WO2014035361A1 - IL-1β INHIBITOR COMPOSITION AND USE THEREOF - Google Patents

Abstract

A therapeutic composition is described that can be used for treating or prevention of diseases association with modulation of activity of human IL-Ιβ. In certain aspects, the disclosed invention is based, on engineering of a heterodimeric protein assembly that is capable of binding to human IL-Ιβ and attenuating its function. The heterodimeric protein assembly comprises an extracellular portions of human IL1-R1 and of human IL-1RAcP, or their functional fragments. Each, the IL1-R1 portion and the IL-1RAcP portion, is fused to a distinct mutant of Fc portion of the human Ig Gamma-1. The two distinct Fc mutants in the heterodimeric protein assembly are engineered as to favor the heteromeric dimer formation between the two Fc mutants over any homomeric assembly. DNA expression vectors and expression systems for overproducing the polypeptides in mammalian cells are also provided for.

Description

IL-ip INHIBITOR COMPOSITION AND USE THEREOF

FIELD OF THE INVENTION

Generally, the invention relates to the field of biological pharmaceuticals as well as their use in conditions associated with inflammatory disorders (e.g rheumatoid arthritis, Crohn's desease, etc.), diabetes, cardiovascular disease and gout. More specifically, the invention relates to a

heterodimeric IL-lRl/IL-lRAcP -derived composition that is capable of inhibiting IL-Ιβ cytokine.

BACKGROUND

The interleukin-1 (IL-1) family of cytokines comprises 11 proteins (IL-1F1 to IL-1F11) encoded by 11 distinct genes in humans and mice. IL-l-type cytokines are major mediators of innate immune reactions, and blockade of the founding members IL-1 or IL-Ιβ by the interleukin- 1 receptor antagonist (IL-1RA) has demonstrated a central role of IL-1 in a number of human autoinfiammatory diseases. IL-1 or IL-Ιβ rapidly increase messenger R A expression of hundreds of genes in multiple different cell types. The potent proinflammatory activities of IL-1 and IL-Ιβ are restricted at three major levels: (i) synthesis and release, (ii) membrane receptors, and (iii) intracellular signal transduction. This pathway summarizes extracellular and intracellular signaling of IL-1 or IL-Ιβ, including positive- and negative-feedback mechanisms that amplify or terminate the IL-1 response. In response to ligand binding of the receptor, a complex sequence of

combinatorial phosphorylation and ubiquitination events results in activation of nuclear factor kappa-B signaling and the TNK and p38 mitogen-activated protein kinase pathways, which, cooperatively, induce the expression of canonical IL-1 target genes (such as IL-6, IL-8, MCP-1, COX-2, IB, IL-1, IL-Ιβ, MKP-1) by transcriptional and posttranscriptional mechanisms. Of note, most intracellular components that participate in the cellular response to IL-1 also mediate responses to other cytokines (IL-18 and IL-33), Toll-like-receptors (TLRs), and many forms of cytotoxic stresses (see Weber A, et al, Sci Signal, 2010 Jan 19;3(105), the entire teachings of which are incorporated by reference herein).

IL-1 and IL-Ιβ independently bind the type I IL-1 receptor (IL-1R1), which is ubiquitously expressed. A third specific ligand, the IL-1 receptor antagonist (IL-IRA), binds the IL-1RI with similar specificity and affinity but does not activate the receptor and trigger downstream signaling. The IL-1 receptor accessory protein (IL-lRAcP) serves as a co-receptor that is required for signal transduction of IL-1/IL-1RI complexes, and this co-receptor is also necessary for activation of IL- 1R1 by other IL-1 family members, in particular IL-18 and IL-33. The type II IL-1 receptor (IL- 1R2) binds IL-1 and IL-1 β but lacks a signaling-competent cytosolic part and thus serves as a decoy receptor. The IL-IRA, the plasma membrane-anchored IL-1R2, and the naturally occurring "shed" domains of each of the extracellular IL-1 receptor chains (termed sIL-lRI, sIL-lRII, and sIL-lRAcP, where "s" stands for soluble) provide inducible negative regulators of IL-1 signaling in the extracellular space whose abundance, which is regulated by a combination of increased transcription and controlled release, can limit or terminate IL-1 effects.

The initial step in IL-1 signal transduction is a ligand-induced conformational change in the first extracellular domain of the IL-1RI that facilitates recruitment of IL-lRacP. Through conserved cytosolic regions called Toll- and IL-lR-like (TIR) domains, the trimeric complex rapidly assembles two intracellular signaling proteins, myeloid differentiation primary response gene 88 (MYD88) and interleukin-1 receptor-activated protein kinase (IRAK) 4. Mice lacking MYD88 or IRAK4 show severe defects in IL-1 signaling. Similarly, humans with mutations in the IRAK4 gene have defects in IL-IRI and Toll-like receptor (TLR) signaling. IL-1, IL-IRI, IL-RAcP, MYD88, and IRAK4 form a stable IL-l-induced first signaling module. This is paralled by the (auto)phosphorylation of IRAK4, which subsequently phosphorylates IRAKI and IRAK2, and then this is followed by the recruitment and oligomerization of tumor necrosis factor-associated factor (TRAF) 6. IRAKI and 2 function as both adaptors and protein kinases to transmit downstream signals. Complexes of IRAKI, IRAK2, and TRAF6 dissociate from the initial receptor complex, and cells lacking these proteins have impaired activation of the transcription factors nuclear factor kappa-B (NF-kappa-B) and activator protein 1 (AP-1).

Overproduction of IL-1 is the cause of many inflammatory disorders. For example, IL-1 has been linked to the pathology of diabetes, cardiovascular disease, gout and certain types of arthritis (e.g. rheumatoid arthritis (RA)).

Rilonacept is an IL-1 antagonist which includes an IL-1 -specific fusion protein which comprises an IL-1 binding portion of the extracellular domain of human ILl-RAcP, an IL-1 binding portion of the extracellular domain of human IL-IRI, and a multimerizing component. This IL-l-specific fusion protein is described in U.S. Pat. No. 6,472,179, U.S. patent publication No. 2003/0143697, published 31 Jul. 2003, U.S. Pat. No. 7,361,350, and U.S. patent publication No. 2005/0197293, published 8 Sep. 2005 (all of which are incorporated by reference herein in their entirety). Rilonacept under the trade name ARCALYST was approved by U.S. Food and Drug Administration (FDA) for the treatment of Cryopyrin- Associated Periodic Syndromes (CAPS), including Familial Cold Auto-inflammatory Syndrome (FCAS) and Muckle-Wells Syndrome (MWS) in adults and children 12 and older. Further clinical trials of rilonacept are currently under way, i.e. for gout.

SUMMARY OF THE INVENTION This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In certain aspects, the present invention provides for a heterodimeric protein composition capable of binding human IL-Ιβ (GenBank: AAH08678.1). The protein composition comprises a first polypeptide which includes a first amino acid sequence which contains amino acids 18 through 333 of human IL1-R1 (GenBank: AAM88423.1), and a second amino acid sequence which contains a first mutant of a Fc portion of human immunoglobulin gamma- 1 Fc (GenBank: J00228.1). The protein composition also comprises a second polypeptide which includes another first amino acid sequence containing amino acids 21 through 358 of human ILl-RAcP (GenBank: BAA25421.1), and another second amino acid sequence which contains a second mutant of the Fc portion of human immunoglobulin gamma- 1 Fc. In the protein composition, the first and second mutants are selected as to favor hetedimeric assembly between the first and second mutants over any homodimeric assembly. The protein composition may be capable of exhibiting human IL-Ιβ binding activity in an ELISA assay with an EC50 of about 50 ng/ml. The first polypeptide of the protein composition may contain amino acid sequence of SEQ ID NO. 1, while the second polypeptide may contain amino acid sequence of SEQ ID NO. 2.

In certain aspects, the present invention provides for a therapeutic composition. The therapeutic composition comprises a heterodimeric protein composition capable of binding human IL-Ιβ. The protein composition comprises a first polypeptide which includes a first amino acid sequence which contains amino acids 18 through 333 of human IL1-R1 , and a second amino acid sequence which contains a first mutant of the Fc portion of human immunoglobulin gamma- 1 Fc. The protein composition also comprises a second polypeptide which includes another first amino acid sequence containing amino acids 21 through 358 of human ILl-RAcP, and another second amino acid sequence which contains a second mutant of the Fc portion of human immunoglobulin gamma- 1 Fc. In the protein composition, the first and second mutants are selected as to favor hetedimeric assembly between the first and second mutants over any homodimeric assembly. The therapeutic composition may exhibit a half-life of the heterodimeric protein composition in systemic circulation in mice after a subcutaneous administration at a dose of 5 mg/kg of at least about 88 hours, as assayed by human Fc ELISA. The therapeutic composition may comprise a heterodimeric protein comprised of a first polypeptide containing amino acid sequence of SEQ ID NO. 1 and a second polypeptide containing amino acid sequence of SEQ ID NO. 2.

In certain aspects, the present invention provides for an isolated nucleic acid encoding a polypeptide comprising amino acid sequence of SEQ ID NO. 3. The codon usage of the nucleic acid may be optimized for high expression of the polypeptide in a mammalian cell. 1. The nucleic acid may contain the sequence of SEQ ID NO. 5. The nucleic acid may comprise an expression vector

In certain aspects, the present invention provides for an isolated nucleic acid encoding a polypeptide comprising amino acid sequence of SEQ ID NO. 4. The codon usage of the nucleic acid may be optimized for high expression of the polypeptide in a mammalian cell. The nucleic acid may contain the sequence of SEQ ID NO. 6. The nucleic acid may comprise an expression vector.

In certain aspects, the present invention provides for an isolated nucleic acid of SEQ ID NO. 7. In certain aspects, the present invention provides for a heterologous expression system. The expression system harbors an expression vector comprising a nucleic acid sequence encoding a first polypeptide containing amino acid sequence of SEQ ID NO. 3 and another nucleic acid sequence encoding a second polypeptide containing amino acid sequence of SEQ ID NO. 4. The expression vector of the expression system may be harbored in a mammalian cell. The mammalian cell may be a CHO cell. The expression system may be capable of expressing a heterodimeric protein comprising a first polypeptide containing amino acid sequence of SEQ ID NO. 1 and a second polypeptide containing amino acid sequence of SEQ ID NO. 2. The level of expression of the heterodimeric protein may be at least 300 mg per liter of cell culture. In certain aspects, the present invention provides for use of a substance for manufacture of a medicament for the treatment or prevention of a disease associated with modulation of activity of human IL-Ιβ . The substance comprises a heterodimeric protein comprised of a first polypeptide containing amino acid sequence of SEQ ID NO. 1 and a second polypeptide containing amino acid sequence of SEQ ID NO. 2. The disease associated with modulation of activity of human IL-Ιβ may be an arthritis, a gout, a rheumatoid arthritis, a Cryopyrin- Associated Periodic Syndromes (CAPS), a scleroderma, a diabetes, a atherosclerosis, a dry eye disease, an ocular allergy, or an uveitis.

In certain aspects, the present invention provides for a method of treating or preventing a disease or condition associated with modulation of activity of human IL-Ιβ. The method comprising administering to a patient in need for treating or preventing a disease associated with modulation of activity of human IL-Ιβ a therapeutically effective amount of a pharmaceutical composition. The pharmaceutical composition comprising a heterodimeric protein comprised of a first polypeptide containing amino acid sequence of SEQ ID NO. 1 and a second polypeptide containing amino acid sequence of SEQ ID NO. 2. The disease associated with modulation of activity of human IL-Ιβ may be an arthritis, a gout, a rheumatoid arthritis, a Cryopyrin- Associated Periodic Syndromes (CAPS), a scleroderma, a diabetes, a atherosclerosis, a dry eye disease, an ocular allergy, or an uveitis.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings and descriptions are provided to aid in the understanding of the invention:

Figure 1 illustratively shows a heterodimeric protein assembly of the present teachings comprising an extracellular portion of IL1-R1 fused with an IgG-Fc domain (Fc-II) via a flexible linker and an extracellular portion of ILl-RAcP fused with another IgG-Fc domain (Fc-V) via another flexible linker;

Figure 2 schematically shows the map of PKN012 plasmid and annotated sequence used in the cloning of the polypeptides of the present invention;

Figure 3 shows representative transfection growth curve obtained in the process of generating stable cell lines for expressing the polypeptides of the present invention; Figure 4 shown a size-exclusion HPLC analytical chromatogram of the sample containing heterodimers comprising polypeptide of SEQ ID NO. 1 and polypeptide of SEQ ID NO. 2 after the anion exchange chromatography purification step;

Figure 5 shown a SDS-PAGE analysis of the sample containing heterodimers comprising polypeptide of SEQ ID NO. 1 and polypeptide of SEQ ID NO. 2 after the anion exchange chromatography purification step; and

Figure 6 shows a typical binding curve of a purified sample containing heterodimers comprising polypeptide of SEQ ID NO. 1 and polypeptide of SEQ ID NO. 2 in an ELISA assay using commercially available human IL-Ιβ.

DETAILED DESCRIPTION OF THE INVENTION

The teachings disclosed herein are based, in part, upon engineering of a heterodimeric protein assembly that is capable of binding to human IL-1 β and attenuating its function. The heterodimeric protein assembly of the present teachings comprises an extracellular portions of IL1-R1 (GenBank: AAM88423.1) and of IL-lRAcP (GenBank: BAA25421.1), or functional fragments thereof. Each, the ILl-Rl portion and the IL-lRAcP portion, is fused to a distinct mutant of Fc portion of the human Ig Gamma-1 (GenBank: J00228.1). The two distinct Fc mutants in the heterodimeric protein assembly are engineered as to favor the heteromeric dimer formation between the two Fc mutants over any homomeric assembly. To enable recombinant production of the heterodimeric protein assembly of the present teachings, a DNA expression vector has been constructed for overproducing the heterodimeric protein assembly in a heterologous protein expression system, and mammalian cells have been prepared stably expressing the heterodimeric protein assembly to a high expression level. A protein purification procedure has been devised allowing obtaining a physiologically relevant substantially pure preparation of the heterodimeric protein assembly of the present teachings. Thus purified protein molecule demonstrates a high degree of specific activity in an in vitro Enzyme-Linked Immunosorbent Assay (ELISA) using human IL- 1β (GenBank: AAH08678.1). Unexpectedly, the protein molecule exhibits an acceptable pharmacokinetics profile upon subcutaneous animal administration, while not resulting in any body weight loss or adverse clinical events. The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification, to provide additional guidance to the

practitioner in describing the compositions and methods of the invention and how to make and use them. The scope or meaning of any use of a term will be apparent from the specific context in which the term is used. "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, preferably within 5- fold and more preferably within 2-fold of a given value.

Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences to each other, including wild-type sequence to one or more mutants (sequence variants). Such comparisons typically comprise alignments of polymer sequences, e.g., using sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few). The skilled artisan can readily appreciate that, in such alignments, where a mutation contains a residue insertion or deletion, the sequence alignment will introduce a "gap" (typically represented by a dash, or "A") in the polymer sequence not containing the inserted or deleted residue.

The methods of the invention may include statistical calculations, e.g. determination of IC50 or EC50 values, etc.. The skilled artisan can readily appreciate that such can be performed using a variety of commercially available software, e.g. PRISM (GraphPad Software Inc, La Jolla, CA, USA) or similar.

"Homologous," in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a "common evolutionary origin," including proteins from superfamilies in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions. However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and may or may not relate to a common evolutionary origin.

The term "sequence similarity," in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.

The terms "protein" and "polypeptide" are used interchangeably. The polypeptides described herein may be comprised of more than one contiguous amino acid chain, thus forming dimers or other oligomeric formations. In general, the polypetides of the present teachings for use in mammals are expressed in mammalian cells that allow for proper post-translational modifications, such as CHO or HEK293 cell lines, although other mammalian expression cell lines are expected to be useful as well. It is therefore anticipated that the polypeptides of the present teachings may be post-translationally modified without substantially effecting its biological function.

In certain aspects, functional variants of the heterodimeric protein assemblies of the present teachings include fusion proteins having at least a biologically active portion of the human ILl-Rl or IL-lRAcP or a functional fragment thereof, and one or more fusion domains. Well known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (e.g., an Fc), maltose binding protein (MBP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For example, the ILl-Rl or IL- IRAcP polypeptide portions may be fused with a domain that stabilizes the ILl-Rl or IL-lRAcP polypeptides in vivo (a "stabilizer" domain), optionally via a suitable peptide linker. The term "stabilizing" means anything that increases the half life of a polypeptide in systemic circulation, regardless of whether this is because of decreased destruction, decreased clearance, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on certain proteins. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains that confer an additional biological function, e.g. promoting accumulation at the targeted site of action in vivo. In certain aspects, the heterodimeric protein assemblies of the present teachings comprise an extracellular portion of ILl-Rl, or a functional fragment thereof, fused with a IgG-Fc domain, and an extracellular portion IL-lRAcP, or a functional fragment thereof, fused with another IgG-Fc domain. The IgG-Fc domain and the another IgG-Fc domain are chosen as to favor a heterodimeric protein assembly over any homodimeric protein assembly. The extracellular portion of IL1-R1 may be fused with the IgG-Fc domain via a flexible linker, while IL-lRAcP, or a functional fragment thereof, may be fused with the another IgG-Fc domain via the flexible linker of the same amino acid sequence or via another flexible linker.

In an example embodiment, illustratively shown in Figure 1, the extracellular portion of IL1-R1 fused with IgG-Fc domain (Fc-II) via a flexible linker may comprise the amino acid sequence of SEQ. ID NO. 1, while IL-lRAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker may comprise the amino acid sequence of SEQ. ID NO. 2. hILl-Rl-hlgGl-Fc polypeptide (SEQ ID NO. 1)

LEADKCKERE EKIILVSSAN EIDVRPCPLN PNEHKGTITW YKDDSKTPVS TEQASRIHQH 60

KEKLWFVPAK VEDSGHYYCV VRNSSYCLRI KISAKFVENE PNLCYNAQAI FKQKLPVAGD 120

GGLVCPYMEF FKNENNELPK LQWYKDCKPL LLDNIHFSGV KDRLIVMNVA EKHRGNYTCH 180

ASYTYLGKQY PITRVIEFIT LEENKPTRPV IVSPANETME VDLGSQIQLI CNVTGQLSDI 240

AYWKWNGSVI DEDDPVLGED YYSVENPANK RRSTLITVLN ISEIESRFYK HPFTCFAKNT 300

HGIDAAYIQL IYPVTNGSGG GDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV 360

TCWVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRWSVLTVL HQDWLNGKEY 420

KCKVSNKALP APIEKTI SKA KGQPREPQVC TLPPSRDELT KNQVSLSCAV KGFYPSDIAV 480

EWESNGQPEN NYKTTPPVLD SDGSFFLVSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK 540

SLSLSPGK 548 hIL-lRAcP-hlgGl-Fc polypeptide (SEQ ID NO. 2)

SERCDDWGLD TMRQIQVFED EPARIKCPLF EHFLKFNYST AHSAGLTLIW YWTRQDRDLE 60

EPINFRLPEN RISKEKDVLW FRPTLLNDTG NYTCMLRNTT YCSKVAFPLE VVQKDSCFNS 120

PMKLPVHKLY IEYGIQRITC PNVDGYFPSS VKPTITWYMG CYKIQNFNNV I PEGMNLSFL 180

IALISNNGNY TCVVTYPENG RTFHLTRTLT VKVVGSPK A VPPVIHSPND HWYEKEPGE 240

ELLI PCTVYF SFLMDSRNEV WWTIDGKKPD DITIDVTINE SISHSRTEDE TRTQILSIKK 300

VTSEDLKRSY VCHARSAKGE VAKAAKVKQK VPAPRYTVGS GGGDKTHTCP PCPAPELLGG 360

PSVFLFPPKP KDTLMISRTP EVTCWVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 420 STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPCRDE 480

LTKNQVSLWC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 540

QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 570

In certain aspects, the present teachings provides for a recombinant DNA molecule having an open reading frame coding for a polypeptide comprising the leading 333 amino acids of the human IL1-R1 fused with IgG-Fc domain (Fc-II) via a flexible linker, and for another recombinant DNA molecule having an open reading frame coding for another polypeptide comprising the leading 358 amino acids of the human IL-lRAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker.

In an example embodiment, the polypeptide comprising the leading 333 amino acids of the human IL1-R1 fused with IgG-Fc domain (Fc-II) via a flexible linker comprises the amino acid sequence of SEQ. ID NO.3. The corresponding to it DNA molecule may comprise the nucleotide sequence of SEQ ID NO.4. The another polypeptide comprises the leading 358 amino acids of the human IL-lRAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker may comprise the amino acid sequence of SEQ. ID NO.5. The corresponding to it DNA molecule may comprise the nucleotide sequence of SEQ ID NO.6. hILl-Rl-hlgGl-Fc polypeptide (SEQ D NO.3)

MKVLLRLICF I ALLI SSLEA DKCKEREEKI ILVSSANEID VRPCPLNPNE HKGTITWYKD 60 DSKTPVSTEQ ASRIHQHKEK LWFVPAKVED SGHYYCVVRN SSYCLRIKIS AKFVENEPNL 120 CYNAQAIFKQ KLPVAGDGGL VCPYMEFFKN ENNELPKLQW YKDCKPLLLD NIHFSGVKDR 180 LIVMNVAEKH RGNYTCHASY TYLGKQYPIT RVIEFITLEE NKPTRPVIVS PANETMEVDL 240 GSQIQLICNV TGQLSDIAYW KWNGSVIDED DPVLGEDYYS VENPANKRRS TLITVLNISE 300 IESRFYKHPF TCFAKNTHGI DAAYIQLIYP VTNGSGGGDK THTCPPCPAP ELLGGPSVFL 360 FPPKPKDTLM ISRTPEVTCV WDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV 420 VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVCTLP PSRDELTKNQ 480 VSLSCAVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSKLTV DKSRWQQGNV 540 FSCSVMHEAL HNHYTQKSLS LSPGK 565 hILl-Rl-hlgGl-Fc DNA (SEQ ID NO. 4)

ATGAAGGTCC TGCTCAGGCT GATCTGCTTC ATTGCCCTGC TCATCAGCAG CCTGGAAGCC 60 GACAAGTGCA AGGAGAGGGA GGAGAAGATC ATCCTCGTCA GCTCCGCCAA CGAGATTGAT 120 GTCAGGCCCT GCCCCCTCAA CCCCAATGAG CACAAGGGCA CAATCACCTG GTACAAGGAC 180 GACAGCAAGA CCCCTGTCTC CACCGAGCAG GCCAGCAGAA TCCACCAGCA CAAAGAGAAG 240 CTGTGGTTCG TGCCTGCCAA GGTGGAAGAC AGCGGCCACT ACTACTGTGT GGTGAGGAAC 300 AGCTCCTACT GCCTCAGGAT CAAGATCTCC GCCAAGTTCG TGGAGAACGA GCCCAACCTC 360 TGTTACAACG CTCAGGCTAT TTTCAAGCAA AAGCTCCCCG TGGCTGGAGA CGGAGGCCTG 420 GTCTGTCCCT ACATGGAGTT CTTCAAGAAT GAGAATAATG AGCTCCCCAA GCTCCAGTGG 480 TACAAGGACT GTAAGCCTCT GCTCCTGGAC AACATCCACT TCTCCGGCGT GAAGGACAGA 540 CTGATCGTCA TGAACGTGGC CGAGAAGCAC AGGGGAAACT ACACCTGTCA CGCCTCCTAC 600 ACCTACCTCG GCAAGCAATA TCCCATCACC AGGGTCATCG AGTTCATCAC CCTCGAAGAG 660 AACAAGCCCA CAAGGCCTGT CATCGTCAGC CCCGCCAATG AAACCATGGA GGTGGACCTC 720 GGCAGCCAGA TCCAGCTGAT CTGCAACGTG ACAGGCCAGC TCAGCGACAT TGCCTACTGG 780 AAGTGGAACG GCTCCGTGAT CGACGAAGAT GATCCCGTGC TGGGCGAGGA CTACTATAGC 840 GTGGAGAACC CCGCCAACAA AAGAAGGAGC ACCCTGATCA CCGTGCTGAA CATCAGCGAG 900 ATCGAGTCCA GATTCTATAA GCATCCTTTC ACCTGCTTTG CCAAGAACAC CCACGGCATC 960 GACGCCGCTT ACATCCAGCT GATCTATCCC GTGACCAACG GATCCGGTGG AGGTGACAAA 1020 ACTCACACAT GCCCACCGTG CCCAGCTCCG GAACTCCTGG GCGGACCGTC AGTCTTCCTC 1080 TTCCCCCCAA AACCCAAGGA CACCCTCATG ATCTCCCGGA CCCCTGAGGT CACATGCGTG 1140 GTGGTGGACG TGAGCCACGA AGACCCTGAG GTCAAGTTCA ACTGGTACGT GGACGGCGTG 1200 GAGGTGCATA ATGCCAAGAC AAAGCCGCGG GAGGAGCAGT ACAACAGCAC GTACCGTGTG 1260 GTCAGCGTCC TCACCGTCCT GCACCAGGAC TGGCTGAATG GCAAGGAGTA CAAGTGCAAG 1320 GTCTCCAACA AAGCCCTCCC AGCCCCCATC GAGAAAACCA TCTCCAAAGC CAAAGGGCAG 1380 CCCCGAGAAC CACAGGTGTG TACCCTGCCC CCATCCCGGG ATGAGCTGAC CAAGAACCAG 1440 GTCAGCCTGA GTTGCGCGGT CAAAGGCTTC TATCCCAGCG ACATCGCCGT GGAGTGGGAG 1500 AGCAATGGGC AGCCGGAGAA CAACTACAAG ACCACGCCTC CCGTGTTGGA CTCCGACGGC 1560 TCCTTCTTCC TCGTCAGCAA GCTCACCGTG GACAAGAGCA GGTGGCAGCA GGGGAACGTC 1620 TTCTCATGCT CCGTGATGCA TGAGGCTCTG CACAACCACT ACACGCAGAA GAGCCTCTCC 1680 CTGTCTCCGG GTAAA 1695 hIL-lRAcP-hlgGl-Fc polypeptide (SEQ ID NO. 5)

MTLLWCWSL YFYGILQSDA SERCDDWGLD TMRQIQVFED EPARIKCPLF EHFLKFNYST 6 0 AHSAGLTLIW YWTRQDRDLE EPINFRLPEN RISKEKDVLW FRPTLLNDTG NYTCMLRNTT 1 20 YCSKVAFPLE VVQKDSCFNS PMKLPVHKLY IEYGIQRITC PNVDGYFPSS VKPTITWYMG 1 80 CYKIQNFNNV I PEGMNLSFL IALISNNGNY TCVVTYPENG RTFHLTRTLT VKVVGSPKNA 2 40 VPPVIHSPND HWYEKEPGE ELLI PCTVYF SFLMDSRNEV WWTI DGKKPD DITIDVTINE 3 00 SISHSRTEDE TRTQILSIKK VTSEDLKRSY VCHARSAKGE VAKAAKVKQK VPAPRYTVGS 3 60 GGGDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCWVDVS HEDPEVKFNW 4 20 YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS 4 80 KAKGQPREPQ VYTLPPCRDE LTKNQVSLWC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV 5 40 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 590 hIL-lRAcP-hlgGl-Fc DNA (SEQ ID NO. 6) ATGACTCTGC TGTGGTGCGT CGTGTCCCTC TACTTCTACG GCATCCTCCA GTCCGACGCC 60 AGCGAGAGGT GCGATGACTG GGGCCTGGAC ACCATGAGGC AGATCCAGGT GTTTGAGGAC 120 GAGCCTGCCA GGATTAAGTG CCCCCTCTTC GAGCACTTTC TGAAGTTCAA CTACAGCACC 180 GCTCACAGCG CTGGCCTGAC ACTGATCTGG TACTGGACAA GGCAGGACAG GGATCTCGAG 240 GAGCCCATCA ACTTCAGGCT GCCCGAAAAC AGAATCAGCA AGGAGAAGGA CGTGCTGTGG 300 TTCAGACCCA CCCTCCTCAA CGACACAGGC AACTACACCT GCATGCTCAG GAACACCACC 360 TACTGCAGCA AGGTGGCCTT CCCTCTCGAG GTGGTCCAGA AGGACAGCTG CTTCAACAGC 420 CCCATGAAGC TGCCCGTCCA TAAACTGTAC ATCGAGTACG GCATCCAGAG GATCACATGC 480 CCCAACGTGG ACGGCTACTT CCCCAGCTCC GTGAAGCCCA CCATCACATG GTACATGGGC 540 TGTTACAAAA TCCAGAACTT TAACAACGTC ATCCCCGAGG GCATGAATCT GTCCTTCCTG 600 ATCGCCCTGA TCAGCAACAA CGGCAATTAC ACCTGCGTCG TGACCTACCC CGAAAACGGC 660 AGGACCTTCC ACCTGACCAG GACCCTGACC GTGAAAGTCG TGGGAAGCCC CAAGAATGCC 720 GTGCCCCCCG TGATCCATTC CCCCAACGAC CACGTGGTGT ACGAGAAGGA GCCTGGAGAG 780 GAGCTGCTGA TCCCCTGCAC AGTGTACTTC TCCTTCCTGA TGGACTCCAG GAATGAAGTG 840 TGGTGGACCA TCGACGGCAA GAAGCCTGAC GACATCACCA TCGATGTGAC CATCAACGAG 900 AGCATCAGCC ACAGCAGGAC CGAGGACGAG ACCAGGACCC AGATCCTGAG CATCAAGAAA 960 GTCACCAGCG AGGACCTCAA GAGAAGCTAC GTCTGTCACG CCAGAAGCGC CAAAGGCGAG 1020 GTGGCCAAGG CTGCTAAGGT GAAACAGAAA GTGCCCGCTC CTAGGTACAC AGTCGGATCC 1080 GGTGGAGGTG ACAAAACTCA CACATGCCCA CCGTGCCCAG CTCCGGAACT CCTGGGCGGA 1140 CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCC TCATGATCTC CCGGACCCCT 1200 GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACC CTGAGGTCAA GTTCAACTGG 1260 TACGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGC CGCGGGAGGA GCAGTACAAC 1320 AGCACGTACC GTGTGGTCAG CGTCCTCACC GTCCTGCACC AGGACTGGCT GAATGGCAAG 1380 GAGTACAAGT GCAAGGTCTC CAACAAAGCC CTCCCAGCCC CCATCGAGAA AACCATCTCC 1440 AAAGCCAAAG GGCAGCCCCG AGAACCACAG GTGTACACCC TGCCCCCATG TCGGGATGAG 1500 CTGACCAAGA ACCAGGTCAG CCTGTGGTGC CTGGTCAAAG GCTTCTATCC CAGCGACATC 1560 GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT ACAAGACCAC GCCTCCCGTG 1620 TTGGACTCCG ACGGCTCCTT CTTCCTCTAC AGCAAGCTCA CCGTGGACAA GAGCAGGTGG 1680 CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG ATGCATGAGG CTCTGCACAA CCACTACACG 1740 CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA 1770

In certain aspects, the present invention provides for a recombinant mamalian expression plasmid for high expression of a polypeptide comprising the leading 333 amino acids of the human ILl-Rl fused with IgG-Fc domain (Fc-II) via a flexible linker, and for another recombinant DNA molecule having an open reading frame coding for another polypeptide comprising the leading 358 amino acids of the human IL-lRAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker. This plasmid comprises two cytomegalovirus (CMV) promoters to drive transcription of the two genes coding for said polypeptide and said another polypeptide, each followed by a transcription termination sequence and a polyadenylation sequence. The plasmid also contains an origin of replication and a gene conferring ampicillin resistance, for supporting plasmid propagation and selection in bacteria. The plasmid further contains a gene for Glutamine synthetase, a selectable marker widely used for establishing stable CHOKl and NSO cell lines. The plasmid of the present invention is illustratively shown in Figure 2.

In an example embodiment, the mammalian expression plasmid of the present teachings comprises the nucleotide sequence of SEQ ID NO. 7. hILl-Rl-hlgGl-Fc-II/ IL-lRAcP- hlgGl-Fc-V expression plasmid (SEQ ID NO. 7) AAGCTTGCCA CCATGAAGGT CCTGCTCAGG CTGATCTGCT TCATTGCCCT GCTCATCAGC 60 AGCCTGGAAG CCGACAAGTG CAAGGAGAGG GAGGAGAAGA TCATCCTCGT CAGCTCCGCC 120 AACGAGATTG ATGTCAGGCC CTGCCCCCTC AACCCCAATG AGCACAAGGG CACAATCACC 180 TGGTACAAGG ACGACAGCAA GACCCCTGTC TCCACCGAGC AGGCCAGCAG AATCCACCAG 240 CACAAAGAGA AGCTGTGGTT CGTGCCTGCC AAGGTGGAAG ACAGCGGCCA CTACTACTGT 300 GTGGTGAGGA ACAGCTCCTA CTGCCTCAGG ATCAAGATCT CCGCCAAGTT CGTGGAGAAC 360 GAGCCCAACC TCTGTTACAA CGCTCAGGCT ATTTTCAAGC AAAAGCTCCC CGTGGCTGGA 420 GACGGAGGCC TGGTCTGTCC CTACATGGAG TTCTTCAAGA ATGAGAATAA TGAGCTCCCC 480 AAGCTCCAGT GGTACAAGGA CTGTAAGCCT CTGCTCCTGG ACAACATCCA CTTCTCCGGC 540 GTGAAGGACA GACTGATCGT CATGAACGTG GCCGAGAAGC ACAGGGGAAA CTACACCTGT 600 CACGCCTCCT ACACCTACCT CGGCAAGCAA TATCCCATCA CCAGGGTCAT CGAGTTCATC 660 ACCCTCGAAG AGAACAAGCC CACAAGGCCT GTCATCGTCA GCCCCGCCAA TGAAACCATG 720 GAGGTGGACC TCGGCAGCCA GATCCAGCTG ATCTGCAACG TGACAGGCCA GCTCAGCGAC 780 ATTGCCTACT GGAAGTGGAA CGGCTCCGTG ATCGACGAAG ATGATCCCGT GCTGGGCGAG 840 GACTACTATA GCGTGGAGAA CCCCGCCAAC AAAAGAAGGA GCACCCTGAT CACCGTGCTG 900 AACATCAGCG AGATCGAGTC CAGATTCTAT AAGCATCCTT TCACCTGCTT TGCCAAGAAC 960 ACCCACGGCA TCGACGCCGC TTACATCCAG CTGATCTATC CCGTGACCAA CGGATCCGGT 1020 GGAGGTGACA AAACTCACAC ATGCCCACCG TGCCCAGCTC CGGAACTCCT GGGCGGACCG 1080 TCAGTCTTCC TCTTCCCCCC AAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAG 1140 GTCACATGCG TGGTGGTGGA CGTGAGCCAC GAAGACCCTG AGGTCAAGTT CAACTGGTAC 1200 GTGGACGGCG TGGAGGTGCA TAATGCCAAG ACAAAGCCGC GGGAGGAGCA GTACAACAGC 1260 ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGG ACTGGCTGAA TGGCAAGGAG 1320 TACAAGTGCA AGGTCTCCAA CAAAGCCCTC CCAGCCCCCA TCGAGAAAAC CATCTCCAAA 1380 GCCAAAGGGC AGCCCCGAGA ACCACAGGTG TGTACCCTGC CCCCATCCCG GGATGAGCTG 1440 ACCAAGAACC AGGTCAGCCT GAGTTGCGCG GTCAAAGGCT TCTATCCCAG CGACATCGCC 1500 GTGGAGTGGG AGAGCAATGG GCAGCCGGAG AACAACTACA AGACCACGCC TCCCGTGTTG 1560 GACTCCGACG GCTCCTTCTT CCTCGTCAGC AAGCTCACCG TGGACAAGAG CAGGTGGCAG 1620 CAGGGGAACG TCTTCTCATG CTCCGTGATG CATGAGGCTC TGCACAACCA CTACACGCAG 1680 AAGAGCCTCT CCCTGTCTCC GGGTAAATAA TAGAATTCAT TGATCATAAT CAGCCATACC 1740 ACATTTGTAG AGGTTTTACT TGCTTTAAAA AACCTCCCAC ACCTCCCCCT GAACCTGAAA 1800 CATAAAATGA ATGCAATTGT TGTTGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA 1860 TAAAGCAATA GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT 1920 GGTTTGTCCA AACTCATCAA TGTATCTTAT CATGTCTGGC GGCCGCCGAT ATTTGAAAAT 1980 ATGGCATATT GAAAATGTCG CCGATGTGAG TTTCTGTGTA ACTGATATCG CCATTTTTCC 2040 AAAAGTGATT TTTGGGCATA CGCGATATCT GGCGATAGCG CTTATATCGT TTACGGGGGA 2100 TGGCGATAGA CGACTTTGGT GACTTGGGCG ATTCTGTGTG TCGCAAATAT CGCAGTTTCG 2160 ATATAGGTGA CAGACGATAT GAGGCTATAT CGCCGATAGA GGCGACATCA AGCTGGCACA 2220 TGGCCAATGC ATATCGATCT ATACATTGAA TCAATATTGG CCATTAGCCA TATTATTCAT 2280 TGGTTATATA GCATAAATCA ATATTGGCTA TTGGCCATTG CATACGTTGT ATCCATATCA 2340 TAATATGTAC ATTTATATTG GCTCATGTCC AACATTACCG CCATGTTGAC ATTGATTATT 2400 GACTAGTTAT TAATAGTAAT CAATTACGGG GTCATTAGTT CATAGCCCAT ATATGGAGTT 2460 CCGCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA CCGCCCAACG ACCCCCGCCC 2520 ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA ATAGGGACTT TCCATTGACG 2580 TCAATGGGTG GAGTATTTAC GGTAAACTGC CCACTTGGCA GTACATCAAG TGTATCATAT 2640 GCCAAGTACG CCCCCTATTG ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA 2700 GTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT 2760 TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT GGATAGCGGT TTGACTCACG 2820 GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT TTGTTTTGGC ACCAAAATCA 2880 ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCG 2940 TGTACGGTGG GAGGTCTATA TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG 3000 ACGCCATCCA CGCTGTTTTG ACCTCCATAG AAGACACCGG GACCGATCCA GCCTCCGCGG 3060 CCGGGAACGG TGCATTGGAA CGCGGATTCC CCGTGCCAAG AGTGACGTAA GTACCGCCTA 3120 TAGAGTCTAT AGGCCCACCC CCTTGGCTTC TTATGCATGC TATACTGTTT TTGGCTTGGG 3180 GTCTATACAC CCCCGCTTCC TCATGTTATA GGTGATGGTA TAGCTTAGCC TATAGGTGTG 3240 GGTTATTGAC CATTATTGAC CACTCCCCTA TTGGTGACGA TACTTTCCAT TACTAATCCA 3300 TAACATGGCT CTTTGCCACA ACTCTCTTTA TTGGCTATAT GCCAATACAC TGTCCTTCAG 3360 AGACTGACAC GGACTCTGTA TTTTTACAGG ATGGGGTCTC ATTTATTATT TACAAATTCA 3420 CATATACAAC ACCACCGTCC CCAGTGCCCG CAGTTTTTAT TAAACATAAC GTGGGATCTC 3480 CACGCGAATC TCGGGTACGT GTTCCGGACA TGGGCTCTTC TCCGGTAGCG GCGGAGCTTC 3540 TACATCCGAG CCCTGCTCCC ATGCCTCCAG CGACTCATGG TCGCTCGGCA GCTCCTTGCT 3600 CCTAACAGTG GAGGCCAGAC TTAGGCACAG CACGATGCCC ACCACCACCA GTGTGCCGCA 3660 CAAGGCCGTG GCGGTAGGGT ATGTGTCTGA AAATGAGCTC GGGGAGCGGG CTTGCACCGC 3720 TGACGCATTT GGAAGACTTA AGGCAGCGGC AGAAGAAGAT GCAGGCAGCT GAGTTGTTGT 3780 GTTCTGATAA GAGTCAGAGG TAACTCCCGT TGCGGTGCTG TTAACGGTGG AGGGCAGTGT 3840 AGTCTGAGCA GTACTCGTTG CTGCCGCGCG CGCCACCAGA CATAATAGCT GACAGACTAA 3900 CAGACTGTTC CTTTCCATGG GTCTTTTCTG CAGTCACCGT CCTTGACACG AAGCTTGCCA 3960 CCATGACTCT GCTGTGGTGC GTCGTGTCCC TCTACTTCTA CGGCATCCTC CAGTCCGACG 4020 CCAGCGAGAG GTGCGATGAC TGGGGCCTGG ACACCATGAG GCAGATCCAG GTGTTTGAGG 4080 ACGAGCCTGC CAGGATTAAG TGCCCCCTCT TCGAGCACTT TCTGAAGTTC AACTACAGCA 4140 CCGCTCACAG CGCTGGCCTG ACACTGATCT GGTACTGGAC AAGGCAGGAC AGGGATCTCG 4200 AGGAGCCCAT CAACTTCAGG CTGCCCGAAA ACAGAATCAG CAAGGAGAAG GACGTGCTGT 4260 GGTTCAGACC CACCCTCCTC AACGACACAG GCAACTACAC CTGCATGCTC AGGAACACCA 4320 CCTACTGCAG CAAGGTGGCC TTCCCTCTCG AGGTGGTCCA GAAGGACAGC TGCTTCAACA 4380 GCCCCATGAA GCTGCCCGTC CATAAACTGT ACATCGAGTA CGGCATCCAG AGGATCACAT 4440 GCCCCAACGT GGACGGCTAC TTCCCCAGCT CCGTGAAGCC CACCATCACA TGGTACATGG 4500 GCTGTTACAA AATCCAGAAC TTTAACAACG TCATCCCCGA GGGCATGAAT CTGTCCTTCC 4560 TGATCGCCCT GATCAGCAAC AACGGCAATT ACACCTGCGT CGTGACCTAC CCCGAAAACG 4620 GCAGGACCTT CCACCTGACC AGGACCCTGA CCGTGAAAGT CGTGGGAAGC CCCAAGAATG 4680 CCGTGCCCCC CGTGATCCAT TCCCCCAACG ACCACGTGGT GTACGAGAAG GAGCCTGGAG 4740 AGGAGCTGCT GATCCCCTGC ACAGTGTACT TCTCCTTCCT GATGGACTCC AGGAATGAAG 4800 TGTGGTGGAC CATCGACGGC AAGAAGCCTG ACGACATCAC CATCGATGTG ACCATCAACG 4860 AGAGCATCAG CCACAGCAGG ACCGAGGACG AGACCAGGAC CCAGATCCTG AGCATCAAGA 4920 AAGTCACCAG CGAGGACCTC AAGAGAAGCT ACGTCTGTCA CGCCAGAAGC GCCAAAGGCG 4980 AGGTGGCCAA GGCTGCTAAG GTGAAACAGA AAGTGCCCGC TCCTAGGTAC ACAGTCGGAT 5040 CCGGTGGAGG TGACAAAACT CACACATGCC CACCGTGCCC AGCTCCGGAA CTCCTGGGCG 5100 GACCGTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC CCTCATGATC TCCCGGACCC 5160 CTGAGGTCAC ATGCGTGGTG GTGGACGTGA GCCACGAAGA CCCTGAGGTC AAGTTCAACT 5220 GGTACGTGGA CGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAG GAGCAGTACA 5280 ACAGCACGTA CCGTGTGGTC AGCGTCCTCA CCGTCCTGCA CCAGGACTGG CTGAATGGCA 5340 AGGAGTACAA GTGCAAGGTC TCCAACAAAG CCCTCCCAGC CCCCATCGAG AAAACCATCT 5400 CCAAAGCCAA AGGGCAGCCC CGAGAACCAC AGGTGTACAC CCTGCCCCCA TGTCGGGATG 5460 AGCTGACCAA GAACCAGGTC AGCCTGTGGT GCCTGGTCAA AGGCTTCTAT CCCAGCGACA 5520 TCGCCGTGGA GTGGGAGAGC AATGGGCAGC CGGAGAACAA CTACAAGACC ACGCCTCCCG 5580 TGTTGGACTC CGACGGCTCC TTCTTCCTCT ACAGCAAGCT CACCGTGGAC AAGAGCAGGT 5640 GGCAGCAGGG GAACGTCTTC TCATGCTCCG TGATGCATGA GGCTCTGCAC AACCACTACA 5700 CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA AATAATAGAA TTCATTGATC ATAATCAGCC 5760 ATACCACATT TGTAGAGGTT TTACTTGCTT TAAAAAACCT CCCACACCTC CCCCTGAACC 5820 TGAAACATAA AATGAATGCA ATTGTTGTTG TTAACTTGTT TATTGCAGCT TATAATGGTT 5880 ACAAATAAAG CAATAGCATC ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA 5940 GTTGTGGTTT GTCCAAACTC ATCAATGTAT CTTATCATGT CTGGATCCTC TACGCCGGAC 6000 GCATCGTGGC CGGCATCACC GGCGCCACAG GTGCGGTTGC TGGCGCCTAT ATCGCCGACA 6060 TCACCGATGG GGAAGATCGG GCTCGCCACT TCGGGCTCAT GAGCGCTTGT TTCGGCGTGG 6120 GTATGGTGGC AGGCCCCGTG GCCGGGGGAC TGTTGGGCGC CATCTCCTTG CATGCACCAT 6180 TCCTTGCGGC GGCGGTGCTC AACGGCCTCA ACCTACTACT GGGCTGCTTC CTAATGCAGG 6240 AGTCGCATAA GGGAGAGCGT CGACCTCGGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC 6300 GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG 6360 GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA 6420 CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC 6480 ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG 6540 TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT 6600 CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA 6660 GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA 6720 CTAGAAGAAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG 6780 TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA 6840 AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG 6900 GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA 6960 AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA 7020 TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG 7080 CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA 7140 TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC 7200 CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC 7260 CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA 7320 GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC TACAGGCATC GTGGTGTCAC 7380 GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT 7440 GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA 7500 GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG 7560 TCATGCCATC CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG 7620 AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AATACGGGAT AATACCGCGC 7680 CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT 7740 CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT 7800 CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG 7860 CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC 7920 AATATTATTG AAGCATTTAT CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA 7980 TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG 8040 TCTAAGAAAC CATTATTATC ATGACATTAA CCTATAAAAA TAGGCGTATC ACGAGGCCCT 8100 GATGGCTCTT TGCGGCACCC ATCGTTCGTA ATGTTCCGTG GCACCGAGGA CAACCCTCAA 8160 GAGAAAATGT AATCACACTG GCTCACCTTC GGGTGGGCCT TTCTGCGTTT ATAAGGAGAC 8220 ACTTTATGTT TAAGAAGGTT GGTAAATTCC TTGCGGCTTT GGCAGCCAAG CTAGATCCGG 8280 CTGTGGAATG TGTGTCAGTT AGGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT 8340 ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCAGGTGTG GAAAGTCCCC AGGCTCCCCA 8400 GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCATAGT CCCGCCCCTA 8460 ACTCCGCCCA TCCCGCCCCT AACTCCGCCC AGTTCCGCCC ATTCTCCGCC CCATGGCTGA 8520

CTAATTTTTT TTATTTATGC AGAGGCCGAG GCCGCCTCGG CCTCTGAGCT ATTCCAGAAG 8580

TAGTGAGGAG GCTTTTTTGG AGGCCTAGGC TTTTGCAAAA AGCTAGCTTG GGGCCACCGC 8640

TCAGAGCACC TTCCACCATG GCCACCTCAG CAAGTTCCCA CTTGAACAAA AACATCAAGC 8700 AAATGTACTT GTGCCTGCCC CAGGGTGAGA AAGTCCAAGC CATGTATATC TGGGTTGATG 8760

GTACTGGAGA AGGACTGCGC TGCAAAACCC GCACCCTGGA CTGTGAGCCC AAGTGTGTAG 8820

AAGAGTTACC TGAGTGGAAT TTTGATGGCT CTAGTACCTT TCAGTCTGAG GGCTCCAACA 8880

GTGACATGTA TCTCAGCCCT GTTGCCATGT TTCGGGACCC CTTCCGCAGA GATCCCAACA 8940

AGCTGGTGTT CTGTGAAGTT TTCAAGTACA ACCGGAAGCC TGCAGAGACC AATTTAAGGC 9000 ACTCGTGTAA ACGGATAATG GACATGGTGA GCAACCAGCA CCCCTGGTTT GGAATGGAAC 9060

AGGAGTATAC TCTGATGGGA ACAGATGGGC ACCCTTTTGG TTGGCCTTCC AATGGCTTTC 9120

CTGGGCCCCA AGGTCCGTAT TACTGTGGTG TGGGCGCAGA CAAAGCCTAT GGCAGGGATA 9180

TCGTGGAGGC TCACTACCGC GCCTGCTTGT ATGCTGGGGT CAAGATTACA GGAACAAATG 9240

CTGAGGTCAT GCCTGCCCAG TGGGAACTCC AAATAGGACC CTGTGAAGGA ATCCGCATGG 9300 GAGATCATCT CTGGGTGGCC CGTTTCATCT TGCATCGAGT ATGTGAAGAC TTTGGGGTAA 9360

TAGCAACCTT TGACCCCAAG CCCATTCCTG GGAACTGGAA TGGTGCAGGC TGCCATACCA 9420

ACTTTAGCAC CAAGGCCATG CGGGAGGAGA ATGGTCTGAA GCACATCGAG GAGGCCATCG 9480

AGAAACTAAG CAAGCGGCAC CGGTACCACA TTCGAGCCTA CGATCCCAAG GGGGGCCTGG 9540

ACAATGCCCG TGGTCTGACT GGGTTCCACG AAACGTCCAA CATCAACGAC TTTTCTGCTG 9600 GTGTCGCCAA TCGCAGTGCC AGCATCCGCA TTCCCCGGAC TGTCGGCCAG GAGAAGAAAG 9660

GTTACTTTGA AGACCGCGGC CCCTCTGCCA ATTGTGACCC CTTTGCAGTG ACAGAAGCCA 9720

TCGTCCGCAC ATGCCTTCTC AATGAGACTG GCGACGAGCC CTTCCAATAC AAAAACTAAT 9780

TAGACTTTGA GTGATCTTGA GCCTTTCCTA GTTCATCCCA CCCCGCCCCA GAGAGATCTT 9840

TGTGAAGGAA CCTTACTTCT GTGGTGTGAC ATAATTGGAC AAACTACCTA CAGAGATTTA 9900 AAGCTCTAAG GTAAATATAA AATTTTTAAG TGTATAATGT GTTAAACTAC TGATTCTAAT 9960

TGTTTGTGTA TTTTAGATTC CAACCTATGG AACTGATGAA TGGGAGCAGT GGTGGAATGC 10020

CTTTAATGAG GAAAACCTGT TTTGCTCAGA AGAAATGCCA TCTAGTGATG ATGAGGCTAC 10080

TGCTGACTCT CAACATTCTA CTCCTCCAAA AAAGAAGAGA AAGGTAGAAG ACCCCAAGGA 10140

CTTTCCTTCA GAATTGCTAA GTTTTTTGAG TCATGCTGTG TTTAGTAATA GAACTCTTGC 10200 TTGCTTTGCT ATTTACACCA CAAAGGAAAA AGCTGCACTG CTATACAAGA AAATTATGGA 10260

AAAATATTCT GTAACCTTTA TAAGTAGGCA TAACAGTTAT AATCATAACA TACTGTTTTT 10320

TCTTACTCCA CACAGGCATA GAGTGTCTGC TATTAATAAC TATGCTCAAA AATTGTGTAC 10380

CTTTAGCTTT TTAATTTGTA AAGGGGTTAA TAAGGAATAT TTGATGTATA GTGCCTTGAC 10440

TAGAGATCAT AATCAGCCAT ACCACATTTG TAGAGGTTTT ACTTGCTTTA AAAAACCTCC 10500 CACACCTCCC CCTGAACCTG AAACATAAAA TGAATGCAAT TGTTGTTGTT AACTTGTTTA 10560

TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC AAATTTCACA AATAAAGCAT 10620

TTTTTTCACT GCATTCTAGT TGTGGTTTGT CCAAACTCAT CAATGTATCT TATCATGTCT 10680

GGATCTAGCT TCGTGTCAAG GACGGTGACT GCAGTGAATA ATAAAATGTG TGTTTGTCCG 10740

AAATACGCGT TTTGAGATTT CTGTCGCCGA CTAAATTCAT GTCGCGCGAT AGTGGTGTTT 10800 ATCGCCGATA GAGATGGCGA TATTGGAAAA ATCGATATTT GAAAATATGG CATATTGAAA 10860

ATGTCGCCGA TGTGAGTTTC TGTGTAACTG ATATCGCCAT TTTTCCAAAA GTGATTTTTG 10920

GGCATACGCG ATATCTGGCG ATAGCGCTTA TATCGTTTAC GGGGGATGGC GATAGACGAC 10980

TTTGGTGACT TGGGCGATTC TGTGTGTCGC AAATATCGCA GTTTCGATAT AGGTGACAGA 11040

CGATATGAGG CTATATCGCC GATAGAGGCG ACATCAAGCT GGCACATGGC CAATGCATAT 11100 CGATCTATAC ATTGAATCAA TATTGGCCAT TAGCCATATT ATTCATTGGT TATATAGCAT 11160

AAATCAATAT TGGCTATTGG CCATTGCATA CGTTGTATCC ATATCATAAT ATGTACATTT 11220

ATATTGGCTC ATGTCCAACA TTACCGCCAT GTTGACATTG ATTATTGACT AGTTATTAAT 11280

AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC 11340

TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG ACGTCAATAA 11400 TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA TGGGTGGAGT 11460

ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA AGTACGCCCC 11520

CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAT 11580 GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC 11640 GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA TTTCCAAGTC 11700 TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA AAATCAACGG GACTTTCCAA 11760 AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA CGGTGGGAGG 11820 TCTATATAAG CAGAGCTCGT TTAGTGAACC GTCAGATCGC CTGGAGACGC CATCCACGCT 11880 GTTTTGACCT CCATAGAAGA CACCGGGACC GATCCAGCCT CCGCGGCCGG GAACGGTGCA 11940 TTGGAACGCG GATTCCCCGT GCCAAGAGTG ACGTAAGTAC CGCCTATAGA GTCTATAGGC 12000 CCACCCCCTT GGCTTCTTAT GCATGCTATA CTGTTTTTGG CTTGGGGTCT ATACACCCCC 12060 GCTTCCTCAT GTTATAGGTG ATGGTATAGC TTAGCCTATA GGTGTGGGTT ATTGACCATT 12120 ATTGACCACT CCCCTATTGG TGACGATACT TTCCATTACT AATCCATAAC ATGGCTCTTT 12180 GCCACAACTC TCTTTATTGG CTATATGCCA ATACACTGTC CTTCAGAGAC TGACACGGAC 12240 TCTGTATTTT TACAGGATGG GGTCTCATTT ATTATTTACA AATTCACATA TACAACACCA 12300 CCGTCCCCAG TGCCCGCAGT TTTTATTAAA CATAACGTGG GATCTCCACG CGAATCTCGG 12360 GTACGTGTTC CGGACATGGG CTCTTCTCCG GTAGCGGCGG AGCTTCTACA TCCGAGCCCT 12420 GCTCCCATGC CTCCAGCGAC TCATGGTCGC TCGGCAGCTC CTTGCTCCTA ACAGTGGAGG 12480 CCAGACTTAG GCACAGCACG ATGCCCACCA CCACCAGTGT GCCGCACAAG GCCGTGGCGG 12540 TAGGGTATGT GTCTGAAAAT GAGCTCGGGG AGCGGGCTTG CACCGCTGAC GCATTTGGAA 12600 GACTTAAGGC AGCGGCAGAA GAAGATGCAG GCAGCTGAGT TGTTGTGTTC TGATAAGAGT 12660 CAGAGGTAAC TCCCGTTGCG GTGCTGTTAA CGGTGGAGGG CAGTGTAGTC TGAGCAGTAC 12720 TCGTTGCTGC CGCGCGCGCC ACCAGACATA ATAGCTGACA GACTAACAGA CTGTTCCTTT 12780 CCATGGGTCT TTTCTGCAGT CACCGTCCTT GACACG 12816

In certain aspects, the present teachings provide for a mammalian expression system for production of a heterodimeric protein assembly comprising a polypeptide comprising amino acid residues 18 through 333 of the human ILl-Rl fused with IgG-Fc domain (Fc-II) via a flexible linker, and another polypeptide comprising amino acid residues 21 through 358 of the human IL- lRAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker.

In an example embodiment, the mammalian expression system of the present invention comprises Chinese hamster ovary cells (CHO-K1) harboring a plasmid comprising nucleotide sequence of SEQ ID NO. 7.

In certain aspects, the present teachings provide for a method of treatment of a mammal effected by the following disorders associated with IL-Ιβ modulation: arthritis, a gout, a rheumatoid arthritis, a Cryopyrin-Associated Periodic Syndromes (CAPS), a scleroderma, a diabetes, a atherosclerosis, a dry eye disease, an ocular allergy, or an uveitis.

EXAMPLES

The following Examples illustrate the forgoing aspects and other aspects of the present teachings. These non- limiting Examples are put forth so as to provide those of ordinary skill in the art with illustrative embodiments as to how the compounds, compositions, articles, devices, and/or methods claimed herein are made and evaluated. The Examples are intended to be purely exemplary of the inventions disclosed herein and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for.

Example 1 : Construction of plasmids for expression of polypeptides of the present invention.

Optimized gene sequences comprising sequences encoding residues amino acid residues 1 through 333 of IL-1R1 or residues 1 through 358 of IL-lRAcP were chemically synthesized. The resulting fragments were cloned in-frame into the recipient intermediate vectors comprising sequences coding for Fc-V or Fc-II, via Hindlll and BspEI restriction sites. Obtained positive clones were verified by DNA sequencing. The resulting constructions were termed PKN001 '-IL- lR-FcV and PKNOOl '-RAcP-FcII, respectively.

The gene encoding IL-lRl-Fc-V was then cloned into a second intermediate vector, termed

PKN002, via Hindlll and EcoRI restriction sites. Positive clones were screened by double digestion and the insertion sequence of the correct plasmids was verified by DNA sequencing. The resulting constructions were termed PKN002-IL-lR-FcV.

Finally, the expression cassette for RAcP-FcII from PKN001 '-RAcP-FcII was integrated into PKN002-IL-lR-FcV plasmid via Notl and Sail restriction sites and yielded a new

recombinant construction that contained expression elements for both RAcP-FcII and IL-lR-FcV. The resulting clones were screened by Notl and Sail double digestion followed by DNA gel electrophoresis, the correct clones were exhibiting one band migrating approximately as an 8kb DNA fragment and another band migrating approximately as a 4kb DNA fragment. The final plasmid was termed PKN012-ILlR-FcV-RAcP-FcII.

Recombinant plasmid PKN012-ILlR-FcV-RAcP-FcII combines expression cassettes for both RAcP-FcII and IL-lR-FcV. The plasmid can be used for co-expressing RAcP-FcII and IL- lR-FcV proteins in a 1 to 1 ratio under the control of a CMV promoter. The majority of these two fusion proteins would then form ILlR-FcV/RAcP-FcII heterodimers after secretion. The plasmid also expresses Glutamine synthetase (GS) protein via a SV40 promoter, which can be used as a selection marker to generate stable cell lines for ILlR-FcV/RAcP-FcII heterodimer production. The plasmid map for PKN012-ILlR-FcV-PvAcP-FcII is illustratively shown in Figure 2.

Example 2: Generation of stable cell lines for expressing polypeptides of the present invention.

A stable clone of CHO-Kl cells co-expressing hILl-Rl-hlgGl-Fc polypeptide of SEQ ID NO. 1 and hIL-lRAcP-hlgGl-Fc polypeptide of SEQ ID NO. 2 has been generated through standard cell biology protocols. The expression plasmid PKN012-ILlR-FcV-RAcP-FcII described in Example 1 was used for generating stable cell lines for high expression of said polypeptides. Expression levels of said polypeptides in a plurality of clones were about or over 100 mg/L in a 7-day batch culture. One clonal cell line showed expression levels of about or over 300 mg/L in shake flask batch culture. Materials

Chinese hamster ovary cells (CHO-Kl) were obtained as frozen stocks from ATCC (CCL- 61™). The cells were adapted in house into a CD CHO media. Media and reagent were obtained from commercial source. Upon full adaptation, the cells were grown to high density for a few passages. The resulting cells were subcloned. One of the resulting clones with a doubling time under 20 hrs and good morphology was selected as parental cell line.

Methods

CHO-Kl cells at passage 4 were cultured in CD-CHO chemically defined media (Invitrogen) containing 6 mM L-Glutamine. The cells were maintained by 1 :3 splits after reaching a cell density of 4xl0⁶ cells/ml. Cells were span down by centrifugation and resuspended into 1 ml of CD-CHO chemically defined media (Invitrogen).

The following electroporation protocol was utilized:

40 ug of plasmid DNA in 100 ul sterilized TE buffer was used for an electroporation; on the day of trans fection the viability of cells was at least 95%;

- cells were centrifuged at 800rpm for 5 min; the super was removed and the cells were resuspended in 10 ml CD CHO media and centrifuged again; - the super was removed and the cells were resuspended in a small vol. of CD CHO media to 1.43xl0⁷ cells/ml;

- 0.7 ml cells (10⁷ cells) were added to the DNA and mixed gently by pipetting, avoiding generating bubbles;

cells were immediately electroporated by delivering a single pulse of 300 volts, 900 uF to each cuvette;

50 ml CD CHO (without L-glutamine) was immediately added to the elecroporated cells and mixed gently;

- the cell suspension was distributed into ten 96 well plates at 50 ul/well; the following day, 150 ul of CD CHO containing 33.3 uM MSX was added to each well.

A number of transfections were carried out in CHO-K1 cells in the process of generation of potential ILlR-FcV-RAcP-FcII expression cell line; a representative transfection growth curve is shown in Figure 3 and the data is shown in Table 1. Protein expression levels were analyzed by SDS-PAGE. Cell lines with high levels of protein overexpression exhibit a strong band with an apparent molecular weight of about 180 kDa. Based on the preliminary analysis four clones were selected for further analysis, where expression levels were further assessed by SDS-PAGE and ELISA. Well expressing clones were inoculated into 125 ml shake flasks, the cells were expanded and frozen.

The chosen highest production cell line was selected and thawed into CD-CHO. Growth curves on the cell line were assessed, and samples were collected daily for cell count, cell viability and ILlR-FcV-RAcP-FcII productivity. Based on these studies, it was determined that selected highest productivity cell line was expressing ILlR-FcV-RAcP-FcII heterodimer in CD-CHO media in amounts necessary to support commercial production. The yield of the heterodimer after purification was at least about 300 mg/L (without any production optimization). Table 1: A Representative Clonal Cell Line Growth curve

Culture period Cell density

Feeding (DAY) (10⁶ cells/ml)

0 0.5

1 1.09

2 1.93

3 2.97 +10% feed B

5 4.13

6 4.55 +10% feed B

7 4.6

8 5.46 +10% feed B

9 4.86

10 2.4

11 1

Example 3: Purification of polypeptides of the present invention. hILl-Rl-hlgGl-Fc polypeptide of SEQ ID NO. 1 and hIL-lRAcP-hlgGl-Fc polypeptide of SEQ ID NO. 2 were co-expressed in CHO-K1 essentially as described in foregoing Example 2. Cells were harvested and lysed utilizing well established protocols. After cell lysate clarification, the supernatant at protein concentration of about 0.4 mg/ml, containing expressed hlLl-Rl-hlgGl- Fc/ hIL-lRAcP-hlgGl-Fc polypeptides, was applied to a Protein A affinity column. The affinity purification step was carried out according to the procedure outlined in Table 2. The Protein A eluate containing hILl-Rl-hlgGl-Fc/ hIL-lRAcP-hlgGl-Fc at pH of about 3.5 - 3.7 is incubated for 45-60 minutes to inactivate potentially existing in the contaminating materials.

After material incubation for about 45 minutes at pH 3.6 at room temperature its pH is adjusted to about 7.9-8.1 with 2 M Tris-HCl pH 9.5 (~ 3.5% v/v of the diluted Pro A eluate). The low pH treated Protein A eluate is pH-adjusted to pH of about 8.0 using 1 M Tris-HCl, pH 9.0. The conductivity is adjusted to about 5.5 mS/cm with deionized water (dH20) if needed. In order to reduce the contents of DNA, HCP, endotoxin and potential viral contaminants, the pH adjusted Protein A column eluate was further purified by anion-exchange chromatography

(AIEX) utilizing Q Sepharose resin. The AIEX step is operated according to a step-elution

procedure outlined in Table 3. Pooled elution peak fractions are concentrated by microfiltration to a concentration of about 20 mg/ml, followed by addition of a 20% Sucrose stock solution to a final Sucrose concentration of about 1% (w/v) and freezing at -80°C.

A sample of thus purified protein was analyzed by size-exclusion HPLC (SEC-HPLC) and SDS-PAGE (reducing and non-reducing). The results of the analysis are presented in Figure 4 and Figure 5, respectively. In Figure 5, lanes 1, 3 show hILl-Rl-hlgGl-Fc/ hIL-lRAcP-hlgGl-Fc SDS-PAGE under non-reducing conditions, while lanes 4, 6 - under reducing conditions. Lanes 2, 5 show molecular weight markers. hILl-Rl-hlgGl-Fc/ hIL-lRAcP-hlgGl-Fc heterodimer has an apparent molecular weight of about 180 kDa, consisting of two di-sulfide linked monomers, each of an apparent molecular weight of about 90 kDa.

The SEC-HPLC operational procedure is outlined in Table 4. Loading sample is diluted with mobile phase to reach protein concentration of about 5 mg/ml. The biologically relevant form of hILl-Rl-hlgGl-Fc/ hIL-lRAcP-hlgGl-Fc heterodimer is represented by the major peak with retention time RT=14.979 min.

Table 2: The operational procedure of Protein A Affinity Chromatography

Step Buffer Vol Flow

CV cm/h

Rinse Before-use dH20 3 150

Equilibration lO mM NaPh, pH 6.0 5 150

Sample Load Cell harvest - 150

Wash 1 lO mM NaPh, pH 6.0 3 150

25 mM NaPh, 0.5 M NaCl, 5%

Wash 2 j 150

Isopropanol pH 7.0

Re-Equilibration lO mM NaPh, pH 6.0 3 150

Elution 20 mM Na-Citrate, pH 3.4 4 150

Re-Equilibration lO mM PB, pH 6.0 3 150

0.1 M NaOH (contact time 15 min),

CIP 1 100 reversed flow, CIP every 5 cycles

Rinse with NaCl lM NaCl 3 150

Rinse After-use dH20 3 150

Storage 20% (v/v) Ethanol, 20 mM NaPh, pH 7.0 3 150 Table 3: The operational procedure of AIEX

Step Buffer Vol Flow

cv cm/h

Rinse Before-use dH20 3 150

Recharge 10 mM Tris-HCl, 1 M NaCl, pH 8 3 150

Equilibration 10 mM Tris-HCl, 50 mM NaCl, pH 8 3 150

Sample Load Prepared Q Load - 150

Wash 10 mM Tris-HCl, 50 mM NaCl, pH 8 3 150

Elution 10 mM Tris-HCl, 0.35 M NaCl, pH 8.0 3 150

CIP 1 M NaOH (contact time 1 hr), 3 40 reversed-flow

Regeneration 10 mM Tris-HCl, 1 M NaCl, pH 8.0 3 150

Rinse After-use dH20 3 150

Storage 20% (v/v) Ethanol 3 150

Table 4: The Operational Procedure of SEC-HPLC

Mobile Phase: 20 mM phosphate, 300 mM NaCl, pH 7.4

Flow Rate: 0.5 mL/min

Column: G2000 SWxl, 7.8mmx300mm, TOSOH Bioscience

Guard column: TSK Guard SWxl,6.0mmx40mm, TOSOH Bioscience

Column Temperature: 25 ^°C

Sampler temperature: Rome temperature

Injection Volume: 10 μΐ

Detector Wavelength: 280 nm

Run Time: 30 min

The activity of thus purified sample, which was expressed and purified essentially as described in this example, was tested in a standard ELISA assay using commercially available human IL-Ιβ (PrimGene, Shanghai, China; Cat#: 101-OlB). A typical binding curve obtained in the assay is shown in Figure 6. Based on the curve analysis, the calculated EC50 value is about 50 ng/ml.

Example 4: Pharmacokinetics (PK) of ILlR-FcV-RAcP-FcII heterodimer after subcutaneous administration in mice.

Polypeptides of ILlR-FcV-RAcP-FcII hetedimer (SEQ ID NO. l and SEQ ID NO. 2) were co-expressed and purified essentially as described in the forgoing examples. For administration into animals, the polypeptides were formulated in the following buffer: 1% w/v Sucrose, lOOmM Sodium Chloride, 20 mM L-Arginine Hydrochloride, 25 mM Sodium Bicarbonate, pH 6.3. The dosing stock concentration used was 0.5 mg/mL of the polypeptide.

Fourteen female Balb/c nu/nu mice were randomized based on body weight into seven groups of two animals on Day 0 of the study. A single treatment of the polypeptides (5 mg/kg) was administered subcutaneously (dorsal) on Day 0 to all groups except mice in Group 1 , which were bled via cardiac puncture for plasma preparation on Day 0 of the study. Blood samples were collected from mice via the orbital sinus in the remaining groups at various times throughout the study for preparation of plasma.

Body weights were recorded for all animals on the treatment day (Day 0) and then three times per week, including the termination day of each group.

Groups of mice were culled at specific time points for plasma preparation. Body weight changes were not measured in groups culled for sample collection at 0 hours and within 36 hours of dose administration. All other mice gained body weight and no adverse clinical signs were reported during the study period.

Following the in-life phase of the study, plasma samples were analyzed by ELISA for Hu-Fc proteins. Quantification of Hu-Fc in mouse plasma samples by ELISA was used as a read-out for circulating levels of the polypeptides. The assay was performed on samples from all mice in the study.

The polypeptides were detected in the plasma of animals at 1 hour post-administration. One Phase Decay Model equation using Prism 5.0c (GraphPad Software Inc, La Jolla, CA, USA) was then used to determine pharmacokinetics of the polypeptides as detected by Hu-Fc ELISA. Peak circulating level of Hu-Fc (Cmax) was determined to be 1.65 μg/mL, and time to peak circulating levels (Tmax) was 36 hours post treatment. The half-life (Tl/2) was 88.15 hours and the rate constant (K) was 0.0079 hr-1. Hu-Fc levels were negligible in the plasma of the untreated Group 1 animals. The results of the study are summarized in Table 6.

Table 6: Mean Human-Fc Protein Concentration ± SEM ^g/mL) at each Time Post- Administration

Bleeding Schedule Mean Human-Fc

Group Treatment (time post- Protein Concentration SEM

administration) [^g/mL]

1 No treatment 0 hours 0.00

2 30 minutes 0.02 0.01

3 1 hour 0.12 0.00

4 2 hours 0.24 0.09

5 4 hours 0.76 0.03

6 8 hours 1.1 1 0.10

7 polypeptide of SEQ 10 hours 1.53 0.08

ID NO. 1 (5 mg/kg,

2 24 hours"

Once only, s.c.) 1.47 0.14

3 36 hours" 1.65 0.1 1

4 96 hours* 1.27 0.01

5 7 days" 0.43 0.01

6 14 days" 0.13 0.07

7 21 days" 0.06 0.04

*SEM unable to be calculated as level of Hu-Fc was below detectable limit of ELISA for one of samples in group.

The Human-Fc Protein Concentration was determined by Prism Software based on the mean absorbance of the triplicate samples

Bleed via orbital sinus

Bleed via terminal cardiac puncture All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

at is claimed is:

A heterodimeric protein composition capable of binding human IL-Ιβ, said protein composition comprising:

a first polypeptide comprising

a first amino acid sequence comprising amino acids 18 through 333 of human IL1-R1, and

a second amino acid sequence comprising a first mutant of a Fc portion of human immunoglobulin gamma- 1 Fc;

a second polypeptide comprising

another first amino acid sequence comprising amino acids 21 through 358 of human ILl-RAcP, and

another second amino acid sequence comprising a second mutant of the Fc portion of human immunoglobulin gamma- 1 Fc; and

wherein said first and second mutants are selected as to favor hetedimeric assembly between said first and second mutants over any homodimeric assembly.

The protein composition of claim 1 , wherein said protein composition exhibits human IL- 1β binding activity in an ELISA assay with an EC50 of about 50 ng/ml.

The protein composition of claim 1 , said first polypeptide comprises amino acid sequence of SEQ ID NO. 1 and said second polypeptide comprises amino acid sequence of SEQ ID NO. 2.

A therapeutic composition, the composition comprising a heterodimeric protein

composition capable of binding human IL-Ιβ, said heterodimeric protein composition comprising: a first polypeptide comprising

a first amino acid sequence comprising amino acids 18 through 333 of human IL1-R1, and a second amino acid sequence comprising a first mutant of a Fc portion of human immunoglobulin gamma- 1 Fc;

a second polypeptide comprising

another first amino acid sequence comprising amino acids 21 through 358 of human ILl-RAcP, and

another second amino acid sequence comprising a second mutant of the Fc portion of human immunoglobulin gamma- 1 Fc; and

wherein said first and second mutants are selected as to favor hetedimeric assembly between said first and second mutants over any homodimeric assembly.

5. The therapeutic composition of claim 4, wherein half-life of said heterodimeric protein composition in systemic circulation in mice after a subcutaneous administration at a dose of 5 mg/kg is at least about 88 hours, as assayed by human Fc ELISA.

6. A therapeutic composition, the composition comprising a heterodimeric protein

composition comprising a first polypeptide comprising amino acid sequence of SEQ ID NO. 1 and a second polypeptide comprising amino acid sequence of SEQ ID NO. 2.

7. An isolated nucleic acid encoding a polypeptide comprising amino acid sequence of SEQ ID NO. 3.

8. An isolated nucleic acid encoding a polypeptide comprising amino acid sequence of SEQ ID NO. 4.

9. The nucleic acid of claims 7 or 8, wherein the codon usage is optimized for high expression of said polypeptide in a mammalian cell.

10. The nucleic acid of claim 7, wherein the nucleic acid sequence comprises the sequence of SEQ ID NO. 5.

11. The nucleic acid of claim 8, wherein the nucleic acid sequence comprises the sequence of SEQ ID NO. 6.

12. The nucleic acid of claims 10 or 11, wherein said nucleic acid comprises an expression vector.

13. An isolated nucleic acid of SEQ ID NO. 7.

14. A heterologous expression system, the expression system harboring an expression vector comprising a nucleic acid sequence encoding a first polypeptide comprising amino acid sequence of SEQ ID NO. 3 and another nucleic acid sequence encoding a second polypeptide comprising amino acid sequence of SEQ ID NO. 4.

15. The expression system of claim 14, wherein said expression vector is harbored in a

mammalian cell.

16. The expression system of claim 15, wherein said mammalian cell is a CHO cell.

17. The expression system of claim 15, wherein said expression system is capable of

expressing a heterodimeric protein comprising a first polypeptide comprising amino acid sequence of SEQ ID NO. 1 and a second polypeptide comprising amino acid sequence of SEQ ID NO. 2, and wherein the level of expression of said heterodimeric protein is at least 300 mg per liter of cell culture.

18. Use of a substance for manufacture of a medicament for the treatment or prevention of a disease associated with modulation of activity of human IL-Ιβ , the substance comprising a heterodimeric protein comprising a first polypeptide comprising amino acid sequence of SEQ ID NO. 1 and a second polypeptide comprising amino acid sequence of SEQ ID NO. 2.

19. The use according to claim 18, wherein said disease is an arthritis.

20. The use according to claim 18, wherein said disease is a gout.

21. The use according to claim 18, wherein said disease is a rheumatoid arthritis.

22. The use according to claim 18, wherein said disease is a Cryopyrin- Associated Periodic Syndromes (CAPS).

23. The use according to claim 18, wherein said disease is a scleroderma.

24. The use according to claim 18, wherein said disease is a diabetes.

25. The use according to claim 18, wherein said disease is atherosclerosis.

26. The use according to claim 18, wherein said disease is a dry eye disease.

27. The use according to claim 18, wherein said disease is an ocular allergy.

28. The use according to claim 18, wherein said disease is an uveitis.

29. A method of treating or preventing a disease or condition associated with modulation of activity of human IL-Ιβ, the method comprising administering to a patient in need for treating or preventing a disease associated with modulation of activity of human IL-Ιβ a therapeutically effective amount of a pharmaceutical composition comprising a heterodimeric protein comprising a first polypeptide comprising amino acid sequence of SEQ ID NO. 1 and a second polypeptide comprising amino acid sequence of SEQ ID NO. 2.

30. The method according to claim 29, wherein said disease is an arthritis.

31. The method according to claim 29, wherein said disease is a gout.

32. The method according to claim 29, wherein said disease is a rheumatoid arthritis.

33. The method according to claim 29, wherein said disease is a Cryopyrin- Associated

Periodic Syndromes (CAPS).

34. The method according to claim 29, wherein said disease is a scleroderma.

35. The method according to claim 29, wherein said disease is a diabetes.

36. The method according to claim 29, wherein said disease is atherosclerosis.

37. The method according to claim 29, wherein said disease is a dry eye disease. The method according to claim 29, wherein said disease is an ocular allergy. The method according to claim 29, wherein said disease is an uveitis.