter sometimes abbreviated to "TNFRII". The first region may consists of at least one (i.e., one, or more than one) extracellular domain of a cytokine receptor.

[0034] <Second Region>

[0035] In the fusion protein of the invention, the second region includes tandemly repeated immunoglobulin Fc domains or tandemly repeated single-chain immunoglobulin Fc domains.

[0036] The immunoglobulin may be any of IgA, IgG, IgD, IgE or IgM, and is preferably IgG from the viewpoints of binding affinity for a Fc receptor, activation potency for complements and retention properties in the body. [0037] The IgG may be any of human IgGl, human IgG2, human IgG3 or humanIgG4, and is preferably human IgGl from the viewpoints of purification by protein A, binding affinity for Fc receptors, activation potency for complements and retention properties in the body.

[0038] In the second region of the fusion protein of the invention, two or more of the immunoglobulin Fc domain are connected in tandem. In the invention, the plural

immunoglobulin Fc domains are immunoglobulin Fc domains derived from the same type of immunoglobulin.

[0039] The second region in the invention may include unmodified immunoglobulin Fc domains. Alternatively, the second region may include single-chain immunoglobulin Fc domains. The term "single-chain immunoglobulin Fc domain" as used herein refers to either one of the two chains constituting an immunoglobulin Fc domain, which may be obtainable by separation from the other chain by cleavage of disulfide bonds linking the two chains.

[0040] In the invention, plural immunoglobulin Fc domains are connected in series. Each of the linker peptides for connecting immunoglobulin Fc domains may be any of the linker peptides described below. That is, the amino acid sequence of the linker peptide may be any amino acid sequence as long as (i) the linker peptide has a flexible structure, (ii) the linker peptide has a certain length, and (iii) the linker peptide is not degraded in the blood.

Examples of the linker peptide include a monomer of glycine-glycine-glycine-glycine-serine, a dimer of glycine-glycine-glycine-glycine-serine, a trimer of

glycine-glycine-glycine-glycine-serine, which is abbreviated as (G₄S)₃, and

SGGGSGG(GSEGGG)n (n is from 1 to 5) (SEQ ID NO: 1 to SEQ ID NO: 5). The linker peptide is more preferably a trimer of glycine-glycine-glycine-glycine-serine (SEQ ID NO: 6). These linkers may be used also in a case in which the second region includes single-chain immunoglobulin Fc domains.

[0041] The number of the immunoglobulin Fc domains included in the protein of the invention is normally from two to five. It is preferable that the protein includes two or three tandemly repeated immunoglobulin Fc domains.

[0042] The number of the single-chain immunoglobulin Fc domains included in the protein of the invention is normally from two to five. It is preferable that the protein includes two or three tandemly repeated single-chain immunoglobulin Fc domains.

[0043] Regardless of whichever of the tandemly repeated immunoglobulin Fc domains or the tandemly repeated single-chain immunoglobulin Fc domains are included in the second region, the tandemly repeated immunoglobulin Fc domains or the tandemly repeated single-chain immunoglobulin Fc domains include a hinge region of immunoglobulin for connecting the first region including an extracellular domain of a cytokine receptor and the second region. The hinge region may be any hinge region of any human immunoglobulin (refer to, for example, J. Immunol. (2006), 177, 1129-1138), and is not limited to a hinge region having a naturally-occurring sequence as long as the function as a hinge region of a human immunoglobulin can be carried out. The second region may consists of tandemly repeated immunoglobulin Fc domains or tandemly repeated single-chain immunoglobulin Fc domains (including the hinge region).

[0044] <Fusion Protein>

[0045] In the invention, the fusion protein is a protein formed through transcription of a gene encoding the first region and a gene encoding the second region in an integrated manner, and an integrated expression of the first and second regions from the transcript.

[0046] The fusion protein of the invention is preferably a fusion protein including a first region including an extracellular domain of TNF a receptor, and a second region which is fused with the first region, and which includes two human IgG Fc domains of that are linked in series via linker peptides each including (G₄S). It is also preferable that the fusion protein of the invention is a fusion protein including a first region including an extracellular domain of TNF a receptor, and a second region which is fused with the first region, and which includes two single-chain human IgG Fc domains that are linked in series via a linker peptide including (G₄S).

[0047] The fusion protein of the invention is more preferably a fusion protein including a first region including an extracellular domain of TNFa receptor II, and a second region which is fused with the first region, and which includes two human IgGl Fc domains that are linked in series via linker peptides each including (G₄S). The fusion protein is more preferably a fusion protein including a first region including an extracellular domain of TNFa receptor II, and a second region which is fused with the first region, and which includes two single-chain human IgGl Fc domains that are linked in series via a linker peptide including (G₄S).

[0048] Here, the fusion protein can be prepared according to known methods, such as genetic recombination methods using a cDNA encoding the above-described protein, and chemical crosslinking methods for proteins. The fusion protein may consist of the first and the second regions that are fused with each other.

[0049] <Therapeutic Agent and Therapeutic Method>

[0050] The therapeutic agent of the invention is a therapeutic agent for treating a

inflammation-related disease which includes the above-described fusion protein.

[0051] The therapeutic method of the invention is a therapeutic method for treating an inflammation-related disease, the method including administering the above-described fusion protein to a patient in need thereof. [0052] Examples of the inflammation-related disease include inflammatory diseases, autoimmune diseases and central demyelinating diseases. Among them, the therapeutic agent and the therapeutic method are more suitable for treating Crohn disease, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, plaque psoriasis, polyarticular juvenile idiopathic arthritis, multiple sclerosis, juvenile idiopathic arthritis, Wegener's granulomatosis, or the like.

[0053] Examples of the Crohn disease include Crohn disease, fistulizing Crohn disease, moderate to severe Crohn disease and ulcerative colitis.

[0054] The therapeutic agent of the invention may further include a pharmaceutically acceptable carrier or additive such as a preservative, a stabilizer, a salt such as sodium chloride, or a buffer such as sodium phosphate. Specific examples thereof include sterile water, physiological saline, stabilizers, excipients, buffers, preservatives, detergents, binders and protein stabilizers such as polyethylene glycol. If necessary, the therapeutic agent of the invention may further include a suspending agent, a solubilizer, an isotonizing agent, an adsorption inhibitor, a diluent, a filler, a pH modifier, a soothing agent, a sulfur-containing reducing agent or an antioxidant. The administration pathway of the therapeutic agent of the invention is not specifically limited, and is preferably via a parenteral route, for example, by injection (such as subcutaneous, intravenous or intramuscular injection).

[0055] The fusion protein of the invention may be purified by a known method, such as protein A affinity chromatography, ion-exchange chromatography, or gel filtration

chromatography, and may be formulated into a drug according to a known method.

[0056] In regard to the dosage of the fusion protein in the invention, a suitable dosage may be determined in accordance with the type of disease to be treated. In order to treat Crohn disease, the dosage in terms of the fusion protein amount per week may be, for example, from 10 mg to 100 mg, and is preferably from 10 mg to 50 mg, and more preferably from 5 mg to 20 mg.

EXAPMLES

[0057] Examples of the invention are described below. However, the invention is not limited by the examples. In the descriptions below, "%" is based on mass unless indicated otherwise.

[0058] The materials employed in Examples 1 to 9 are as follows.

[0059] <Materials >

[0060] [Cell lines]

[0061] CHO-DG44 cells were purchased from Invitrogen (Carlsbad, CA) and cultured in IMDM containing 10% fetal bovine serum (FBS). L929 cells and KHYG-1 cells were kindly provided by the Institute of Development, Aging and Cancer, Tohoku University (Sendai, Miyagi). L929 cells were cultured in DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, and KYHG-1 cells were cultured in RPMI1640 containing 10% FBS, 10 ng/ml IL-2 (Wako Pure Chemical Industries, Osaka), 100 U/ml penicillin and 100 μg/ml streptomycin.

[0062] [Construction of cDNAs, production and purification of TNFRII-Fc and

TNFRII-Fc-Fc]

[0063] The cDNAs encoding the TNFRII-Fc and the TNFRII-Fc-Fc were constructed using PCR and restriction enzymes. The cDNA encoding the extracellular domain of TNFRII was assembled with the cDNA of the human IgGl Fc region (from hinge to CH3 domain) to construct the cDNA of TNFRII-Fc in pcDNA3.1/Zeo (Invitrogen, Carlsbad, CA) as shown in Fig. 1 A. To generate TNFRII-Fc-Fc, the cDNA of the tandemly repeated Fc was connected to the downstream of the cDNA encoding the extracellular domain of TNFRII. The gene encoding the flexible peptide linker (G₄S)3 was inserted between the two Fc cDNAs. The linearized expression vectors were transfected into CHO-DG44 cells with TransFast

Transfection Reagent (Promega, Madison, WI), and the transfected cells were cultured at 37°C in IMDM containing 2% FBS and 100 μ§/τα\ zeocin (Invitrogen) in flat-bottomed BD Falcon 96-well microplates (BD Biosciences, San Jose, CA) for a week. After the cells were cloned by limited dilution, the cloned cells were cultured in CD CHO medium (Invitrogen). TNFRII-Fc and TNFRII-Fc-Fc were purified from the culture supernatants by a protein A-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) column chromatography and subsequent gel-filtration HPLC on a TSK-GEL G3000SWXL (Tosoh, Tokyo) as previously described (Mol Immunol (2008) 45:2752-2763). The purified Fc fusion proteins were concentrated by ultrafiltration using Amicon Ultra-4 50,000 MWCO (Millipore, Billerica, MA) in 20 mM phosphate buffer (pH7.4) containing 0.14 M NaCl (PBS). The purity of Fc fusion proteins were analyzed by SDS-PAGE under reducing and non-reducing conditions.

[0064] <Example 1> Biochemical analyses of Fc fusion proteins

[0065] The biochemical properties of the Fc fusion proteins were analyzed by gel-filtration HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (Mol Immunol (2008) 45:2752-2763). Briefly, the fusion proteins were analyzed by gel-filtration HPLC on a Protein Pak G3000SWXL column (Tosoh, Tokyo, Japan) at a flow rate of 1 ml/min in 0.1 M sodium phosphate (pH 6.8). In addition, the fusion proteins were analyzed by SDS-PAGE under reducing and non-reducing conditions on 10% and 6% polyacrylamide gels, respectively. [0066] The number of free sulfhydryl residues in the individual fusion proteins was measured using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) according to the manufacturer's instructions. The protein concentrations were determined by measurement of the absorbance at 280 nm. The protein solutions were allowed to react with 4 mM DTNB in 0.1 M phosphate buffer (pH 8.0) containing 1 mM EDTA at 37°C for 15 min. The number of free sulfhydryl residues was determined by measurement of the absorbance at 412 nm.

[0067] Human KH YG- 1 cells stably expressing human FcyRIIIA (KH YG- 1 / FcyRIIIA) were developed by culturing of the cells with the culture supernatants of PL AT- A cells that were transfected with retroviral vector pMXs-puro containing the FcyRIIIA cDNA. Cells highly expressing FcyRIIIA were sorted by flow cytometry and were cultured in a similar manner to the parental KHYG-1 cells.

[0068] The structures of TNFRII-Fc and TNFRII-Fc-Fc are shown schematically in Fig. IB, and the calculated MWs are approximately 150kDa and 200kDa, respectively. The results are shown in Figs. 2A, 2B, 2C, 2D, 2E and 2F. TNFRII-Fc(2A) is separated by gel-filtration HPLC. TNFRII-Fc-Fc (Protein A-purified) proteins (2B) are purified by protein A-agarose and separated by gel-filtration HPLC. TNFRII-Fc-Fc proteins (2C) and TNFRII-Fc-Fc (single) proteins (2D) are separated by gel-filtration HPLC. These proteins were

electrophoresed under reducing (2E) and non-reducing (2F) conditions. Lane 1 : TNFRII-Fc, lane 2: TNFRII-Fc-Fc and lane 3: TNFRII-Fc-Fc (single).

[0069] TNFRII-Fc (Fig. 2A) and TNFRII-Fc-Fc (Fig. 2B) were purified from the culture supernatants of the respective transformed cells by means of protein A affinity

chromatography and consequent gel-filtration HPLC. Gel-filtration HPLC revealed two TNFRII-Fc-Fc proteins of different molecular weights (Fig. 2B); one larger and the other smaller than TNFRII-Fc. Those two TNFRII-Fc-Fc proteins were analyzed by SDS-PAGE. Under reducing conditions, both proteins showed a single band of 100 kDa, which is equal to the calculated value of a single chain of TNFRII-Fc-Fc (Fig. 2C). Under non-reducing conditions, the large and small TNFRII-Fc-Fc molecules showed a molecular weight of about 200 kDa and 80 kDa, respectively (Fig. 2D). These results suggest that the smaller molecule is a half molecule of TNFRII-Fc-Fc without disulfide bonds between the two H chains at the hinge region. The smaller molecule, which is herein designated as "TNFRII-Fc-Fc (single)", has a structure that is illustrated schematically at the right end of Fig. IB. The free sulfhydryl residues of these molecules were measured using DTNB, and TNFRII-Fc,

TNFRII-Fc-Fc and TNFRII-Fc-Fc (single) had 0.64, 0.96 and 0.46 free sulfhydryl residues per molecule, respectively. The results suggest that cysteine residues of TNFRII-Fc-Fc (single) would form intrachain disulfide bonds instead of interchain disulfide bonds. [0070] <Example 2> sTNF-a binding assay by ELISA

[0071] The binding activities of these Fc fusion proteins (TNFRII-Fc, TNFRII-Fc-Fc and TNFRII-Fc-Fc (single)) to sTNF-a were measured by ELISA. Flat-bottomed BD Falcon 96-well flexible PVC microplates (BD Biosciences) were coated with 50 μΐ of 1 μg/ml TNF-a (Wako Pure Chemical Industries) in PBS at 4°C for 18 hours. After the solution was discarded, the plates were blocked with 150 μΐ of PBS containing 0.1% bovine serum albumin (BSA) at room temperature for 2 hours. The blocking solution was then discarded, 50 μΐ of serially diluted Fc fusion proteins were incubated at room temperature for 2 hours. After the plates were washed three times with 150 μΐ of PBS containing 0.05% Tween 20 (PBS-T), the plates were incubated with 50 μΐ of 0.2 μg/ml HRP-conjugated goat anti-human IgG gamma chain (Tago, Burlingame, CA) in PBS containing 0.1% BSA. After the plates were washed three times with PBS-T again, 50 μΐ of ELISA Substrate Buffer (1 mM

3,3',5,5'-tetramethylbenzidine, 1.3 mM Ν,Ν'-dimethylformamide, 1 mM benzene sulfonic acid sodium salt, 20 mM acetic acid, 0.03% hydrogen peroxide, pH5.0) was added into individual wells and the plates were allowed to stand at room temperature for 10 min under protection from light. The reaction was stopped by the addition of 50 μΐ of 0.5 M H2SO4. Absorbance at 450 nm was measured using a microplate reader Model 550 (BIO-RAD, Hercules, CA). Samples were analyzed in triplicate and the values were expressed as the mean and S.D.

[0072] Binding activities of the Fc fusion proteins to a soluble form of TNF-a (sTNF-a) were determined by ELISA using sTNF-a-coated plates (Fig. 3 A). The dose response curve of TNFRII-Fc-Fc was very close to that of TNFRII-Fc, implying that multimerization of Fc domain does not affect its TNF-a binding activity. However, TNFRII-Fc-Fc (single) gave an absorbance of 0.6 at the concentration that was 10 to 20 times higher than that of

TNFRII-Fc-Fc, suggesting its low affinity for sTNF-a. In Fig. 3 A, "Human Fc" means a Fc fragment obtained by cleaving human IgG (available from Takara Bio Inc.) with papain, and the same applies hereinafter.

[0073] <Example 3> TNF-a neutralization assay

[0074] Neutralizing activities of the Fc fusion proteins against TNF-a-dependent cytotoxicity were measured using murine fibrosarcoma L929 cells as described previously (J Control Release (2008) 129:179-186). Briefly, 50 μΐ of 2 ^χ 10⁵ cells/ml L929 cells (1 ^χ 10⁴ cells/well) in RPMI1640 were dispensed in flat-bottomed BD Falcon 96-well microplates, and the plates were then incubated at 37°C for 24 hours. 50 μΐ of 40 ng/ml TNF-a, 50 μΐ of serially diluted solutions of the Fc fusion proteins or human IgG Fc fragments as a control and 50 μΐ of 4 μg/ml actinomycin D (Wako Pure Chemical Industries, Ltd.) were added in individual wells, and then the plates were incubated at 37°C for 24 hours. The plates were centrifuged at 300 x g for 15 min, and then 50 μΐ of the supematants were transferred into flat-bottomed BD Falcon 96-well flexible PVC microplates. Lactate dehydrogenase activities in the individual supematants were assessed with Cytotoxicity Detection kit (Roche Diagnostics Corporation, Mannheim, Germany) in accordance with the manufacturer's instructions. The absorbance at 490 nm was measured with a microplate reader Model 550 (BIO-RAD Laboratories, Hercules, CA). The neutralizing activities (%) were calculated according to the following formula: neutralizing activity = 1 - {100 ^χ (sample absorbance - background absorbance) / ( 1 % Triton-X 100 absorbance - background absorbance) } . All samples were analyzed in triplicate, and the values were expressed as the means and S.D.

[0075] Their activities to neutralize the cytotoxicity of sTNF-a were determined in L929 assay system (J Control Release (2008) 129:179-186) to examine whether the neutralizing activity is influenced by Fc multimerization (Fig. 3B). The dose response curve of

TNFRII-Fc-Fc was close to that of TNFRII-Fc. On the other hand, TNFRII-Fc-Fc (single) gave 50% neutralizing activity at the concentration that was 10 to 20 times higher than that of TNFRII-Fc-Fc, suggesting its low neutralizing activity for sTNF-a. Therefore, these results indicate that the binding and neutralizing activities of TNFRII for sTNF-a is not influenced by Fc multimerization.

[0076] <Example 4> tmTNF- binding assay by flow cytometry

[0077] The CHO-DG44 stably expressing tmTNF-a was established by expression of tmTNF-a that has two mutations (R77T and S78T) to resist to TACE-mediated cleavage (J Immunol (2001) 166:130-136). The cDNA encoding the mutated tmTNF-a was cloned into pcDNA3.1/Zeo expression vector and transfected into CHO-DG44 with TransFast

Transfection Reagent (Promega). The CHO-DG44 stably expressing tmTNF-a

(CHO-DG44/tmTNF-a) was cloned and established by the method described above.

[0078] CHO-DG44/tmTNF-a was used to evaluate the binding activities of Fc fusion proteins to the tmTNF-a on the cell surface. 1.33 ^χ 10⁵ cells/ml CHO-DG44/tmTNF-a was blocked in Cytometry buffer (PBS, 0.4% BSA, 3 mM EDTA) at 4°C for 30 min. After the centrifugation at 600 ^χ g at 4°C for 5 min, the supematants were discarded and the cells were allowed to react with 1 μg/ml Fc fusion proteins in 100 μΐ of Cytometry buffer at 4°C for 30 min. After washing with 500 μΐ of Cytometry buffer twice, the cells were allowed to react with 7.8 μg/ml FITC-conjugated goat anti-human IgG F(ab')₂ (Tago) in 100 μΐ of Cytometry buffer at 4°C for 30 min. The cells were washed twice again and then suspended in 500 μΐ of Cytometry buffer. The fluorescence intensities of the individual samples were measured with Gemini XPS (Molecular Devices, Sunnyvale, CA). [0079] The binding activities of Fc fusion proteins to tmTNF-a were determined by flow cytometry with CHO-DG44 cells that had been transfected with a TACE-resistant form of human tmTNF-a. In Fig. 4A, CHO-DG44 cells that exhibit relatively high expression of tmTNF-a were used, and, in Fig. 4B, CHO-DG44 cells that exhibit relatively low expression of tmTNF-a were used. TNFRII-Fc and TNFRII-Fc-Fc bound to tmTNF-a at the same intensities in both of the cell lines with high and low expressions of tmTNF-a. However, the binding activity of TNFRII-Fc-Fc (single) to tmTNF-a was lower than that of TNFRII-Fc-Fc. These results suggest that Fc multimerization does not affect the binding activity of TNFRII to tmTNF-a on the surface of cells, and the formation of the disulfide-linked dimmers is important for the binding activity to tmTNF-a.

[0080] <Example 5> CDC assay

[0081 ] CDC assay was performed using CHO-DG44/tmTNF-a and a low-expressing CHO-DG44/tmTNF-a as target cells. After the CHO-DG44/tmTNF-a were centrifuged at 300 x g at room temperature for 5 min, the cells were then suspended at 10 x 10⁵ cells/ml in CDC Buffer (RPMI1640 containing 0.1% BSA, 20 mM HEPES (pH7.2), 100 U/ml penicillin and 100 μg/ml streptomycin). 50 μΐ of cell suspensions were dispensed into individual wells of uClear fluorescence black plates with transparent bottom, and 50 μΐ of serially diluted Fc fusion proteins or human IgG Fc were added to the cell suspensions. After 50 μΐ of 5-fold diluted fresh baby rabbit complement (Cedarlane Laboratories, Hornby, Ontario) was added into individual wells, the plates were incubated at 37°C for 2 hours. Then, 50 μΐ of Alamar Blue (TREK Diagnostic Systems, Cleveland, Ohio) was dispensed into the wells and the plates were further incubated at 37°C for 16 hours to measure the living cells. The fluorescence of the individual wells were measured using ARVO SX 1420 multilabel counter at the excitation of 530 nm and at the emission of 590 nm. CDC cytotoxicity (%) was calculated according to the following formula: CDC cytotoxicity (%) = 100 x (background fluorescence - sample fluorescence) / background fluorescence. The cytotoxic activities were measured in triplicate, and the results were expressed as the mean and SD.

[0082] CDC activities of Fc fusion proteins were determined using baby rabbit complement and tmTNF-a-expressing CHO-DG44 cells. In Fig. 5A, CHO-DG44 cells highly expressing tmTNF-a were used, and, in Fig. 5B, CHO-DG44 cells with a low expression of tmTNF-a were used. TNFRII-Fc-Fc mediated CDC against CHO-DG44/tmTNF-a cells at the concentrations equal to or more than 2.2 nM while human IgG Fc alone did not at all (Fig. 5A). TNFRII-Fc-Fc at 2.2 nM was equivalent to TNFRII-Fc at 6.6 nM, implying that TNFRII-Fc-Fc would be 3 times as potent as TNFRII-Fc in CDC. TNFRII-Fc-Fc (single) was less active than TNFRII-Fc-Fc and close to TNFRII-Fc in CDC. Similar results were found with CHO-DG44 of lower tmTNF-a expression (Fig. 5B). These results suggest that Fc multimenzation enhances the CDC activity of Fc fusion protein.

[0083] <Example 6> Fey receptor binding assay by ELISA

[0084] Binding activities of Fc fusion proteins to individual Fey receptors were measured by ELISA as previously describe (Mol Immunol (2008) 45:2752-2763). Briefly, FcyRIA, FcyRIIA, FcyRIIB, FcyRIIIA (V158) and FcyRIIIA (F158) in PBS were coated on 96-well microplates in the forms of their extracellular domains fused with glutathione S-transferase, and HRP-conjugated goat anti-human IgG gamma chain was used as the secondary antibody. The enzyme activities were measured with 3,3',5,5'-tetramethylbenzidine.

[0085] Binding activities of Fc fusion proteins to Fey receptors were determined by ELISA to examine whether Fc multimenzation augments the binding activity (Figs. 6A to 6E). The binding activity of TNFRII-Fc-Fc to FcyRIIIA (VI 58) (Fig. 6D) was about 20 times higher than that of TNFRII-Fc, and the binding activities of TNFRII-Fc-Fc to FcyRIA (Fig. 6 A), FcyRIIA (Fig. 6B), FcyRIIB (Fig. 6C) and FcyRIIIA (F158) (Fig. 6E) were about 10 times higher than those of TNFRII-Fc. On the other hand, TNFRII-Fc-Fc (single) was weaker than TNFRII-Fc-Fc, and as potent as TNFRII-Fc in binding activities to all Fey receptors.

Considering that FcyRIIIA has two genotypes at amino acid 158 and that FcyRIIIA (VI 58) binds to human IgG more strongly than FcyRIIIA (F158) (J Immunol (1993) 151:6429-6439), the enhancement of binding activity to FcyRIIIA by Fc multimenzation has nothing to do with the genotype. Taking together, these results suggest that Fc multimerization augments the binding activities of an Fc fusion protein to all Fey receptors.

[0086] <Example 7> ADCC assay

[0087] CHO-DG44/tmTNF-a cells were used as the target cells of ADCC. The cells were suspended in HBSS-F (Hanks Balanced Salt Solution containing 10 mM HEPES and 5% FBS), and the cell suspension was centrifuged at 600 ^χ g at 4°C for 5 min. The supernatant was discarded, and the cells were resuspended in 500 μΐ of HBSS-F. 12.5 μΐ of 1 mM Calcein-AM (Dojindo Molecular Technologies, Inc., Kumamoto) was then added to the cell suspension and the cells were incubated at 37°C for 30 min. After the cells were twice washed with ADCC Buffer (RPMI1640, 2 mM L-glutamine, 10 mM HEPES at pH 7.2, lOOU/ml penicillin and 100 μg/ml streptomycin), the cells were suspended in ADCC Buffer at 2 x 10⁵ cells/ml. 50 μΐ CHO-DG44/tmTNF-a stained with Calcein-AM suspensions (0.1 10⁵ cells/well) were mixed with 50 μΐ serially diluted Fc fusion proteins or human IgG Fc in the wells of round-bottomed BD Falcon 96-well microplates (BD Biosciences), and the plates were incubated at 37°C for 30 min. Then, effector cells or KHYG-1/ FcyRIIIA cells were added to the target cell suspensions. KHYG-1 /FcyRIIIA cells were developed by transfection of human FcyRIIIA to human NK cell line KHYG-1 and selection of stably expressing cells (Kobayashi et al., 2009). 50 μΐ of 50 ^χ 10⁵, 25 10⁵ or 12.5 ^χ 10⁵ cells/ml KH YG- 1 /FcyRIII A (2.5 ^χ 10⁵, 1.25 ^χ 10⁵ or 0.625 ^χ 10⁵ cells/well) was added to the individual wells. The ratios of effector cells to target cells (E/T) were 25/1, 12.5/1 and 6.25/1. The plates were incubated at 37°C for 4 hours and then centrifuged at 300 x g for 10 min. The supernatants (50 μΐ) were transferred into individual wells of uClear fluorescence black plates with transparent bottom (Greiner Bio-one, Frickenhausen, Germany). The fluorescence intensities of the Calcein-AM released from the damaged CHO-DG44/tmTNF-a were measured using ARVO SX 1420 multilabel counter (Perkin Elmer, Waltham, MA) at the excitation of 485 nm and at the emission of 538 run. ADCC cytotoxicities (%) were calculated according to the following formula. The fluorescence intensity of the well treated with 1% Triton-XlOO was used as the maximum cytotoxicity. ADCC cytotoxicity (%) = 100 x (sample fluorescence - background fluorescence) / (1% Triton-XlOO fluorescence - background fluorescence). ADCC activities were measurements in triplicate, and the results were expressed as the mean and SD.

[0088] ADCC activities of Fc fusion proteins were determined using KHYG-1 cells transfected FcyRIIIA (KH YG- 1 /FcyRIII A) and tmTNF-a-expressing CHO-DG44 cells (CHO-DG44/tmTNF-a). Fig. 7A shows that the ADCC activities of three Fc fusion proteins against tmTNF-a highly expressing CHO-DG44 by KHYG-1 /FcyRIIIA at the E/T ratio of 25/1. Human IgG Fc alone did not bind to target cells and showed no significant

cytotoxicity. TNFRII-Fc-Fc was found to be cytotoxic at the concentrations of 0.74 nM and above, and TNFRII-Fc at the concentrations of 6.67 and 20 nM showed the cytotoxicity as much as TNFRII-Fc-Fc of 0.74 nM. TNFRII-Fc-Fc (single) was as potent as TNFRII-Fc in ADCC. Also at the lower E/T ratios (12.5/1 and 6.25/1), the ADCC activity of

TNFRII-Fc-Fc was more potent than TNFRII-Fc at every concentration (Fig. 7B).

CHO-DG44 of lower tmTNF-a expression as well showed higher susceptibility to ADCC with TNFRII-Fc-Fc than with TNFRII-Fc (Fig. 7C). Therefore, TNFRII-Fc-Fc would exert more potent ADCC activity than the ordinary TNFRII-Fc against tmTNF-a-expressing target cells.

[0089] <Example 8> Measurement of half-life of Fc fusion protein in blood by ELISA [0090] 10 μg/body of a fusion protein selected from TNFRII-Fc or TNFRII-Fc-Fc (single) was administered to the tail veins of mice. When a certain time had passed thereafter, 10 μΙ_< of blood was collected from the tip of the tail of each mouse. Directly after the blood collection, 90 μΐ of 1 mM EDTA / PBS was added to dilute the blood 10-fold, the resultant solution was centrifuged at 4 °C and 13200 rpm for 30 minutes, and only blood plasma portion was collected.

[0091] Thereafter, blood collection from the mice was carried out 12 hours, 24hours, 48 hours (2 days), 72 hours (3 days), 96 hours (4 days), 120 hours (5 days), 168 hours (7 days), 192 hours (8 days), 216 hours (9 days) and 264 hours (11 days) after the administration of the Fc fusion protein, and the collected blood was processed in a manner similar to the above.

[0092] Goat anti human TNFRII polyclonal antibody in PBS (final 1.0 μ^ί) was added, in an amount of 50 μίΛνεΙΙ, into the individual wells of an ELISA plate, and was allowed to stand at 4 °C overnight, thereby immobilizing the antibody.

[0093] 0.25% BSA in PBS 150 was added into the wells of the ELISA plate, and was allowed to stand at 37 °C for 2 hours, whereby blocking was carried out. The plasma, which had been stored at -80 °C, was diluted 25-folds with PBS, and was added, in an amount of 50 μΙ,ΛνβΙΙ, into the wells of the ELISA plate. After the ELISA plate was allowed to stand at 37 °C for 2 hours, each well was washed with 150 μΐ of PBST (PBS to which 0.05% Tween 20 was added) three times. 50 μΕΛνεΙΙ of 27,000-fold dilution solution of HRP-Goat anti human IgG (Fc-specific) F(ab')₂ was added thereto as a detection antibody, and the ELISA plate was allowed to stand at 37 °C for 2 hours. Each well was washed with 150 μΐ_^ of PBST three times, and 50 μίΛνεΙΙ of a chromogenic substrate (TMBZ/0.03 % H₂0₂) was added to the wells. 10 minutes later, 30 μΕΛνεΙΙ of 0.5 M H₂S0₄ was added to the wells, to terminate the reaction. Fluorescence at 450 nm was measured with a fluorescence plate reader. The results are shown in Table 1.

[0094] Table 1

[0095] As is clear from the results shown in Table 1, when TNFRII-Fc and TNFRII-Fc-Fc (single) are compared, the half-life of TNFRII-Fc-Fc (single) is clearly elongated. As compared to TNFRII-Fc, TNFRII-Fc-Fc (single) has an enhanced CDC activity and an enhanced ADCC activity, as well as an elongated half-life. Therefore, TNFRII-Fc-Fc (single) is considered to be more effective as a therapeutic agent for inflammation-related diseases, as compared to TNFRII-Fc.

[0096] Incidentally, etanercept, which is a TNFRII-Fc type fusion protein, has been reported to have a half-life of from about 70 hours to about 100 hours in human (J Pharm Pharmacol. (2005) ,57 (11), 1407-13). It is known that the half-life of drugs in human blood is longer than that in mouse blood, and it is estimated that the TNFRII-Fc-Fc (single) according to the invention has a half-life in human of from about 100 hours to about 200 hours. The high efficacy of the fusion protein of the invention is also demonstrated by this result.

[0097] <Example 9> Study on therapeutic effects using DSS-induced colitis model

[0098] TNF-a is known to be involved in ulcerative colitis induced by DSS administration (J. Clin. Invest. (2008) 118, 560-570). In view of this, a study was carried out using a

DSS-induced colitis model in order to confirm the therapeutic effects of TNFRII-Fc -Fc and TNFRII-Fc -Fc (single), which are fusion proteins according to the invention.

[0099] Mice (BALB/c, male, 5-month age) were randomly divided into 14 groups, each group consisting of 5 mice. The weights thereof on Day 0 were measured. Thereafter, the weights of the mice were measured every 24 hours, and the recording of the weights was continued until Day 21. Each mouse was allowed to freely ingest potable water or potable water containing 5%(w/v) dextran sodium sulfate (DSS) dissolved therein, for 7 days.

[0100] Etanercept, TNFRII-Fc, TNFRII-Fc -Fc and TNFRII-Fc -Fc (single) were

individually dissolved in 100 μΐ, of PBS (phosphate buffered saline), to prepare fusion protein solutions.

[0101] From Day3, one selected from PBS and the fusion protein solutions described above was administered to the tail veins of mice. The fusion protein solution was administered in an amount of 0.13 nmol/body. The administration was carried out from Day 3 to Day 7 (five times in total).

[0102] The results are shown in Fig. 8. Six week-old BALB/c mice (five mice per group) received i.v. injections of PBS (trigona), 0.13 nmol/body of etanercept (square), 0.13 nmol/body of TNFRII-Fc-Fc (crosses) or 0.13 nmol/body of TNFRII-Fc-Fc (single) (round) five times over a 21 -day treatment period. In Fig. 8, diamond marks indicate a change of the weight of mice that did not ingest DSS. In Fig. 8, samples were analyzed in triplicate, and the mean values ± S.D thereof are shown.

[0103] A change of the weight of the mice that did not ingest DSS (DSS(-)), and the mice to which PBS was administered instead of a fusion protein solution (DSS(+)) is shown in Fig. 8.

[0104] The mice that did not ingest DSS exhibited an about 20% increase in body weight within the 21 days during which the experiment was carried out. In contrast, the mice which ingested DSS, and to which PBS was administered exhibited a decrease in body weight from around Day 5 of the DSS ingest, and exhibited the minimum weight on Day 11.

[0105] On Day 11, the average body weight had decreased to about 75% of the body weight at the time of starting the experiment. Thereafter, the body weight started to increase, and, on Day 21 after the initiation of the experiment, the body weight returned to the weight at the time of starting the experiment. Since a decrease in body weight reflects exacerbation of inflammation, and an increase in body weight reflects recovery from inflammation, in this experimental model, the first 11 days are considered to indicate exacerbation of inflammation, and the subsequent days up to Day 21 are considered to indicate recovery from the

inflammation.

[0106] Changes in body weights in cases in which various fusion protein solutions were administered are also shown (Fig. 8). Etanercept could not sufficiently suppress a decrease in body weight caused by the DSS ingestion. In a case in which TNFPJI-Fc -Fc (single) was administered, a decrease in body weight due to the DSS digestion was observed; however, the recovery of the body weight was more rapid than the group of mice to which etanercept was administered. In the group to which TNFRII-Fc-Fc was administered, an effect with respect to remarkably alleviating a decrease in body weight caused by the DSS ingestion was observed.

[0107] <Example 10> Study on cell-killing effects on lymphocytes and macrophages

[0108] Measurement of cytotoxicity to the T cells and macrophages using TNFRII-Fc, TNFRII-Fc -Fc and TNFRII-Fc -Fc (single)

[0109] (Example 10- 1 )CDC assay using mtTNF-a-transfected Jurkat cells or PMA-activated THP-1 cells

[0110] CDC assay is performed using mtTNF-a-transfected Jurkat cells or THP-1 cells activated with PMA (phorbol myristate acetate) as target cells. After these cells are centrifuged at 300 x g at room temperature for 5 min, the cells are then suspended at 10 ^χ 10⁵ cells/ml in CDC Buffer (RPMI1640 containing 0.1% BSA, 20 mM HEPES (pH7.2), 100 U/ml penicillin and 100 μg/ml streptomycin). 50 μΐ of cell suspensions are dispensed into individual wells of uClear fluorescence black plates with transparent bottom and 50 μΐ of serially diluted Fc fusion proteins are added to the cell suspensions. After 50 μΐ of 5-fold diluted fresh baby rabbit complement is added into individual wells, the plates are incubated at 37°C for 2 hours. Then, 50 μΐ of Alamar Blue is dispensed into the wells and the plates are further incubated at 37°C for 16 hours to measure the living cells. The fluorescence of the individual wells are measured using ARVO SX 1420 multilabel counter at the excitation of 530 nm and at the emission of 590 nm. CDC cytotoxicity (%) is calculated according to the following formula: CDC cytotoxicity (%) = 100 ^χ (background fluorescence - sample fluorescence) / background fluorescence. The cytotoxic activities are measured in triplicate, and the results are expressed as the means and Standard deviations. CDC activities of Fc fusion proteins are determined using baby rabbit complement and tmTNF-a-expressing Jurkat cells or activated THP-1 cells [0111] (Example 10-2) ADCC assay using mtTNF-ct-transfected Jurkat cells or PMA-activated THP-1 cells

[0112] MtTNF-a-transfected Jurkat cells or PMA-activated THP-1 cells are used as the target cells of ADCC. The cells are suspended in HBSS-F (Hanks Balanced Salt Solution containing 10 mM HEPES and 5% FBS), and the cell suspension is centrifuged at 600 ^χ g at 4°C for 5 min. The supernatant is discarded, and the cells are resuspended in 500 μΐ of HBSS-F. 12.5 μΐ of 1 mM Calcein-AM (Dojindo Molecular Technologies, Inc., Kumamoto) is then added to the cell suspension and the cells are incubated at 37°C for 30 min. After the cells are twice washed with ADCC Buffer (RPMI1640, 2 mM L-glutamine, 10 mM HEPES at pH 7.2, lOOU/ml penicillin and 100 μg/ml streptomycin), the cells are suspended in ADCC Buffer at 2 x 10⁵ cells/ml. 50 μΐ cell suspension of mtTNF-a-transfected Jurkat cells or PMA-activated THP-1 cells that are stained with Calcein-AM suspensions (0.1 x 10⁵ cells/well) are mixed with 50 μΐ serially diluted Fc fusion proteins or human IgG Fc in the wells of round-bottomed BD Falcon 96- well microplates, and the plates are incubated at 37°C for 30 min. Then, effector cells or KHYG-1/ FcyRIIIA cells are added to the target cell suspensions. KH YG- 1 /FcyRIII A cells were developed by transfection of human FcyRIIIA to human NK cell line KHYG-1 and selection of stably expressing cells (Kobayashi et al., 2009). 50 μΐ of 50 x 10⁵, 25 x 10⁵ or 12.5 ^χ 10⁵ cells/ml KHYG-1 /FcyRIII A (2.5 10⁵, 1.25 10⁵ or 0.625 x 10⁵ cells/well) is added to the individual wells. The ratios of effector cells to target cells (E/T) were 25/1 , 12.5/1 and 6.25/1. The plates are incubated at 37°C for 4 hours and then centrifuged at 300 x g for 10 min. The supernatants (50 μΐ) are transferred into individual wells of uClear fluorescence black plates with transparent bottom (Greiner Bio-one, Frickenhausen, Germany). The fluorescence intensities of the Calcein-AM released from the damaged tagete cells are measured using ARVO SX 1420 multilabel counter (Perkin Elmer, Waltham, MA) at the excitation of 485 nm and at the emission of 538 nm. ADCC

cytotoxicities (%) are calculated according to the following formula. The fluorescence intensity of the well treated with 1% Triton-XlOO is used as the maximum cytotoxicity.

ADCC cytotoxicity (%) = 100 x (sample fluorescence - background fluorescence) / (1% Triton-XlOO fluorescence - background fluorescence). ADCC activities are measurements in triplicate, and the results are expressed as the mean and SD.

[0113] ADCC activities of Fc fusion proteins are determined using KHYG-1 cells

transfected with FcyRIII A (KHYG-1 /FcyRIII A) as effector cells and mtTNF-a-transfected Jurkat cells or PMA-activated THP-1 cells as target cells.

[0114] The TNFRII-Fc -Fc and TNFRII-Fc -Fc (single) according to the invention exhibits cytotoxic activity against macrophages and T cells, each of which expresses tmTNFa and strongly induces inflammation, via CDC or ADCC. In immune inflammatory diseases, these T cells play a pivotal role in the pathogenecis (Rheumatology (2010), 49, 1215-1228).

Therefore, not only neutralization of soluble TNF-abut also removal of these cells with tmTNF-aby CDC and ADCC would be desirable.

[0115] TNFRII-Fc-Fc and TNFRII-Fc-Fc (single) have strong activity, and are expected to not only simply neutralize secreted TNFa, but also to injure cells that produces inflammatory proteins such as TNFa, thereby suppressing production of inflammatory proteins.

[0116] The present application claims the benefits of priority to U.S. application Ser. No. 61/359,828, filed Jun 30, 2010 and U.S. application Ser. No. 61/425,773, filed Dec 22, 2010. The contents of that application are incorporated herein by reference in their entirety. All publications, patent applications, and technical standards mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or technical standard was specifically and individually indicated to be

incorporated by reference.

Claims

1. A fusion protein comprising:

a first region comprising an extracellular domain of a cytokine receptor; and a second region comprising plural immunoglobulin Fc domains or plural single-chain immunoglobulin Fc domains, and fused with the first region,

wherein the plural immunoglobulin Fc domains or plural single-chain

2. The fusion protein according to claim 1 , wherein the cytokine receptor is a TNFa receptor.

3. The fusion protein according to claim 2, wherein the TNFa receptor is a TNFa receptor II.

4. The fusion protein according to any one of claims 1 to 3, wherein the

immunoglobulin is IgG

5. The fusion protein according to claim 4, wherein the IgG is human IgGl .

6. The fusion protein according to any one of claims 1 to 5, wherein the tandemly repeated immunoglobulin Fc domains or the tandemly repeated single-chain immunoglobulin Fc domains are linked to each other via a linker peptide.

7. The fusion protein according to claim 6, wherein each of the at least one linker peptide is a trimer of glycine-glycine-glycine-glycine-serine.

8. The fusion protein according to any one of claims 1 to 7, wherein the tandemly repeated immunoglobulin Fc domains or the tandemly repeated single-chain immunoglobulin Fc domains are composed of two or three immunoglobulin Fc domains that are connected in series, or of two or three single-chain immunoglobulin Fc domains that are connected in series.

9. A therapeutic agent for treating an inflammation-related disease, the therapeutic agent comprising the fusion protein of any one of claims 1 to 8.

10. The therapeutic agent according to claim 9, wherein the inflammation-related disease is Crohn disease.

11. A method for treating an inflammation-related disease, comprising

administering the fusion protein of any one of claims 1 to 8 to a patient in need thereof.

12. The method for treating an inflammation-related disease according to claim 11 , wherein the inflammation-related disease is Crohn disease.