IL1-R1-derived inhibitors of IL-1β and uses thereof

Abstract

Therapeutic compositions applicable to the treatment or prevention of diseases associated with modulation of human IL-1β activity are described. In one aspect, the described compositions are based on the design of heterodimeric protein structures capable of binding to and reducing the function of human IL-1β. The heterodimeric protein structures comprise the extracellular portions of human IL1-R1 and human IL-1RAcP, or functional fragments thereof. Each IL1-R1 portion and IL-1RAcP portion is fused to a different variant of the Fc portion of human Ig gamma 1. The two different Fc variants in the heterodimeric protein structure are designed to favor heterodimer formation over any homomeric structure between the two Fc variants. The therapeutic compositions are formulated for administration to humans and animals.

Description

[Technical field]

[0001] Technology Areas The present invention is generally in the field of biopharmaceuticals and their use in conditions related to inflammatory disorders (e.g., rheumatoid arthritis, Crohn's disease, etc.), diabetes, cardiovascular disease and gout. More specifically, the present invention relates to compositions derived from the heterodimeric IL-1R1 / IL-1RAcP capable of inhibiting the IL-1β cytokine. [Background technology]

[0002] Background technology The interleukin (IL-1) family of cytokines comprises 11 proteins (IL-1F1 to IL-1F11) encoded by 11 independent genes in humans and rats. IL-1-type cytokines are the main mediators of innate immune responses, and blockade of the founding members IL-1 or IL-1β by interleukin 1 receptor antagonist (IL-1RA) indicates a major role for IL-1 in various human autoinflammatory diseases. IL-1 or IL-1β rapidly increases messenger RNA expression of hundreds of genes in several different cell types. The potent proinflammatory activity of IL-1 and IL-1β is restricted at three major levels: (i) synthesis and release, (ii) membrane receptors, and (iii) intracellular signaling. This pathway brings together the extracellular and intracellular signaling of IL-1 or IL-1β, including positive or negative feedback mechanisms that amplify or halt the IL-1 response. In response to receptor binding ligand, a complex series of phosphorylation and ubiquitination events activates the nuclear factor kappa B signaling pathway and the JNK and p38 mitogen-activated protein kinase pathways, which cooperate to induce the expression of canonical IL-1 target genes (IL-6, IL-8, MCP-1, COX-2, IB, IL-1, IL-1β, MKP-1, etc.) by transcriptional and post-transcriptional mechanisms. Notably, most of the intracellular components participating 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 stress (see Weber A, et al., Sci Signal., 2010 Jan 19;3(105), the entire teachings of which are incorporated herein by reference).

[0003] IL-1 and IL-1β bind independently to the ubiquitously expressed IL-1 receptor type I (IL-1R1). A third specific ligand, IL-1 receptor antagonist (IL-1RA), binds IL-1RI with similar specificity and affinity but does not activate the receptor and trigger downstream signaling. The IL-1 receptor accessory protein (IL-1RAcP) functions as a coreceptor required for signaling of the IL-1 / IL-1RI complex, and this coreceptor is also required for activation of IL-1R1 by other IL-1 family members, notably IL-18 and IL-33. The IL-1 receptor type II (IL-1R2) binds IL-1 and IL-1β but lacks a signaling cytoplasmic portion, and therefore functions as a decoy receptor. The naturally occurring "skinned" domains of IL-1RA, the cell membrane-anchored IL-1R2 and the extracellular IL-1 receptor chain, respectively (termed sIL-1RI, sIL-1RII and sIL-1RAcP, where "s" stands for soluble), naturally provide inducible negative regulators of IL-1 signaling in the extracellular space, the amount of which can be regulated by a combination of increased transcription and controlled release to limit or terminate the effects of IL-1.

[0004] The first step in IL-1 signaling is a ligand-induced conformational change in the first extracellular domain of IL-1RI that promotes the recruitment of IL-1RAcP. Through conserved cytoplasmic regions termed Toll- and IL-1R-like (TIR) ​​domains, the trimeric complex rapidly aggregates with two intracellular signaling proteins, myeloid differentiation primary response gene (MYD88) and interleukin-1 receptor-activated protein kinase (IRAK)4. Mice lacking MYD88 or IRAK4 exhibit severe defects in IL-1 signaling. Similarly, humans with IRAK4 gene mutations have defects in IL-1RI and Toll-like receptor (TLR) signaling. IL-1, IL-1RI, IL-RAcP, MYD88, and IRAK4 form a stable IL-1-induced first signaling module. This occurs in parallel with the (auto)phosphorylation of IRAK4, which in turn phosphorylates IRAK1 and IRAK2, which are then followed by the recruitment and oligomerization of tumor necrosis factor (TRAF)6. IRAK1 and 2 function as both adaptors and protein kinases to transmit downstream signals. The IRAK1, IRAK2 and TRAF6 complex dissociates from the initial receptor complex, and cells lacking these proteins are impaired in the activation of the nuclear factor kappa B (NF-kappa-B) and activator protein 1 (AP-1) transcription factors.

[0005] Overproduction of IL-1 is the cause of many inflammatory diseases. For example, IL-1 is associated with conditions such as diabetes, cardiovascular disease, gout, certain types of arthritis (e.g., rheumatoid arthritis (RA)), and less widespread autoimmune diseases such as familial Mediterranean fever (FMF) and Behçet's disease (Ozen S, Bilginer Y. “A clinical guide to autoinflammatory diseases: familial Mediterranean fever and next-of-kin”, Nat. Rev. Rheumatol. 2014 Mar;10(3):135-47).

[0006] Rilonacept is an IL-1 antagonist comprising an IL-1 specific fusion protein comprising an IL-1 binding portion of the extracellular domain of human IL1-RAcP, an IL-1 binding portion of the extracellular domain of human IL-1RI, and a multimeric component, as described in U.S. Patent No. 6,472,179, U.S. Patent Publication No. 2003 / 0143697 published July 31, 2003, U.S. Patent No. 7,361,350, and U.S. Patent Publication No. 2005 / 0197293 published September 8, 2005 (all of which are expressly incorporated herein in their entirety). Rilonacept, under the brand name ARCALYST, has been approved by the United States Food and Drug Administration (FDA) for the treatment of cryopin-associated periodic syndromes (CAPS), including familial cold autoinflammatory syndrome (FCAS) and Muckle-Wells syndrome (MWS), in adults and children aged 12 years and older. Further clinical studies of rilonacept are currently underway, including as a gout medication. Summary of the Invention

[0007] Summary of the Invention This Summary is provided to introduce some concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential characteristics of the claimed subject matter, nor is it intended for use as an aid in determining the scope of the claimed subject matter.

[0008] In one aspect, the present invention provides a heterodimeric protein composition capable of binding to human IL-1β (GenBank: AAH08678.1). The protein composition comprises a first polypeptide comprising a first amino acid sequence comprising amino acids 18 to 333 of human IL1-R1 (GenBank: AAM88423.1) and a second amino acid sequence comprising a first variant of the Fc portion of human immunoglobulin gamma 1 Fc (GenBank: J00228.1). The protein composition also comprises a second polypeptide comprising another first amino acid sequence comprising amino acids 21 to 358 of human IL1-RacP (GenBank: BAA25421.1) and another second amino acid sequence comprising a second variant of the Fc portion of human immunoglobulin gamma 1 Fc. In the protein composition, the first and second variants are selected such that a heterodimeric structure is favored between the first and second variants over any homomeric structure. The protein composition comprises approximately 10 -11 The first polypeptide of the protein composition may comprise the amino acid sequence of SEQ ID NO:1 and the second polypeptide may comprise the amino acid sequence of SEQ ID NO:2.

[0009] In one aspect, the present invention provides a heterodimeric protein composition comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:8 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:9.

[0010] In one aspect, the present invention provides a therapeutic composition comprising a heterodimeric protein composition comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 8 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 9. The therapeutic composition may also comprise approximately 6% (weight / volume) sucrose, approximately 3% (weight / volume) polyethylene glycol having an average molecular weight of 3350 Da, approximately 50 mM sodium chloride, approximately 20 mM L-histidine at a pH of approximately 4.5 to approximately 7.0. The pH value may be approximately 6.5.

[0011] In one aspect, the present invention provides a therapeutic composition. The therapeutic composition comprises a heterodimeric protein composition capable of binding to human IL-1β. The protein composition comprises a first polypeptide comprising a first amino acid sequence comprising amino acids 18 to 333 of human IL1-R1 and a second amino acid sequence comprising a first variant of the Fc portion of human immunoglobulin gamma 1 Fc. The protein composition also comprises a second polypeptide comprising another first amino acid sequence comprising amino acids 21 to 358 of human IL1-RacP and another second amino acid sequence comprising a second variant of the Fc portion of human immunoglobulin gamma 1 Fc. In the protein composition, the first and second variants are selected such that a heterodimeric structure between the first and second variants is favored over any homomeric structure.

[0012] The protein composition is approximately 10 -11 The therapeutic composition may be capable of exhibiting human IL-1β / IL-1F2 binding activity with a Kd value of less than or equal to M. The therapeutic composition may exhibit a half-life of the heterodimeric protein composition in the systemic circulation of mice of at least about 97 hours following subcutaneous administration at a dose of 5 mg / kg, as assayed by human Fc ELISA.

[0013] The therapeutic composition may exhibit a half-life of the heterodimeric protein composition in the systemic circulation of cynomolgus monkeys of at least about 3 days after subcutaneous administration at a dose of 10 mg / kg as assayed by human Fc ELISA. The therapeutic composition may comprise a heterodimeric protein composed of a first polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 2. The therapeutic composition may also comprise about 6% (weight / volume) sucrose, about 3% (weight / volume) polyethylene glycol having an average molecular weight of 3350 Da, about 50 mM sodium chloride, about 20 mM L-histidine at pH 6.5.

[0014] In one aspect, the present invention provides a therapeutic composition comprising a heterodimeric protein composition comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 8 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 9. The therapeutic composition may also comprise approximately 6% (weight / volume) sucrose, approximately 3% (weight / volume) polyethylene glycol having an average molecular weight of 3350 Da, approximately 50 mM sodium chloride, approximately 20 mM L-histidine at a pH of approximately 4.5 to approximately 7.0. The pH value may be approximately 6.5.

[0015] In certain aspects, the present teachings provide a method for treating a variety of conditions, including arthritis, gout, rheumatoid arthritis, cryopin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergies, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST-segment elevation myocardial infarction (STEMI), acute respiratory distress syndrome / cytokine release storm (ARSD / CRS), Schnitzler syndrome, post-operative incision pain, chronic kidney disease (CKD), PFAPA (periodic fever, aphthous pulmonary arthritis), and / or other conditions. The present invention provides a substance or composition comprising a heterodimeric protein structure comprising a polypeptide of SEQ ID NO:8 and another polypeptide of SEQ ID NO:9 for use in the treatment of a disorder or disease associated with modulation of IL-1β, including, but not limited to, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), pyoderma gangrenosum, Kawasaki disease, acne vulgaris, atopic dermatitis, Behcet's disease, breast cancer, non-small cell lung cancer, or stroke.

[0016] In one aspect, the present teachings provide a method for treating or preventing a disease or condition associated with the modulation of human IL-1β activity, comprising administering to a patient in need of treatment or prevention of a disease associated with the modulation of human IL-1β activity a therapeutically effective amount of a pharmaceutical composition comprising a heterodimeric protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:8 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:9. Diseases associated with modulation of IL-1β include, but are not limited to, arthritis, gout, rheumatoid arthritis, cryopin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergies, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST-segment elevation myocardial infarction (STEMI), acute respiratory distress syndrome / cytokine release storm (ARSD / CRS), Schnitzler syndrome, post-operative incision pain, chronic kidney disease (CKD), PFAPA (Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis) syndrome, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), pyoderma gangrenosum, Kawasaki disease, acne vulgaris, atopic dermatitis, Behcet's disease, breast cancer, non-small cell lung cancer, or stroke.

[0017] To aid in understanding the invention, the following figures and explanations are provided. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows a schematic representation of a heterodimeric protein structure of the present teachings that includes the extracellular portion of IL1-R1 fused to an IgG-Fc domain (Fc-II) via a flexible linker and the extracellular portion of IL1-RAcP fused to another IgG-Fc domain (Fc-V) via another flexible linker. [Diagram 2] FIG. 2 shows a representative series of buffer-normalized sensograms at various concentrations of IL-1β / IL-1F2, with the lowest curve representing an IL-1β / IL-1F2 concentration of 0.919 nM, followed by subsequent curves representing 1.838, 3.676, 7.35, 14.7, and 29.4 nM, respectively. [Diagram 3]Figure 3 shows representative IL1 binding data, where the relative responses were calculated by subtracting the "buffer only" background and the error bars reflect the standard deviation values ​​calculated by the Bioacore T200 Evaluation Software package. [Figure 4] FIG. 4 shows a representative "response vs. concentration" curve with IL-1β / IL-1F2 concentration in Mol on the X-axis and response in RU on the Y-axis. [Diagram 5] FIG. 5 shows the concentration (in ng / ml) of IL1R-FcV-RAcP-FcII heterodimer in serum of an initial set of three Cynomolgus monkeys after a single subcutaneous dose of 10 mg / kg (vertical bars represent standard deviation values ​​at each time point). [Figure 6] FIG. 6 shows the concentration (in ng / ml) of the IL1R-FcV-RAcP-FcII heterodimer in serum of a follow-up set of three cynomolgus monkeys after a single subcutaneous dose of 10 mg / kg; the three curves shown represent measurements obtained from three individual animals designated F1290, F1269, and F1254. [Figure 7] FIG. 7 shows the IL1R-FcV-RAcP-FcII heterodimer titration curves of mouse IL-1B / IL-1F2-induced mouse IL6 secretion in MEFs, and the inset table shows the results of curve fitting using a 4-parameter algorithm and curve interpolation for the determination of IC50 values. [Figure 8] FIG. 8 shows the IL1R-FcV-RAcP-FcII heterodimer titration curves of human IL-1B / IL-1F2-induced human IL6 secretion in MRC5 cells, and the inset table shows the results of curve fitting using a 4-parameter algorithm and curve interpolation for the determination of IC50 values. [Figure 9]FIG. 9 shows the IL1R-FcV-RAcP-FcII heterodimer titration curves of human IL6 secretion induced by M. Rhesus IL-1B / IL-1F2 in MRC5 cells, and the inset table shows the results of curve fitting using a 4-parameter algorithm and curve interpolation for determination of IC50 values. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description of the Invention The teachings disclosed herein are based in part on the engineering of heterodimeric protein assemblies that can bind and attenuate the function of human IL-1β. The heterodimeric protein structures of the present teachings include the extracellular portions of IL1-R1 (GenBank: AAM88423.1) and IL-1RAcP (GenBank: BAA25421.1), or functional fragments thereof. Each of the IL1-R1 and IL-1RAcP portions is fused to a different variant of the Fc portion of human Ig gamma-1 (GenBank: J00228.1). The two different Fc variants in the heterodimeric protein structure are designed such that heterodimer formation is favored over any homomeric structure between the two Fc variants. To enable recombinant production of the heterodimeric protein structure of the present teachings, a DNA expression vector for overproducing the heterodimeric protein structure in a heterologous protein expression system was constructed, and mammalian cells were generated that stably express the heterodimeric protein structure to high expression levels. A protein purification procedure was devised to obtain a physiologically relevant, substantially pure preparation of the heterodimeric protein structure of the present teachings. Thus, the 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 pharmacokinetic profile upon subcutaneous administration in animals without causing weight loss or clinical adverse events. The design, preparation, and preliminary characterization of compositions of the present teachings are disclosed in part in International Patent Application Publication No. WO / 2014 / 035361, published March 6, 2014, and International Patent Application Serial No. PCT / US / 2013 / 026349, filed February 15, 2013, both of which are incorporated by reference in their entireties.

[0020] The terms used herein generally have their ordinary meaning in the art, within the context of the present invention and in the specific context in which each term is used. Certain terms are explained below or elsewhere herein to provide additional guidance to the skilled artisan in describing the compositions and methods of the present invention, and the methods of making and using them. The scope or meaning of any use of a term will be clear from the specific context in which the term is used. "About" and "approximately" generally refer to the degree of error that is acceptable to one of ordinary skill in the art for a measured quantity given the nature or precision of the measurement. 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, particularly in biological systems, the terms "about" and "approximately" can refer to values ​​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 otherwise specified, meaning that the terms "about" or "approximately" can be inferred when not explicitly stated.

[0021] The method of the present invention may include comparing sequences with each other, including wild-type sequences and one or more mutations (sequence variants). Such comparison typically includes alignment of polymer sequences, for example, using sequence alignment programs and / or algorithms well known in the art (e.g., BLAST, FASTA and MEGALIGN, to name a few). Those skilled in the art can easily understand that in such alignment, if the mutation includes insertion or deletion of a residue, the sequence alignment introduces a "gap" (typically represented by a dash, or "A") in the polymer sequence that does not include the inserted or deleted residue.

[0022] The methods of the present invention may include statistical calculations, such as determining IC50 or EC50 values, which the skilled artisan can readily appreciate can be performed using a variety of commercially available software, such as PRISM (GraphPad Software Inc, La Jolla, Calif., USA).

[0023] "Homologous," in all grammatical forms and spelling variations, refers to the relationship of two proteins that have a "common evolutionary origin," including superfamilies present in the same organism, as well as homologous proteins from organisms of different species. Such proteins (and the nucleic acids encoding them) have sequence homology, as reflected by sequence similarity in terms of percent identity, or by the presence of specific residues or motifs and conserved positions. However, in common usage and in this application, the term "homologous," when modified with an adverb such as "highly," refers to sequence similarity, which may or may not be related to a common evolutionary origin.

[0024] 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.

[0025] The terms "protein" and "polypeptide" are used interchangeably. The polypeptides described herein may be composed of more than one contiguous amino acid chain, thus forming dimeric or other oligomeric structures. Generally, polypeptides of the present teachings for mammalian use are expressed in mammalian cells that allow for appropriate post-translational modifications, such as CHO or HEK293 cell lines, although other mammalian expression cell lines are expected to be useful as well. Thus, it is expected that the polypeptides of the present teachings can be post-translationally modified without substantially affecting their biological function.

[0026] In certain aspects, functional variants of the heterodimeric protein structure of the present teachings include fusion proteins having at least a biologically active portion of human IL1-R1 or IL-1RAcP 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, immunoglobulin heavy chain constant region (e.g., Fc), maltose binding protein (MBP), or human serum albumin. The fusion domain may be selected to confer a desired property. For example, an IL1-R1 or IL-1RAcP polypeptide portion may be fused to a domain that stabilizes the IL1-R1 or IL-1RAcP polypeptide in vivo (a "stabilizer" domain), optionally via a suitable peptide linker. The term "stabilization" refers to something that increases the half-life of the polypeptide in the systemic circulation, whether due to reduced destruction, reduced clearance, or other pharmacokinetic effects. Fusion with the Fc portion of an immunoglobulin is known to confer desirable pharmacokinetic properties to certain proteins. Similarly, fusion with human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerization (e.g., dimerization, tetramerization) domains and functional domains that confer additional biological functions (e.g., promoting accumulation at the target site of action in vivo).

[0027] In certain aspects, the heterodimeric protein structure of the present teachings comprises an extracellular portion of IL1-R1 or a functional fragment thereof fused to an IgG-Fc domain, and an extracellular portion IL-1RAcP or a functional fragment thereof fused to another IgG-Fc domain. The IgG-Fc domain and the other IgG-Fc domain are selected to favor the heterodimeric protein structure over any homodimeric protein structure. The extracellular portion of IL1-R1 may be fused to the IgG-Fc domain via a flexible linker, while IL-1RAcP or a functional fragment thereof may be fused to another IgG-Fc domain via a flexible linker of the same amino acid sequence or via another flexible linker.

[0028] In an embodiment exemplarily shown in FIG. 1, the extracellular portion of IL1-R1 fused to an IgG-Fc domain (Fc-II) via a flexible linker may comprise the amino acid sequence of SEQ ID NO:1, while IL-1RAcP fused to another IgG-Fc domain (Fc-V) via a flexible linker may comprise the amino acid sequence of SEQ ID NO:2. hIL1-R1-hIgG1-Fc Polypeptide (SEQ ID NO:1) [ka] hIL-1RAcP-hIgG1-Fc Polypeptide (SEQ ID NO:2) [ka]

[0029] In certain aspects, the present teachings provide a recombinant DNA molecule having an open reading frame encoding a polypeptide comprising the first 333 amino acids of human IL1-R1 fused via a flexible linker to an IgG-Fc domain (Fc-II), and another recombinant DNA molecule having an open reading frame encoding another polypeptide comprising the first 358 amino acids of human IL-1RAcP fused via a flexible linker to another IgG-Fc domain (Fc-V).

[0030] In an exemplary embodiment, a polypeptide comprising the first 333 amino acids of human IL1-R1 fused to an IgG-Fc domain (Fc-II) via a flexible linker comprises the amino acid sequence of SEQ ID NO: 3. The corresponding DNA molecule may comprise the nucleotide sequence of SEQ ID NO: 4. Another polypeptide comprising the first 358 amino acids of human IL-1RAcP fused to another IgG-Fc domain (Fc-V) via a flexible linker may comprise the amino acid sequence of SEQ ID NO: 5. The corresponding DNA molecule may comprise the nucleotide sequence of SEQ ID NO: 6. hIL1-R1-hIgG1-Fc Polypeptide (SEQ ID NO:3) [ka] hIL1-R1-hIgG1-Fc DNA (SEQ ID NO:4) [ka] hIL-1RAcP-hIgG1-Fc Polypeptide (SEQ ID NO:5) [ka] hIL-1RAcP-hIgG1-Fc DNA (SEQ ID NO:6) [ka]

[0031] In a particular aspect, the present invention provides a recombinant mammalian expression plasmid for high expression of a polypeptide comprising the first 333 amino acids of human IL1-R1 fused to an IgG-Fc domain (Fc-II) via a flexible linker, and another recombinant DNA molecule having an open reading frame encoding another polypeptide comprising the first 358 amino acids of human IL-1RAcP fused to another IgG-Fc domain (Fc-V) via a flexible linker. The plasmid comprises two cytomegalovirus (CMV) promoters for driving the transcription of two genes encoding said polypeptide and said other polypeptide, each followed by a transcription termination sequence and a polyadenylation sequence. The plasmid also comprises an origin of replication and a gene conferring ampicillin resistance to aid in the propagation and selection of the plasmid in bacteria. In addition, the plasmid comprises a gene for glutamine synthetase, a selection marker widely used to establish stable CHOK1 and NSO cell lines.

[0032] In an exemplary embodiment, a mammalian expression plasmid of the present teachings comprises the nucleotide sequence of SEQ ID NO:7. hIL1-R1-hIgG1-Fc-II / IL-1RAcP-hIgG1-Fc-V expression plasmid (SEQ ID NO: 7) [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0033] In certain aspects, the present teachings provide a mammalian expression system for the production of a heterodimeric protein structure comprising a polypeptide comprising amino acid residues 18 to 333 of human IL1-R1 fused via a flexible linker to an IgG-Fc domain (Fc-II) and another polypeptide comprising amino acid residues 21 to 358 of human IL-1RAcP fused via a flexible linker to another IgG-Fc domain (Fc-V).

[0034] In an exemplary embodiment, a mammalian expression system of the present teachings comprises Chinese hamster ovary cells (CHO-K1) harboring a plasmid comprising the nucleotide sequence of SEQ ID NO:7.

[0035] In certain aspects, the mammalian expression system of the present teachings produces a heterodimeric protein structure comprising a polypeptide of SEQ ID NO:8 and another polypeptide of SEQ ID NO:9. hIL1-R1-hIgG1-Fc Polypeptide (SEQ ID NO:8) [ka] hIL-1RAcP-hIgG1-Fc Polypeptide (SEQ ID NO:9) [ka]

[0036] In certain aspects, the present teachings are directed to treating arthritis, gout, rheumatoid arthritis, cryopin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergies, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST-segment elevation myocardial infarction (STEMI), acute respiratory distress syndrome / cytokine release storm (ARSD / CRS), Schnitzler syndrome, post-operative incision pain, chronic kidney disease (CKD), PFAPA (periodic fever, aphthous pulmonary arthritis), and other conditions. The present invention provides a substance or composition comprising a heterodimeric protein structure comprising a polypeptide of SEQ ID NO:8 and another polypeptide of SEQ ID NO:9 for use in the treatment of a disorder or disease associated with modulation of IL-1β, including, but not limited to, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), pyoderma gangrenosum, Kawasaki disease, acne vulgaris, atopic dermatitis, Behcet's disease, breast cancer, non-small cell lung cancer, or stroke.

[0037] In certain aspects, the present teachings provide a method for treating or preventing a disease or condition associated with the modulation of human IL-1β activity, comprising administering to a patient in need of treatment or prevention of a disease associated with the modulation of human IL-1β activity a therapeutically effective amount of a pharmaceutical composition comprising a heterodimeric protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:8 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:9. Diseases associated with modulation of IL-1β include, but are not limited to, arthritis, gout, rheumatoid arthritis, cryopin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergies, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST-segment elevation myocardial infarction (STEMI), acute respiratory distress syndrome / cytokine release storm (ARSD / CRS), Schnitzler syndrome, post-operative incision pain, chronic kidney disease (CKD), PFAPA (Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis) syndrome, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), pyoderma gangrenosum, Kawasaki disease, acne vulgaris, atopic dermatitis, Behcet's disease, breast cancer, non-small cell lung cancer, or stroke. The present disclosure relates, for example, to the following: [Section 1] A heterodimeric protein composition, comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO:8; and A second polypeptide comprising the amino acid sequence of SEQ ID NO:9. A protein composition comprising: [Section 2] 1. A therapeutic composition, comprising a heterodimeric protein composition, the protein composition comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO:8; and A second polypeptide comprising the amino acid sequence of SEQ ID NO:9. A therapeutic composition comprising: [Section 3] The therapeutic composition of paragraph 2, further comprising approximately 6% (weight / volume) sucrose, approximately 3% (weight / volume) polyethylene glycol having an average molecular weight of 3350 Da, approximately 50 mM sodium chloride, approximately 20 mM L-histidine at a pH of approximately 4.5 to approximately 7.0. [Section 4] Item 3. The therapeutic composition of item 2, wherein the pH is approximately 6.5. [Section 5] A composition comprising a heterodimeric protein structure comprising a polypeptide of SEQ ID NO:8 and another polypeptide of SEQ ID NO:9 for use in the treatment of a disease associated with IL-1β modulation. [Section 6] Item 6. The composition according to item 5, wherein the disease is arthritis. [Section 7] Item 6. The composition according to item 5, wherein the disease is gout. [Section 8] Item 6. The composition according to item 5, wherein the disease is rheumatoid arthritis. [Section 9] Item 6. The composition of item 5, wherein the disease is cryopin-associated periodic syndrome (CAPS). [Section 10] Item 6. The composition according to item 5, wherein the disease is scleroderma. [Section 11] Item 6. The composition according to item 5, wherein the disease is diabetes. [Section 12] Item 6. The composition according to item 5, wherein the disease is atherosclerosis. [Section 13] Item 6. The composition according to item 5, wherein the disease is dry eye syndrome. [Section 14] Item 6. The composition of item 5, wherein the disease is an eye allergy. [Section 15] Item 6. The composition according to item 5, wherein the disease is uveitis. [Section 16] Item 6. The composition according to item 5, wherein the disease is recurrent pericarditis. [Section 17] The composition of item 5, wherein the disease is Familial Mediterranean Fever (FMF). [Section 18] Item 6. The composition according to item 5, wherein the disease is ST-segment elevation myocardial infarction (STEMI). [Section 19] The composition of item 5, wherein the disease is acute respiratory distress syndrome / cytokine release storm (ARSD / CRS). [Section 20] Item 6. The composition according to item 5, wherein the disease is Schnitzler syndrome. [Section 21] Item 6. The composition according to item 5, wherein the disease is incision pain after surgery. [Section 22] Item 6. The composition according to item 5, wherein the disease is chronic kidney disease (CKD). [Section 23] Item 6. The composition according to item 5, wherein the disease is PFAPA (periodic fever, aphthous stomatitis, pharyngitis, adenitis) syndrome. [Section 24] Item 6. The composition of item 5, wherein the disease is hemophagocytic lymphohistiocytosis (HLH). [Section 25] The composition of item 5, wherein the disease is macrophage activation syndrome (MAS). [Section 26] Item 6. The composition according to item 5, wherein the disease is pyoderma gangrenosum. [Section 27] Item 6. The composition according to item 5, wherein the disease is Kawasaki disease. [Section 28] Item 6. The composition according to item 5, wherein the disease is acne vulgaris. [Section 29] Item 6. The composition according to item 5, wherein the disease is atopic dermatitis. [Section 30] Item 6. The composition according to item 5, wherein the disease is Behcet's disease. [Section 31] A method for treating or preventing a disease or condition associated with modulation of human IL-1β activity, the method comprising administering to a patient in need of treatment or prevention of a disease associated with modulation of human IL-1β activity a therapeutically effective amount of a pharmaceutical composition comprising a heterodimeric protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 8 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 9. [Section 32] 32. The method of treatment and prevention according to claim 31, wherein the disease is arthritis. [Section 33] Item 32. The method for treatment and prevention according to item 31, wherein the disease is gout. [Section 34] 32. The method of treatment and prevention according to claim 31, wherein the disease is rheumatoid arthritis. [Section 35] Item 32. The method for treatment and prevention of item 31, wherein the disease is cryopin-associated periodic syndrome (CAPS). [Section 36] 32. The method of treatment and prevention according to claim 31, wherein the disease is scleroderma. [Section 37] 32. The method of treatment and prevention according to claim 31, wherein the disease is diabetes. [Section 38] Item 32. The method for treatment and prevention of item 31, wherein the disease is atherosclerosis. [Section 39] Item 32. The method for treatment and prevention according to Item 31, wherein the disease is dry eye syndrome. [Section 40] 32. The method of treatment and prevention according to claim 31, wherein the disease is an ocular allergy. [Section 41] Item 32. The method for treatment and prevention of item 31, wherein the disease is uveitis. [Section 42] 32. The method of treatment and prevention according to claim 31, wherein the disease is recurrent pericarditis. [Section 43] 32. The method of treatment and prevention according to claim 31, wherein the disease is Familial Mediterranean Fever (FMF). [Section 44] Item 32. The method for treatment and prevention according to Item 31, wherein the disease is ST-segment elevation myocardial infarction (STEMI). [Section 45] The method for treatment and prevention of item 31, wherein the disease is acute respiratory distress syndrome / cytokine release storm (ARSD / CRS). [Section 46] Item 32. The method for treatment and prevention of item 31, wherein the disease is Schnitzler's syndrome. [Section 47] Item 32. The method for treatment and prevention according to Item 31, wherein the disease is incision pain after surgery. [Section 48] Item 32. The method for treatment and prevention of item 31, wherein the disease is chronic kidney disease (CKD). [Section 49] Item 32. The method for treatment and prevention of item 31, wherein the disease is PFAPA (periodic fever, aphthous stomatitis, pharyngitis, adenitis) syndrome. [Section 50] Item 32. The method of treatment and prevention according to item 31, wherein the disease is hemophagocytic lymphohistiocytosis (HLH). [Section 51] 32. The method of treatment and prevention according to claim 31, wherein the disease is macrophage activation syndrome (MAS). [Section 52] Item 32. The method for treatment and prevention according to item 31, wherein the disease is pyoderma gangrenosum. [Section 53] Item 32. The method for treatment and prevention of item 31, wherein the disease is Kawasaki disease. [Section 54] Item 32. The method of treatment and prevention according to item 31, wherein the disease is acne vulgaris. [Section 55] Item 32. The method for treatment and prevention according to Item 31, wherein the disease is atopic dermatitis. [Section 56] Item 32. The method for treatment and prevention according to item 31, wherein the disease is Behcet's disease. [Section 57] 32. The method of treatment and prevention according to claim 31, wherein the disease is breast cancer. [Section 58] Item 32. The method of treatment and prevention according to item 31, wherein the disease is non-small cell lung cancer. [Section 59] Item 32. The method for treatment and prevention according to Item 31, wherein the disease is stroke. EXAMPLES

[0038] Working Example The following examples illustrate the aforementioned aspects and other aspects of the present teachings. These non-limiting examples are presented to provide those skilled in the art with exemplary embodiments of how to make and evaluate the compounds, compositions, articles, devices, and / or methods claimed herein. The examples are intended to be purely illustrative of the inventions disclosed herein and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure accuracy with respect to numerical values ​​(e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for.

[0039] Example 1 Construction of the Polypeptides of the Invention The hIL1-R1-hIgG1-Fc polypeptide of SEQ ID NO:1 and the hIL-1RAcP-hIgG1-Fc polypeptide of SEQ ID NO:2 were co-expressed in CHO-K1 using molecular biology, cell culture and protein biochemistry techniques known in the art, as described in PCT Publication WO / 2014 / 035361 and PCT Application Serial No. PCT / US / 2013 / 026349. Essentially, CHO-K1 cells expressing the polypeptides were harvested and lysed using well-established protocols. After washing the cell lysate, the supernatant containing the expressed polypeptides was first applied to a Protein A affinity column. The pH-adjusted Protein A column eluate was further purified by anion exchange chromatography (AIEX) using Q-Sepharose resin. The AIEX flow-through was analyzed by size-exclusion HPLC (SEC-HPLC), SDS-PAGE and other analytical techniques as appropriate.

[0040] For subsequent testing, therapeutic compositions containing hIL1-R1-hIgG1-Fc and hIL-1RAcP-hIgG1-Fc polypeptides were formulated to contain 40 mg / ml polypeptide, 6% (m / v) sucrose, 3% (m / v) polyethylene (PEG) 3350, 50 mM sodium chloride, and 20 mM L-histidine pH from about 4.5 to about 7.0, preferably about 6.5.

[0041] The sequences of the polypeptides contained in the final product were analyzed as outlined in the Examples below. Unexpectedly, the polypeptides in the final product predominantly contained the hIL1-R1-hIgG1-Fc polypeptide of SEQ ID NO:8 and the hIL-1RAcP-hIgG1-Fc polypeptide of SEQ ID NO:9.

[0042] Example 2 Peptide mapping and characterization of the polypeptides of the invention Three samples of the product prepared essentially as described in the above examples were analyzed as follows: First, the molecular weights of the two intact polypeptides contained in each sample were determined by liquid chromatography-mass spectrometry (LC-MS). Second, peptide mapping was performed by liquid chromatography tandem mass spectrometry (LC-MS / MS). Finally, the terminal peptides were sequenced.

[0043] For intact peptide mass measurement by LC / MS, protein samples were reduced and deglycosylated according to protocols well established in the art.

[0044] Peptide mapping was performed essentially as follows. The samples were digested with LysC, trypsin, and chymotrypsin, and each sample was analyzed by LC-MS / MS. 1) 40 μg of sample was denatured, reduced, and digested with trypsin (Cat# VS280, Promega Corporation, Madison, WI) according to established protocols. 2) 40 μg of sample was denatured, reduced, and digested with LysC according to established protocols (Cat# VA1170, Promega Corporation, Madison, WI). 3) 40 μg of each sample was denatured, reduced, and digested with chymotrypsin according to established protocols (Cat# VA106A, Promega Corporation, Madison, WI). 4) High pressure liquid chromatography using an Agilent 1900 UPLC system (Agilent Technologies, Santa Clara, CA) was performed as follows. Column: Analytical column - Waters ACQUITY UPLC BEH C18, 1,7 uM, 2.1×150 mm Column temperature: 45℃ Sample volume: 10μl Solvent A: H2O containing 0.1% formic acid Solvent B: Acetonitrile (ACN) containing 0.1% formic acid Flow: 300 μL / min (min) Operating conditions: 98% A, 2% B; 0 (initial state) 70% A, 30% B; 0 - 35 min (linear gradient) 5% A, 95% B; 35 - 46 min (linear gradient) 95% A, 2% B; 46 - 50 min (linear gradient) 98% A, 2% B; 50 - 60 minutes (equilibration) Data system: PC controlled data acquisition system 5) Tandem mass spectrometry - Spectra were acquired using a QTOF 6550 mass spectrometer (Agilent Technologies, Santa Clara, CA). The mass spectrometer was operated in positive ion mode. Mass spectra were acquired over m / z 350-2000 with a resolution of 20,000 (m / z 1521), and data-dependent acquisition selected the top 10 most abundant precursor ions for tandem mass spectrometry by CID fragmentation using an isolation width of 4.0 Da and a collision energy of (slope)*(m / z) / 100 + offset. Dynamic exclusion was used to minimize redundancy in MS / MS acquisition and maximize peptide identification. 6) Data Analysis - Raw data were extracted and searched by using Spectrum Mill v5.01 and Hunter (Agilent Technologies). Collected MS and MS / MS spectra were analyzed against protein and decoy sequence databases. Enzyme parameters were limited with a maximum miscleavage of 2 for trypsin, 2 for LysC, and 5 for chymotrypsin. An additional non-enzymatic search was performed for N-terminal peptides. All other search parameters were set to Spectrum Mill default settings (carbamidomethylation of cysteines, + / - 20 ppm for precursor ions, + / - 50 ppm for fragment ions, and a minimum matched percent scored peak intensity (SPI%) of 50%). A concatenated forward-reverse database was constructed and the in situ false discovery rate (FDR) was calculated. Cutoff scores were dynamically assigned to each dataset to maintain the false discovery rate below 0.1% at the peptide level. Manual inspection was also applied to the uniquely identified peptides for each sample analyzed.

[0045] In the intact mass spectrum, two well-resolved major peaks (each with a purity of 98% or higher) were detected, the first corresponding to MW = 61,505.9 ± 0.1 Da and the second to MW = 64,753.3 ± 0.1 Da. The first peak corresponds to SEQ ID NO:8 (theoretical MW 61,495 Da) and the second peak corresponds to SEQ ID NO:9 (theoretical MW 64,743 Da). The difference in MW (~11 Da) is likely due to deamination of Asn residues after deglycosylation.

[0046] Peptide mapping and C-terminal sequence analysis further confirmed these sequences with high confidence.

[0047] Example 3: Assessing the affinity binding of polypeptides of the present teachings to RANKL using a surface plasmon resonance (SPR) assay The binding affinity of the prepared polypeptide of IL1R-FcV-RAcP-FcII heterodimer to IL-1β / IL-1F2 (NCBI Accession # NP_000567) was measured using a specially designed surface plasmon resonance (SPR) assay. The assay was performed using a capture method in which anti-human IgG was cross-linked to the surface of a sensor chip to capture the IL1R-FcV-RAcP-FcII heterodimer via its IgG (Fc) fragment. A series of different concentrations of IL-1β / IL-1F2 were used for the calculation of the dissociation constant (Kd).

[0048] Reagents and equipment: device: BiaCore T200, Instrument No. 12108, GE Healthcare, with Biacore T200 Control and Evaluation Software Package

[0049] reagent: IL1R-FcV-RAcP-FcII heterodimer stock solution, polypeptide 20 mg / ml, sucrose 6% (m / v), PEG3350 3% (m / v), sodium chloride 50 mM, and L-histidine 20 mM pH 6.5. IL-1β / IL-1F2, human recombinant, from E. Coli, Ala117-Ser269, accession no. NP_000567, R&D systems, catalog #201-LB, lot #AD1412111. Sensor Chip CM5, Series S, GE Healthcare BR-1005-30, Lot# 10189577 Human Antibody Capture Kit, GE Healthcare, Cat# BR-1008-39, Lot# 10202616; HBS-EP+ 10x Running Buffer, GE Healthcare, Cat# BR-1006-69;

[0050] procedure : Anti-human IgG conjugation: The anti-human IgG (Fc) conjugation procedure was performed according to the manufacturer's protocol using the following conditions: 1. CM5 sensor chip was placed in the instrument and primed with Biacore running buffer, 1x HBS-EP for 6 min at 10 μl / min, repeated twice. All steps were performed at 25°C. Channels 1 and 2 were used for the experiment, channels 3 and 4 were kept as backup. 2. Anti-human IgG from the kit, 0.5 mg / ml in 0.15 M NaCl, was diluted 20-fold in fixation buffer (10 mM Na-acetate pH 5.0) to a final concentration of 25 μg / ml. 3. Reagents for the immobilization procedure were prepared as follows: EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide)-0.4 M in Milli-Q water; NHS (N-hydroxysuccinimide)-0.1 M in Milli-Q water; 1M ethanolamine-HCL pH 8.5 in Milli-Q water; 4. Standard protocols for surface activation and immobilization were used. 5. Activation: EDC and NHS were mixed in a 1:1 ratio and injected over the chip at 10 μl / min for 7 min. 6. Immobilization: Anti-human IgG was injected over the chip at 10 μl / min for 5 min. 7. Deactivation: Unreacted active groups were blocked by injection of 1M ethanolamine-HCL at 10 μl / min for 7 min. 8. After antibody binding, the chip was washed twice with 1x HBS-EP at 10 μl / min for 6 min, followed by two "dry" working cycles without the addition of protein components. The working cycle consisted of a 1 min ligand (IL1R-FcV-RAcP-FcII heterodimer) loading step, a 3 min wash step, a 1 min sample (IL-1) loading step, a 16.7 min wash step, a 1 min chip regeneration step, and 3 M MgCl2. All steps were performed at 10 μl / min, except for the sample loading step, which was performed at 30 μl / min.

[0051] Experimental Data : Affinity evaluation of the IL1R-FcV-RAcP-FcII heterodimer / IL-1β / IL-1F2 interaction The purpose of this experiment was to determine the binding constants for the IL1R-FcV-RAcP-FcII heterodimer and IL-1β / IL-1F2. Anti-human IgG was covalently immobilized onto a CM5 sensor chip and the IL1R-FcV-RAcP-FcII heterodimer was loaded, followed by various concentrations of human IL-1β / IL-1F2. A series of sensorgrams was generated and used to calculate Kd values.

[0052] Experimental setup : 1. In a series of preliminary experiments, several different concentrations (1, 10 and 100 μg / ml) of the ILlR-FcV-RAcP-FcII heterodimer were prepared and tested for binding to immobilized anti-human IgG. At 1 μg / ml, the ILlR-FcV-RAcP-FcII heterodimer was found to produce a sufficient signal in the range of ∼100 RU, and this concentration was used throughout the assay. 2. The parameters of the association / dissociation cycle were optimized in a series of pilot runs and are summarized in Table 1. 3. Human IL-1β / IL-1F2 was used at the concentrations shown in Table 2, with a concentration of 3.676 nM run independently in duplicate as an internal control for instrument reproducibility. 4. A series of sensorgrams corresponding to different concentrations of IL-1β / IL-1F2 were generated. The data were normalized by subtracting the "buffer only" sensorgram. The buffer-normalized sensorgrams are shown in Figure 2 and the corresponding data are shown in Table 2. Table 1 BiaCore cycling parameters [Table 1]

[0053] Analysis of experimental data The experimental conditions were optimized to allow accurate use of the curve-fitting algorithm. As is evident from the sensorgrams (Figure 2), all concentrations of IL-1β / IL-1F2 tested showed dose-dependent binding curves. However, due to the very high affinity of the IL1R-FcV-RAcP-FcII heterodimer / IL-1β / IL-1F2 interaction, there was no detectable dissociation within 1000 seconds. Therefore, calculation of Kd values ​​using a kinetic model could not be performed accurately.

[0054] As an alternative method for calculating Kd, equilibrium data analysis using a 1:1 Langmuir binding model was used. According to this method, Kd is calculated from a series of plots of steady-state analyte binding levels (Req) versus concentration. The resulting data are summarized in Table 2.

[0055] The experimental data is summarized in Table 3 and shown in Figure 3. A four-parameter curve-fitting algorithm was used and the resulting curve response vs. concentration is shown in Figure 4. The equation for this curve was used for Kd calculation and was implemented by the Biacore T200 Evaluation Software. Table 2. Kd values ​​for IL1R-FcV-RAcP-FcII heterodimer and human IL-1β / IL-1F2 calculated from the equilibrium model. [Table 2] Table 3. IL-1β / IL-1F2 concentration and binding (relative response). Standard deviation values ​​% were calculated by Biacore T200 evaluation software and then converted to standard deviation by multiplying by Rmax*StDev %. StDev values ​​are plotted as error bars on Figure 3. [Table 3]

[0056] Example 4 : Pharmacokinetics (PK) of IL1R-FcV-RAcP-FcII heterodimer after subcutaneous injection in mice The IL1R-FcV-RAcP-FcII heterodimer (SEQ ID NO:1 and SEQ ID NO:2) polypeptides were co-expressed and purified substantially as described in the above examples. For administration to animals, the polypeptides were formulated in the following buffer: 1% w / v sucrose, 100 mM sodium chloride, 20 mM L-arginine hydrochloride, 25 mM sodium bicarbonate, pH 6.3. The administration stock concentration used was 0.5 mg / mL of polypeptide.

[0057] On study day 0, 14 male DBA / 1 mice were randomly divided into 7 groups of 2 mice each according to body weight. On study day 0, a single dose of IL1R-FcV-RAcP-FcII heterodimer (5 mg / kg in 10 ml / kg) was administered subcutaneously (dorsally) to 6 mice in a group. Mice in the remaining groups were kept untreated and bled via cardiac puncture for plasma preparation on study day 0. Plasma was prepared from blood samples taken from mice in the treated groups via the orbital sinus or terminal cardiac puncture at predetermined times during the study. Body weight was recorded for all animals on the day of treatment (day 0) and then three times weekly, including the termination day of each group. No change in body weight was measured in groups culled for sample collection at 0 and 36 hours after dosing. The average weight loss from day 0 to termination for groups culled between 96 hours and 21 days after dosing was minimal. No mice lost weight beyond the ethical limit. After the in-life phase of the study, plasma samples were analyzed for Hu-Fc protein by enzyme-linked immunosorbent assay (ELISA). Quantification of Hu-Fc in mouse plasma samples by ELISA was used as a readout for circulating levels of IL1R-FcV-RAcP-FcII heterodimers. This assay was performed on samples from all mice in the study.

[0058] The polypeptide (detected as human Fc protein) was detected in the plasma of the animals at all time points after dosing. The pharmacokinetics of the polypeptide, as detected by Hu-Fc ELISA, was then determined using the One Phase Decay Model formula using Prism 5.0c (GraphPad Software Inc, La Jolla, CA, USA). The peak circulating level (Cmax) of Hu-Fc was determined to be 1.284 μg / mL, and the time to peak circulating level (Tmax) was determined to be 24 hours after dosing. The half-life (T1 / 2) was determined to be 97 hours 31 minutes, and the rate constant (K) was determined to be 0.0071 hr-1. Hu-Fc was below the level of detection in the plasma of untreated animals. The results of this study are summarized in Table 4. Table 4. Mean human Fc protein concentrations ± SEM (μg / mL) at each time point after administration. [Table 4] *0.040 is the limit of detection for this assay. Human Fc protein concentrations were determined by Prism software based on the average absorbance of triplicate samples. ^ Blood flow through the orbital sinus # Blood via terminal cardiac puncture

[0059] Example 5 : Pharmacokinetics (PK) of IL1R-FcV-RAcP-FcII heterodimer after subcutaneous administration in primates For this study, three naive male cynomolgus monkeys were initially used. The animals were approximately 2-4 years old and weighed approximately 2 kg. The animals received a single dose of overexpressed and purified IL1R-FcV-RAcP-FcII heterodimer (SEQ ID NO: 1 and SEQ ID NO: 2), formulated essentially as described in Example 1 above, via subcutaneous administration at a dose level of 10 mg / kg on day 1 of the study. Bioanalysis results from the first set of three animals are shown in FIG. 5. To further meet the study objectives, an additional three male cynomolgus monkeys received a single dose of IL1R-FcV-RAcP-FcII heterodimer via subcutaneous administration at a dose level of 10 mg / kg on day 1, and blood samples were collected at predefined time points up to day 21. Bioanalysis results from an additional set of three follow-up animals are shown in FIG. 5. All animals were observed once daily for any response to treatment during the study. Body weights were measured and recorded prior to dosing. Blood samples for pharmacokinetic analysis were collected at the indicated time points. Collected serum samples were stored at -80°C for bioanalysis. Measurement of plasma concentrations of polypeptides was performed using ELISA method.

[0060] There were no clinical signs noted during the study period. The body weight profile was satisfactory. Results of the initial PK data analysis showed that the Cmax and Tmax after a single subcutaneous dose of 10 mg / kg were approximately 24-48 hours. PK results from an additional three monkeys showed that the IL1R-FcV-RAcP-FcII heterodimer was quantifiable in plasma for at least 10 days in two of the three monkeys and up to 14 days in one monkey. Pharmacokinetic parameters for a follow-up set of three monkeys were determined using a noncompartmental model in the WinNonLin 6.3 software package and are summarized in Table 5. Table 5. Summary of PK parameters from single subcutaneous administration studies in cynomolgus monkeys (values ​​in parentheses are mean CV%) [Table 5] 1 harmonic mean 2 Median [min-max] *Estimated from AUC extra>20%

[0061] All animals were extensively exposed to the IL1R-FcV-RAcP-FcII heterodimer. The observed inter-individual variability was relatively high with a CV% of about 60%. The latter was explained by the lowest drug exposure found in animal F1290 (Figure 6), which had at least 5-fold less exposure to the IL1R-FcV-RAcP-FcII heterodimer than the remaining two animals. The maximum concentration (Cmax) was reached on day 1 to day 2. The estimated T1 / 2 was assessed to be about 4 days.

[0062] Example 6: Interspecies specific activity of IL1R-FcV-RAcP-FcII heterodimer IL1R-FcV-RAcP-FcII is a heterodimer composed of soluble portions of human IL-1R and IL-1RAcP linked to a unique IgG1 Fc portion, respectively. Sequence alignment of the 333 amino acid portion of human IL-1R with related portions from several species shows only slight sequence identity (~64%) with the IL-1R portion from rodents (mouse, rat). However, there is a much higher sequence identity between the human IL-1R and the IL-1R of other primates (e.g., 91% with marmoset monkey). Protein binary sequence alignment of the 358 amino acid portion of human IL-1RAcP forming part of the IL1R-FcV-RAcP-FcII heterodimer molecule with related portions from several species is further shown below. The sequence identity across species of this portion of the IL1R-FcV-RAcP-FcII heterodimer is rather high. Higher sequence identity is observed between a 358 amino acid portion of human IL-1RAcP and its orthologue from Macaca mulatta (92%) versus Mus musculus (85%).

[0063] The following studies were performed to comparatively evaluate the functional (inhibitory) properties of the novel drug candidates IL1R-FcV-RAcP-FcII heterodimers (SEQ ID NO: 1 and SEQ ID NO: 2). Assays were performed using human, Macaca Rhesus, and mouse IL-1β IL-1F2 orthologues. Human vs. mouse IL-1β / IL-1F2 were compared in mouse embryonic fibroblasts. Human vs. rhesus IL-1β / IL-1F2 were compared in MRC5 human lung fibroblasts. As functional comparators, a previously characterized mouse monoclonal antibody against human IL-1β IL-1F2 and a goat polyclonal antibody against mouse IL-1β IL-1F2 were used. Quantification of IL-1β IL-1F2-induced IL-6 production by MRC5 cells or MEFs was used to determine the inhibitory properties (IC50 values) of all three orthologues.

[0064] Materials and Reagents cell MRC5 cells, human lung fibroblasts, ATCC Cat # CCL-171, Lot # 59474707. Mouse embryonic fibroblasts (MEFs) were used in the experiments.

[0065] Culture medium DMEM, Dulbecco's Modified Eagle's Medium, High Glucose (4.5g / L), supplemented with L-glutamine and 1x penicillin / streptomycin and 10% Benchmark fetal bovine serum, Invitrogen, Cat # 11995-065, Lot # 1237317, Gemini Bioproducts, Cat # 100-106, Lot # A78D00E

[0066] reagent Preparation of IL1R-FcV-RAcP-FcII heterodimer, 1.5 mg / ml. IL-1β IL-1F2, human recombinant, derived from E. coli, Ala117-Ser269, Accession # NP_000567, R&D systems, Cat # 201-LB, Lot # AD1412111 IL-1β IL-1F2, Rhesus monkey recombinant, from E. coli, Ala117-Ser269, Accession # P48090, R&D systems, Cat # 1318-RL, Lot # GUG0110111 IL-1β / IL-1F2, Mouse recombinant, E. coli derived, Vla118-Ser269, Accession # NP_032387, R&D systems, Cat # 401-ML-005, Lot # BN0713032 Mouse monoclonal antibody against human IL-1β / IL-1F2, clone #8516, R&D systems, Cat # MAB201, Lot # AWE1011081 Goat polyclonal antibody against mouse IL-1β / IL-1F2, clone #8516, R&D systems, Cat # AF-401-NA, Lot # NP2812121 IL-6 Quantakine Immunoassay, R&D systems, Cat # D6050, Lot # 308916 Mouse IL-6 Quantakine Immunoassay, R&D systems, Cat # M6000B, Lot # 309487

[0067] procedure Cell maintenance The supernatant is centrifuged at 300 xg for 10 minutes and the clarified supernatant is aliquoted and used for ELISA either as is (MEF) or diluted 1 / 5 (MRC5) depending on the needs of the pilot experiment.

[0068] ELISA The assay uses a quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-6 is precoated onto a microplate. Standards and samples are dispensed into the wells and any IL-6 present is bound by the immobilized antibody. After washing away unbound material, an enzyme-linked polyclonal antibody specific for IL-6 is added to the wells. After washing to remove unbound antibody-enzyme reagent, a substrate solution is added to the wells and color is developed in proportion to the amount of IL-6 bound in the first step. Color development is stopped and the color intensity measured.

[0069] Experimental Data The aim of the three series of experiments was to identify a suitable cell line for measuring IL6 secretion upon treatment with human and mouse orthologues of IL-1β / IL-1F2. Several preliminary pilot experiments were performed to identify mouse cells that respond to mouse-IL-1β / IL-1F2 treatment with robust secretion of IL6. Based on these preliminary experiments, MEFs were selected as a model cell line for IL1R-FcV-RAcP-FcII heterodimer titration experiments. The IL1R-FcV-RAcP-FcII heterodimer titration curve of mouse IL6 secretion induced by mouse IL-1B / IL-1F2 in MEFs is shown in Figure 7. IL6 production data was calculated from the calibration curve shown in Figure 9. The inset table shows the results of curve fitting using a four-parameter algorithm and curve interpolation for the determination of IC50 values. The calculated IC50 value of the IL1R-FcV-RAcP-FcII heterodimer against mouse IL-1B / IL-1F2 is >210 ng / ml.

[0070] The experimental data show that the IL1R-FcV-RAcP-FcII heterodimer is an efficient inhibitor of the human IL-1β / IL-1F2 signaling pathway, but not of the mouse IL-1B / IL-1F2 signaling pathway. The IL1R-FcV-RAcP-FcII heterodimer IC50 values ​​were 0.19 ng / ml for human IL-1B / IL-1F2 and >200 ng / ml for mouse IL-1B / IL-1F2 (0.95 pM and >1000 pM, respectively, assuming the molecular weight of the IL1R-FcV-RAcP-FcII heterodimer is 200 kDa). The IL1R-FcV-RAcP-FcII heterodimer titration curves of human IL-1B / IL-1F2-induced human IL6 secretion in MRC5 cells are shown in FIG. 8. The calculated IC50 value of the IL1R-FcV-RAcP-FcII heterodimer against human IL-1B / IL-1F2 (X-column in the curve interpolation table) is 0.22 ng / mL. The IL1R-FcV-RAcP-FcII heterodimer titration curve of human IL6 secretion induced by rhesus IL-1B / IL-1F2 in MRC5 cells is shown in FIG. 9. The calculated IC50 value of the IL1R-FcV-RAcP-FcII heterodimer against human IL-1B / IL-1F2 is 0.38 ng / ml. The IL-6 recovery from the IL1R-FcV-RAcP-FcII heterodimer preparation at a final concentration of 200 ng / ml was 95%. The IL1R-FcV-RAcP-FcII heterodimer is an efficient inhibitor of both human and rhesus IL-1B / IL-1F2 signaling pathways. The IC50 values ​​of the IL1R-FcV-RAcP-FcII heterodimer are 0.19 ng / ml for human IL-1B / IL-1F2 and 0.38 ng / ml for rhesus IL-1B / IL-1F2 (1.1 pM and 1.9 pM, respectively). The IL-6 recovery from the IL1R-FcV-RAcP-FcII heterodimer preparation at a final concentration of 200 ng / ml was 92%.

[0071] Therefore, stimulation of IL-6 production upon treatment of mouse or human cells with IL-1B / IL-1F2 was used for functional testing of the inhibitory properties of the novel drug candidate IL1R-FcV-RAcP-FcII heterodimer against the human, mouse, and rhesus orthologues of IL-1B / IL-1F2. Appropriate cell lines were identified and experimental conditions, including cell density, treatment time, and linear range of IL6 detection, were optimized for all three orthologues. The obtained data are summarized in Table 6. Table 6. IC50 values ​​of IL1R-FcV-RAcP-FcII heterodimers against human, mouse, and rhesus orthologues of IL-1B / IL-1F2 [Table 6]

[0072] All publications and patents mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0073] While certain aspects 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 that follow. 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

1. 1. A therapeutic composition for use in treating a disease associated with IL-1β modulation, the composition comprising a heterodimeric protein, the protein comprising: A first polypeptide consisting of the amino acid sequence of SEQ ID NO:8, and A second polypeptide consisting of the amino acid sequence of SEQ ID NO:

9. Including, the disease associated with IL-1β modulation is selected from the group consisting of arthritis, gout, rheumatoid arthritis, cryopin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergies, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST-segment elevation myocardial infarction (STEMI), acute respiratory distress syndrome / cytokine release storm (ARSD / CRS), Schnitzler syndrome, post-operative incision pain, chronic kidney disease (CKD), PFAPA (Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis) syndrome, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), pyoderma gangrenosum, Kawasaki disease, acne vulgaris, atopic dermatitis, Behcet's disease, breast cancer, non-small cell lung cancer, and stroke; Therapeutic composition.

2. 2. The therapeutic composition of claim 1, additionally comprising 6% (wt / vol) sucrose, 3% (wt / vol) polyethylene glycol having an average molecular weight of 3350 Da, 50 mM sodium chloride, and 20 mM L-histidine at a pH of 4.5 to 7.

0.

3. 3. The therapeutic composition of claim 2, wherein the pH is 6.5.

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