Antibodies directed against interleukin 36 receptor (il-36r)

Abstract

To provide IL-36R-binding agents, such as IL-36R antagonists (e.g., antibodies), that bind to IL-36R with high affinity and efficiently neutralize IL-36R activity.SOLUTION: The invention relates to an isolated immunoglobulin heavy chain polypeptide and an isolated immunoglobulin light chain polypeptide that bind to a protein encoded by the interleukin 36 receptor (IL-36R). The invention provides an IL-36R-binding agent that comprises the aforementioned immunoglobulin heavy chain polypeptide and immunoglobulin light chain polypeptide. The invention also provides related vectors, compositions, and methods of using the IL-36R-binding agent to treat a disorder or disease that is responsive to IL-36R inhibition, such as cancer, an infectious disease, or an autoimmune disease.SELECTED DRAWING: Figure 1A

Description

[Background technology]

[0001] Incorporation by reference of electronically submitted property The computer-readable nucleotide / amino acid sequence listing, filed herewith and identified as follows, is incorporated herein by reference in its entirety: One 70,258 byte ASCII (text) file entitled "723558_ST25.txt", created on April 13, 2016.

[0002] Background of the Invention The interleukin-36 (IL-36) cytokines IL-36α, IL-36β, and IL-36γ (formerly IL-1F6, IL-1F8, and IL-1F9) are members of the interleukin-1 (IL-1) family that bind to the IL-36 receptor (IL-36R) (formerly IL-1Rrp2 or IL-1RL2) and use the IL-1 receptor accessory protein (IL-1RAcP) as a coreceptor to stimulate intracellular signals similar to those induced by IL-1 (Towne et al., J. Biol. Chem., 279(14): 13677-13688 (2004)). IL-1F5 is a member of the IL-1 family that has been shown to act as an antagonist of IL-36R, now called IL-36Ra (Dinarello et al., Nat. Immunol., 11(11): 973 (2010)).

[0003] IL-36α, IL-36β, and IL-36γ are expressed in several tissues, including internal epithelial tissues that have been exposed to pathogens. IL-36Ra and IL-36α expression was significantly upregulated in IL-1β / TNF-α-stimulated human keratinocytes, and IL-36Rα and IL-36γ mRNA were significantly upregulated in these tissues. IL-36R is overexpressed in psoriatic skin lesions. Elevated IL-36α mRNA and protein expression has also been observed in chronic kidney disease (Ichii et al., Lab Invest., 90(3): 459-475 (2010)). Both murine bone marrow-derived dendritic cells (BMDCs) and CD4+ T lymphocytes constitutively express IL-36R and promote the growth of psoriasis by producing proinflammatory cytokines (e.g., IL-12, IL-1β, IL-6, TNF-α, and IL-23) that induce a more potent stimulatory effect than other IL-1 cytokines. It reacts directly with IL-36α, IL-36β, and IL-36γ (Vigne et al., Blood, 118(22): 5813-5823 (2011)).

[0004] Transgenic mice overexpressing IL-36α in keratinocytes exhibited the following abnormalities at birth: Mice exhibited a transient inflammatory skin disorder, resembling human psoriasis, characterized by 12-O-tetrahydropyridine (TTA). IL-36R-deficient mice are highly susceptible to tradecanoylphorbol-13-acetate-induced skin lesions (Blumberg et al., J. Exp. Med., 204(11): 2603-2614 (2007); and Blumberg et al., J. Immunol., 185(7):4354-4362 (2010)). Furthermore, IL-36R-deficient mice are protected from imiquimod-induced psoriasis-like dermatitis (Tortola et al., J. Clin. Invest., 122(11): 3965-3976 (2012)). These results strongly suggest a role for IL-36 in certain cutaneous inflammatory disorders.

[0005] IL-36 cytokines have also been associated with certain severe forms of psoriasis, including pustular psoriasis, generalized pustular psoriasis (GPP), and palmoplantar pustulosis (PPP) (see, e.g., Town, JE and Sims, JE, Curr. Opin. Pharmacol., 12(4): 486-90 (2012); and Naik, HB and Cowen, EW, Dermatol Clin., 31(3): 405-425 (2013)). Pustular psoriasis is a rare form of psoriasis characterized by white pustules surrounded by red skin. Generalized pustular psoriasis is a severe, generalized form of pustular psoriasis with a high risk of mortality, while palmoplantar pustulosis is a chronic form of pustular psoriasis that affects the palms and soles of the feet. Current treatments for pustular psoriasis, GPP, and PPP include oral retinoids and topical steroids, but these treatments are poorly effective and have serious side effects. Summary of the Invention [Problem to be solved by the invention]

[0006] There is a need for IL-36R antagonists (e.g., antibodies) that bind to IL-36R with high affinity and efficiently neutralize IL-36R activity. The present invention provides such IL-36R-binding agents. [Means for solving the problem]

[0007] Brief summary of the invention The present invention provides an isolated immunoglobulin light chain polypeptide, comprising: Gln Val Gln Xaa1 Xaa2 Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser Tyr Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Xaa3 Leu Glu Trp Met Gly Trp Ile Tyr Pro Gly Asp Xaa4 Ser Thr Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Xaa5 Asp Xaa6 Ser Ala Xaa7 Thr Ala Tyr Met Glu Leu Xaa8 Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Xaa9 Cys Thr Arg Ser Phe Tyr Thr Met Amino acid sequence of Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 56) (a) Xaa1 is leucine (Leu) or phenylalanine (Phe), (b) Xaa2 is valine (Val), methionine (Met), or leucine (Leu), (c) Xaa3 is arginine (Arg) or glycine (Gly), (d) Xaa4 is glycine (Gly), serine (Ser), or alanine (Ala), (e) Xaa5 is arginine (Arg) or alanine (Ala), (f) Xaa6 is threonine (Thr) or lysine (Lys), and (g) Xaa7 is serine (Ser) or asparagine (Asn). (h) Xaa8 is serine (Ser) or alanine (Ala), and (i) Xaa9 is tyrosine (Tyr). or phenylalanine (Phe). do.

[0008] The present invention provides an isolated immunoglobulin heavy chain polypeptide, comprising: Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Xaa1 Met Xaa2 Trp Val Arg Gln Ala Pro Xaa3 Gln Gly Leu Glu Trp Met Gly Met Phe Xaa4 Pro Xaa5 Xaa6 Xaa7 Val Thr Arg Leu Asn Gln Lys Phe Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Thr Ser Met Ile Ile Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 15) wherein (a) Xaa1 is tryptophan (Trp) or tyrosine (Tyr); (b) Xaa2 is histidine (His), asparagine (Asn), or tyrosine (Tyr); (c) Xaa3 is glycine (Gly) or arginine (Arg); (d) Xaa4 is aspartic acid (Asp); (e) Xaa5 is serine (Ser), threonine (Thr), or tyrosine (Tyr); and (f) Xaa6 is asparagine (Asn) or glycine (Gly). and (g) Xaa7 is serine (Ser), alanine (Ala), or aspartic acid (Asp).

[0009] The present invention provides an isolated immunoglobulin light chain polypeptide, comprising: Xaa1 Xaa2 Gln Xaa3 Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Xaa4 Xaa5 Tyr Ser Ile Thr Xaa6 Asp Phe Ala Trp Asn Trp Ile Arg Gln Xaa7 Pro Gly Xaa8 Xaa9 Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Asp Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Xaa10 Xaa11 Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Xaa12 Tyr Xaa13 Cys Ala Ile Arg Gly Pro Tyr Ser Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Xaa14 (SEQ ID NO: 57), wherein Xaa1 is glutamine (Gln) or aspartic acid (Asp). Xaa2 is valine (Val) or leucine (Leu); Xaa3 is leucine (Leu) or fluoro Xaa4 is threonine (Thr) or serine (Ser); Xaa5 is Xaa6 is serine (Ser) or alanine (Ala); Xaa7 is proline (Pro) or phenylalanine (Phe); Xaa8 is lysine (Lys) or asparagine (Asn); Xaa9 is glycine (Gly) or lysine (Lys); Xaa10 is , serine (Ser) or threonine (Thr); Xaa11 is valine (Val) or arginine (Arg) Xaa12 is threonine (Thr) or valine (Val); Xaa13 is tyrosine (Tyr) or is phenylalanine (Phe); and Xaa14 is alanine (Ala) or absent The present invention provides an isolated immunoglobulin light chain polypeptide.

[0010] The present invention provides an isolated immunoglobulin heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35.

[0011] The present invention provides an isolated immunoglobulin light chain polypeptide, comprising: Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Xaa1 Asn Thr Tyr Leu Tyr Trp Xaa2 Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Xaa3 Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Phe Thr Phe Gly An isolated immunoglobulin light chain polypeptide is provided, comprising the amino acid sequence of Gln Gly Thr Lys Leu Glu Ile Lys (SEQ ID NO: 36), wherein (a) Xaa1 is glycine (Gly) or alanine (Ala), (b) Xaa2 is phenylalanine (Phe) or tyrosine (Tyr), and (c) Xaa3 is tyrosine (Tyr) or serine (Ser).

[0012] The present invention provides isolated immunoglobulin light chain polypeptides, comprising: Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Xaa1 Asn Xaa2 Ile Thr Tyr Phe Tyr Trp Tyr Leu Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys (c) Xaa3 is glutamine (Gln) or histidine (His).

[0013] The present invention provides an isolated immunoglobulin light chain polypeptide, comprising: Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Xaal Ile Asn Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Xaa2 Leu His Ser Gly Val Pro Ser Arg Phe Ser Xaa3 Ser Gly Ser Gly Xaa4 Asp Xaa5 Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Val (c) Xaa3 is glycine (Gly), serine (Ser), or proline (Pro); (d) Xaa4 is threonine (Thr) or asparagine (Asn); and (e) Xaa5 is phenylalanine (Pt). (f) Xaa6 is arginine (Arg) or absent; and (g) Xaa7 is threonine (Thr) or absent.

[0014] The present invention provides an isolated immunoglobulin light chain polypeptide comprising the amino acid sequence of SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50.

[0015] Furthermore, the present invention relates to isolated or purified nucleic acid sequences encoding said immunoglobulin polypeptides, vectors comprising such nucleic acid sequences, IL-36R-binding agents comprising said immunoglobulin polypeptides, nucleic acid sequences encoding such IL-36R-binding agents, vectors comprising such nucleic acid sequences, or Also provided are isolated cells containing such vectors, compositions comprising such IL-36R-binding agents or such vectors together with a pharmaceutically acceptable carrier, and methods for treating disorders responsive to inhibition of IL-36R by administering an effective amount of such a composition to a mammal. [Brief explanation of the drawings]

[0016] Brief description of each figure in the drawing [Figure 1A] FIG. 1A is a graph showing the results of a HEK human IL-36R / IL-8 luciferase reporter assay, as described in Example 1, upon stimulation of cells with hIL-36γ. [Figure 1B] FIG. 1B is a graph showing the results of a HEK human IL-36R / IL-8 luciferase reporter assay described in Example 1 upon stimulation of cells with hIL-36β. [Figure 1C] FIG. 1C is a graph showing the results of a HEK human IL-36R / IL-8 luciferase reporter assay, as described in Example 1, upon stimulation of cells with hIL-36α. [Figure 1D] FIG. 1D is a graph showing the results of a HEK human IL-36R / IL-8 luciferase reporter assay described in Example 1 upon stimulation of cells with 50 ng / mL hIL-36α. [Figure 1E] FIG. 1E is a graph showing the results of a HEK human IL-36R / IL-8 luciferase reporter assay described in Example 1 upon stimulation of cells with 20 ng / mL hIL-36β. [Figure 1F] FIG. 1F is a graph showing the results of a HEK human IL-36R / IL-8 luciferase reporter assay described in Example 1 upon stimulation of cells with 600 ng / mL hIL-36γ. [Figure 2A] FIG. 2A is a graph showing the results of a HEK cynomolgus IL-36R / IL-8 luciferase reporter assay described in Example 1 upon stimulation of cells with 2 μg / mL cynoIL-36α. [Figure 2B] FIG. 2B is a graph showing the results of a HEK cynomolgus IL-36R / IL-8 luciferase reporter assay described in Example 1 upon stimulation of cells with 10 μg / mL cynoIL-36β. [Figure 2C] FIG. 2C is a graph showing the results of a HEK cynomolgus IL-36R / IL-8 luciferase reporter assay described in Example 1 upon stimulation of cells with 300 ng / mL cynoIL-36γ. [Figure 3A] FIG. 3A is a graph showing experimental data illustrating the curve for binding of an antibody designated APE5281 to human IL-36R as determined by the KINEXA™ assay described in Example 2. [Figure 3B] FIG. 3B is a graph showing experimental data illustrating the curve for binding of an antibody designated APE6194 to human IL-36R as determined by the BIACORE™ assay described in Example 2. [Figure 3C] FIG. 3C is a graph showing experimental data illustrating the curve for binding of an antibody designated APE7247 to human IL-36R as determined by the KINEXA™ assay described in Example 2. [Figure 4A] FIG. 4A is a graph showing the results of an IL-8 secretion assay in primary human keratinocytes described in Example 3 using 10 ng / mL hIL-36α. [Figure 4B] FIG. 4B is a graph showing the results of an IL-8 secretion assay in primary human keratinocytes described in Example 3 using 1 ng / mL hIL-36β. [Figure 4C] FIG. 4C is a graph showing the results of an IL-8 secretion assay in primary human keratinocytes described in Example 3 using 100 ng / mL hIL-36γ. [Figure 4D] FIG. 4D is a graph showing the results of an IL-8 secretion assay in primary human keratinocytes described in Example 3 using 10 ng / mL hIL-36α. [Figure 4E] FIG. 4E is a graph showing the results of an IL-8 secretion assay in primary human keratinocytes as described in Example 3 using 1 ng / mL hIL-36β. [Figure 4F]FIG. 4F is a graph showing the results of an IL-8 secretion assay in primary human keratinocytes described in Example 3 using 100 ng / mL hIL-36γ. [Figure 4G] FIG. 4G is a graph showing the results of an IL-8 secretion assay in primary human keratinocytes as described in Example 3 using 10 ng / mL hIL-36α. [Figure 4H] FIG. 4H is a graph showing the results of an IL-8 secretion assay in primary human keratinocytes as described in Example 3 using 1 ng / mL hIL-36β. [Figure 4I] FIG. 4I is a graph showing the results of an IL-8 secretion assay in primary human keratinocytes described in Example 3 using 100 ng / mL hIL-36γ. [Figure 5A] FIG. 5A is a graph showing the results of an IL-8 secretion assay in primary cynomolgus monkey keratinocytes as described in Example 4 using 50 ng / mL of cynoIL-36α. [Figure 5B] FIG. 5B is a graph showing the results of an IL-8 secretion assay in primary cynomolgus monkey keratinocytes as described in Example 4 using 10 ng / mL of cynoIL-36β. [Figure 5C] FIG. 5C is a graph showing the results of an IL-8 secretion assay in primary cynomolgus monkey keratinocytes as described in Example 4 using 250 ng / mL of cynoIL-36γ. [Figure 5D] FIG. 5D is a graph showing the results of an IL-8 secretion assay in primary cynomolgus monkey keratinocytes as described in Example 4 using 50 ng / mL of cynoIL-36α. [Figure 5E] FIG. 5E is a graph showing the results of an IL-8 secretion assay in primary cynomolgus monkey keratinocytes as described in Example 4 using 10 ng / mL of cynoIL-36β. [Figure 5F]FIG. 5F is a graph showing the results of an IL-8 secretion assay in primary cynomolgus monkey keratinocytes as described in Example 4 using 250 ng / mL of cynoIL-36γ. [Figure 6A] FIG. 6A is a graph showing the results of an IL-8 secretion assay in primary human monocytes described in Example 5 using 5 ng / mL of IL-36β. [Figure 6B] FIG. 6B is a graph showing the results of an IL-8 secretion assay in primary human monocytes described in Example 5 using 500 ng / mL of IL-36β. [Figure 7A] FIG. 7A is a graph showing the results of an IL-8 secretion assay in primary human peripheral blood mononuclear cells (PBMCs) as described in Example 6 using 10 ng / mL of IL-36α. [Figure 7B] FIG. 7B is a graph showing the results of an IL-8 secretion assay in primary human peripheral blood mononuclear cells (PBMCs) as described in Example 6 using 1 ng / mL of IL-36β. [Figure 7C] FIG. 7C is a graph showing the results of an IL-8 secretion assay in primary human peripheral blood mononuclear cells (PBMCs) as described in Example 6 using 100 ng / mL of IL-36γ. [Figure 8A] FIG. 8A is a graph showing the results of the antibody / antigen cross-competition binding assay described in Example 8, as determined by a BIACORE™ assay using APE5100 as the primary antibody. [Figure 8B] FIG. 8B is a graph showing the results of the antibody / antigen competitive binding assay described in Example 8, as determined by a BIACORE™ assay using APE6155 as the primary antibody. [Figure 9A] FIG. 9A is a graph showing the results of a competitive binding assay described in Example 9 using CHO-K cells stably co-expressing human IL-36R and human IL-1RAcP. [Figure 9B]FIG. 9B is a graph showing the results of a competitive binding assay described in Example 9 using CHO-K cells stably co-expressing cynomolgus IL-36R variant 1 and cynomolgus IL-1RAcP. [Figure 10A] FIG. 10A is a graph showing the results of a luciferase reporter assay described in Example 1 using HEK cynomolgus monkey IL-36R variant 2 / IL-8 cells stimulated with 20 ng / mL cynoIL-36γ. [Figure 10B] FIG. 10B is a graph showing the results of a luciferase reporter assay described in Example 1 using HEK cynomolgus monkey IL-36R variant 1 / IL-8 cells stimulated with 300 ng / mL cynoIL-36γ. [Figure 10C] FIG. 10C is a graph showing the results of a luciferase reporter assay described in Example 1 using HEK cynomolgus monkey IL-36R variant 3 / IL-8 cells stimulated with 100 ng / mL cynoIL-36γ. [Figure 10D] FIG. 10D is a graph showing the results of a luciferase reporter assay described in Example 1 using HEK cynomolgus monkey IL-36R variant 2 / IL-8 cells stimulated with 300 ng / mL cynoIL-36γ. [Figure 10E] FIG. 10E is a graph showing the results of a luciferase reporter assay described in Example 1 using HEK cynomolgus monkey IL-36R variant 3 / IL-8 cells stimulated with 300 ng / mL cynoIL-36γ. [Figure 10F] FIG. 10F is a graph showing the results of a luciferase reporter assay described in Example 1 using HEK cynomolgus monkey IL-36R variant 4 / IL-8 cells stimulated with 300 ng / mL cynoIL-36γ. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention The present invention provides isolated immunoglobulin heavy chain polypeptides and / or isolated immunoglobulin light chain polypeptides, or fragments thereof (e.g., antigen-binding fragments). As used herein, the term "immunoglobulin" or "antibody" refers to a protein found in the blood or other bodily fluids of vertebrates, and is used by the immune system to identify and neutralize foreign substances, such as bacteria and viruses. The polypeptide is "isolated" in that it is removed from its natural environment. In preferred embodiments, an immunoglobulin or antibody is a protein that includes at least one complementarity-determining region (CDR). CDRs are the amino acid sequence of an antibody. The "hypervariable regions" of the heavy (H) chains form the "hypervariable regions" of the immunoglobulin, which are involved in antigen binding (described further below). A whole immunoglobulin typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N Variable End (V H ) region and three C-terminal constant (C H 1. C H 2 and C H 3) region, and each light chain contains one N-terminal variable (V L ) region and one C-terminal constant (C L ) region. The light chains of an antibody contain Based on the amino acid sequence of their constant domains, they can be assigned to one of two distinct types: either kappa (κ) or lambda (λ). In a typical immunoglobulin, each light chain is linked to a heavy chain by a disulfide bond, and the two heavy chains are linked together by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chain are aligned with each other.

[0018] The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. H and V L The regions have the same overall structure, and each region contains four framework (FW or FR) regions. As used herein, the term "framework region" refers to the relatively conserved amino acid sequences within the variable domains that are located between the hypervariable or complementarity-determining regions (CDRs). There are four framework regions in the FR1 protein, designated FR1, FR2, FR3, and FR4. The framework region forms a β-sheet that provides the structural framework for the variable region (see, e.g., CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001).

[0019] The framework regions are connected by three complementarity determining regions (CDRs). As mentioned above, the three CDRs (known as CDR1, CDR2, and CDR3) form the "hypervariable region" of an antibody, which is responsible for antigen binding. The CDRs are bound by a β-sheet structure formed by the framework regions. The constant regions of the light and heavy chains form loops that connect (and in some cases include part of) the antibody structure. The constant regions of the light and heavy chains are not directly involved in binding the antibody to an antigen, but the constant regions can influence the positioning of the variable regions. The constant regions also play a role in various effector functions (e.g., antibody-dependent complement-mediated lysis through interactions with effector molecules and cells). or involvement in antibody-dependent cellular toxicity).

[0020] The isolated immunoglobulin heavy chain polypeptides and isolated immunoglobulin light chain polypeptides of the present invention desirably bind to the interleukin-36 receptor (IL-36R), formerly known as IL-1Rrp2. IL-36R is a receptor of the IL-1R family and binds to the IL-36α ligand. IL-36α, IL-36β, and IL-36γ are members of the IL-1 family of cytokines and bind to IL-36α, IL-36β, and IL-36γ. Binds to L-36R and uses IL-1 receptor accessory protein (IL-1RAcP) as a coreceptor IL-36R stimulates intracellular signals similar to those induced by IL-1 (Towne et al., J. Biol. Chem., 279(14): 13677-13688 (2004)). , is highly expressed by keratinocytes and other epithelial cell types as well as dendritic cells and naive CD4+ T cells (Towne et al., supra; Vigne et al., Blood, 118(22): 5813-5823 (2011); and Vigne et al., Blood, 120(17): 3478-3487 (2012)).

[0021] The isolated immunoglobulin heavy chain polypeptides of the present invention and the isolated immunoglobulin light chain polypeptides of the present invention can form agents that bind to IL-36R and another antigen, resulting in a "dual reactive" binding agent (e.g., a dual reactive antibody). .

[0022] Certain other antibodies that bind to IL-36R, and components thereof, are known in the art (see, e.g., U.S. Patent Application Publication No. 2013 / 0236471). Anti-IL-36R antibodies are also commercially available from sources such as, for example, Abcam (Cambridge, MA) and R&D Systems, Inc. (Minneapolis, MN). There are.

[0023] The present invention provides an isolated immunoglobulin light chain polypeptide, comprising: Gln Val Gln Xaa1 Xaa2 Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser Tyr Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Xaa3 Leu Glu Trp Met Gly Trp Ile Tyr Pro Gly Asp Xaa4 Ser Thr Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Xaa5 Asp Xaa6 Ser Ala Xaa7 Thr Ala Tyr Met Glu Leu Xaa8 Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Xaa9 Cys Thr Arg Ser Phe Tyr Thr Met Amino acid sequence of Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 56) wherein (a) Xaa1 is leucine (Leu) or phenylalanine (Phe); (b) Xaa2 is valine (Val); (c) Xaa3 is arginine (Arg) or glycine (Gly), (d) Xaa4 is glycine (Gly), serine (Ser), or alanine (Ala), (e) Xaa5 is arginine (Arg) or alanine (Ala), (f) Xaa6 is threonine (Thr) or lysine (Lys), (g) Xaa7 is serine (Ser) or asparagine (Asn), and (h) Xaa8 is and (i) Xaa9 is serine (Ser) or alanine (Ala), and (ii) Xaa9 is tyrosine (Tyr) or phenylalanine (Phe). In one embodiment, the isolated immunoglobulin heavy chain polypeptide comprises: Gln Val Gln Xaa1 Xaa2 Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser Tyr Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Xaa3 Leu Glu Trp Met Gly Trp Ile Tyr Pro Gly Asp Xaa4 Ser Thr Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Xaa5 Asp Xaa6 Ser Ala Ser Thr Ala Tyr Met Glu Leu Xaa7 Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Amino acid sequence of Val Ser Ser (SEQ ID NO: 1) wherein (a) Xaa1 is leucine (Leu) or phenylalanine (Phe); (b) Xaa2 is valine (Val); (c) Xaa3 is arginine (Arg) or glycine (Gly), (d) Xaa4 is glycine (Gly), serine (Ser), or alanine (Ala), (e) Xaa5 is arginine (Arg) or alanine (Ala), (f) Xaa6 is threonine (Thr) or lysine (Lys), (g) Xaa7 is serine (Ser) or alanine (Ala), and (h) Xaa8 is Tyrosine (Tyr) or phenylalanine (Phe).

[0024] The heavy chain polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 56 or SEQ ID NO: 1, or The immunoglobulin may consist of, or consist essentially of, the amino acid sequence, and may have any one of the amino acid substitutions described above in any suitable combination. The heavy chain polypeptides are as set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: Sequence number 7, sequence number 8, sequence number 9, sequence number 10, sequence number 11, sequence number 12, sequence number 13, and SEQ ID NO: 14, or comprising or consisting of the amino acid sequence of any one of SEQ ID NO: 14, or consists essentially of said amino acid sequence.

[0025] The present invention also provides an isolated immunoglobulin heavy chain polypeptide, comprising: Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Xaa1 Met Xaa2 Trp Val Arg Gln Ala Pro Xaa3 Gln Gly Leu Glu Trp Met Gly Met Phe Xaa4 Pro Xaa5 Xaa6 Xaa7 Val Thr Arg Leu Asn Gln Lys Phe Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Thr Ser Met Ile Ile Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 15), wherein (a) Xaa1 is tryptophan (Trp) or tyrosine (Tyr), (b) Xaa2 is histidine (His), asparagine (Asn), or tyrosine (Tyr), (c) Xaa3 is glycine (Gly) or arginine (Arg), (d) Xaa4 is aspartic acid (Asp), glutamic acid (Glu), or histidine (His), and (e) Xaa5 is serine (Ser), threonine (Thr), or threonine (Thr). (f) Xaa6 is asparagine (Asn) or glycine (Gly), and (g) Xaa7 is serine (Ser), alanine (Ala), or aspartic acid (Asp).

[0026] The heavy chain polypeptides of the invention may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 15, with one of the amino acid substitutions described above being any suitable amino acid substitution. In one embodiment, the immunoglobulin heavy chain polypeptide comprises or has the amino acid sequence of any one of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24. The amino acid sequence may consist of or consist essentially of said amino acid sequence.

[0027] The present invention also provides an isolated immunoglobulin light chain polypeptide, comprising: Xaa1 Xaa2 Gln Xaa3 Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Xaa4 Xaa5 Tyr Ser Ile Thr Xaa6 Asp Phe Ala Trp Asn Trp Ile Arg Gln Xaa7 Pro Gly Xaa8 Xaa9 Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Asp Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Xaa10 Xaa11 Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Xaa12 Tyr Xaa13 Cys Ala Ile Arg Gly Pro Tyr Ser Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Xaa14 (SEQ ID NO: 57) Xaa1 is glutamine (Gln) or aspartic acid (Asp); Xaa2 is valine (Val) or leucine (Leu); Xaa3 is leucine (Leu) or phenylalanine (Phe); Xaa4 is threonine (Thr) or serine (Ser); and Xaa5 is glycine (Gly). or arginine (Arg); Xaa6 is serine (Ser) or alanine (Ala); Xaa7 is Xaa8 is proline (Pro) or phenylalanine (Phe); Xaa9 is lysine (Lys) or asparagine (As). Xaa9 is glycine (Gly) or lysine (Lys); Xaa10 is serine (Ser) or threonine (Thr); Xaa11 is valine (Val) or arginine (Arg); Xaa12 Xaa13 is tyrosine (Tyr) or phenylalanine (Phe); and Xaa14 is alanine (Ala) or absent. In some embodiments, the isolated heavy chain immunoglobulin polypeptides are selected from the group consisting of Xaa1 Val Gln, Xaa2 Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val, Xaa3 Gly Tyr Ser Ile Thr Ser Asp Phe Ala Trp Asn Trp Ile Arg Gln, Xaa4 Pro Gly, Xaa5 Xaa6 Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Asp Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Xaa a7 Xaa8 Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Xaa9 Cys Ala Ile Arg Gly Pro Tyr Ser Phe Thr Tyr Trp Gly Gln Gly or the amino acid sequence of Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 25). (c) Xaa3 is threonine (Thr) or serine (Ser); (d) Xaa4 is proline (Pro) or phenylalanine (Phe); and (e) Xaa5 is lysine (Lys) or asparagine (Asn). (f) Xaa6 is glycine (Gly) or lysine (Lys), and (g) Xaa7 is serine (Ser) or (h) Xaa8 is valine (Val) or arginine (Arg), and (i) Xaa9 is tyrosine (Tyr) or phenylalanine (Phe).

[0028] The heavy chain polypeptides of the invention can comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 25, and can have one or more of the amino acid substitutions described above in any suitable combination. The chain polypeptide comprises, consists of, or essentially has the amino acid sequence of any one of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54. It consists of columns.

[0029] In another embodiment, the invention provides an isolated immunoglobulin heavy chain polypeptide comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35.

[0030] The immunoglobulin heavy chain polypeptide of the present invention is essentially any of SEQ ID NO: 1 to SEQ ID NO: 35. When the heavy chain polypeptide of the present invention is directed to IL-36R, the heavy chain polypeptide of the present invention is directed to IL-36R. Additional components may be included in a polypeptide that do not materially affect the polypeptide (by affecting affinity for the polypeptide). Examples of such components include, for example, protein moieties such as biotin that facilitate purification or isolation, passenger mutations, sequences that do not have problem sites containing free cysteines, additional glycosylation sites, and sites with high potential for deamidation or isomerization.

[0031] When the immunoglobulin heavy chain polypeptide of the present invention consists of the amino acid sequence of any one of SEQ ID NOs: 1 to 35, the polypeptide does not contain any additional components (i.e., components that are not endogenous to the immunoglobulin heavy chain polypeptide of the present invention).

[0032] The present invention relates to a nucleic acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NO: 1 to SEQ ID NO: 35. The present invention provides an isolated immunoglobulin heavy chain polypeptide comprising an amino acid sequence (a sequence of which is a sequence of interest or a reference sequence). As described herein, nucleic acid or amino acid sequence "identity" can be determined by comparing a nucleic acid or amino acid sequence of interest with a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (i.e., identical) between the sequence of interest and the reference sequence, divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer). Between two or more sequences, Numerous mathematical algorithms for obtaining optimal alignment and calculating identity are known and are incorporated into many available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for aligning nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions), and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searching). Sequence alignment algorithms are also known. and also disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009); Durbin et al., eds., Biological Sequence Analysis: Probabalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(7): 951-960 (2005); Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

[0033] In another embodiment, the invention provides an immunoglobulin light chain polypeptide, comprising: Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Xaa1 Asn Thr Tyr Leu Tyr Trp Xaa2 Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Xaa3 Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Phe Does it contain the amino acid sequence of Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys (SEQ ID NO: 36)? , consisting of, or consisting essentially of, the amino acid sequence, wherein (a) Xaa1 is (b) Xaa2 is phenylalanine (Phe) or tyrosine (Tyr), and (c) Xaa3 is tyrosine (Tyr) or serine (Ser).

[0034] The light chain polypeptides of the present invention may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 36, optionally with one or more of the amino acid substitutions set forth above. In one embodiment, the isolated immunoglobulin light chain polypeptide may have the amino acid sequence of any one of SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39. The amino acid sequence of the present invention may comprise, consist of, or consist essentially of the amino acid sequence of the present invention.

[0035] The present invention also provides an immunoglobulin light chain polypeptide, comprising: Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Xaa1 Asn Xaa2 Ile Thr Tyr Phe Tyr Trp Tyr Leu Xaa3 Lys Pro Gly Gln Pro Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys (SEQ ID NO: 40) or the amino acid sequence (b) Xaa2 is glycine (Gly) or alanine (Ala), and (c) Xaa3 is a nucleotide sequence selected from the group consisting of: provides an immunoglobulin light chain polypeptide, which is glutamine (Gln) or histidine (His).

[0036] The light chain polypeptides of the invention may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 40, optionally with one or more of the amino acid substitutions described above. In one embodiment, the isolated immunoglobulin light chain polypeptide may have any one of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 44. It comprises, consists of, or consists essentially of an amino acid sequence.

[0037] The present invention provides an isolated immunoglobulin light chain polypeptide, comprising: Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Xaal Ile Asn Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Xaa2 Leu His Ser Gly Val Pro Ser Arg Phe Ser Xaa3 Ser Gly Ser Gly Xaa4 Asp Xaa5 Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Xaa6 Xaa7 (SEQ ID NO: 58) (b) Xaa2 is arginine (Arg) or methionine (Met); (c) Xaa3 is glycine (Gly), serine (Ser), or proline (Pro); and (d) Xaa4 is threonine (Tf), threonine (Tf), or threonine (Tf). (e) Xaa5 is phenylalanine (Phe) or tyrosine (Tyr), (f) Xaa6 is arginine (Arg) or absent, and (g) Xaa7 is threonine (Thr) or absent. In some embodiments, the immunoglobulin light chain polypeptide is Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Xaa1 Ser Gly Ser Gly Thr Asp Xaa2 Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys (SEQ ID NO: 45), wherein (a) Xaa1 is serine (Ser) or proline (Pro), and (b) Xaa2 is phenylalanine (Phe) or tyrosine (Tyr).

[0038] The light chain polypeptides of the invention can comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO:58 or SEQ ID NO:45, and can have one or more of the amino acid substitutions described above in any suitable combination. The globulin light chain polypeptide is any one of SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 55. The amino acid sequence of the present invention may comprise, consist of, or consist essentially of the amino acid sequence of

[0039] In another embodiment, the invention provides an isolated immunoglobulin light chain polypeptide comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50.

[0040] When the immunoglobulin light chain polypeptide of the present invention consists essentially of the amino acid sequence of any one of SEQ ID NOs: 36 to 50, the polypeptide can be substantially The immunoglobulin light chain polypeptide of the present invention may comprise an amino acid sequence of any one of SEQ ID NOs: 36 to 50. In some cases, the polypeptide does not include any additional components (ie, components that are not endogenous to the immunoglobulin light chain polypeptide of the present invention).

[0041] The present invention relates to a nucleic acid sequence that is at least 90% identical to any one of SEQ ID NOs: 36 to 50 (e.g., , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical The present invention provides an isolated immunoglobulin light chain polypeptide comprising an amino acid sequence which is: Nucleic acid or amino acid sequence "identity" can be determined using methods described herein.

[0042] One or more amino acids of the immunoglobulin heavy chain polypeptide and / or light chain polypeptide can be replaced or substituted with a different amino acid. A "replacement" or "substitution" refers to a substitution of a given amino acid sequence within a polypeptide sequence. substitution of one amino acid at a position or residue by another amino acid at the same position or residue It means exchange.

[0043] Amino acids are broadly classified as either "aromatic" or "aliphatic." Aromatic amino acids are "Aromatic" amino acids contain an aromatic ring. Examples of "aromatic" amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acids are broadly classified as "aliphatic." Examples of "aliphatic" amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine ​​(C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gln), lysine (K or Lys), and arginine (R or Arg).

[0044] Aliphatic amino acids can be subdivided into four subgroups: large aliphatic nonpolar subgroups The "aliphatic slightly polar subgroup" consists of valine, leucine, and isoleucine. The "aliphatic slightly polar subgroup" consists of methionine, serine, threonine, and cysteine. The "aliphatic polar / charged subgroup" consists of glutamic acid, aspartic acid, asparagine, glutamine, lysine, and arginine. The "small residue subgroup" consists of glycine and alanine. The group of charged / polar amino acids can be subdivided into three subgroups: lysine and alanine. a "positively charged subgroup" consisting of glutamic acid and aspartic acid, and a "polar subgroup" consisting of asparagine and glutamine.

[0045] The aromatic amino acids can be subdivided into two subgroups: histidine and tryptophan. and the "phenyl subgroup" consisting of phenylalanine and tyrosine.

[0046] Amino acid replacements or substitutions may be conservative, semi-conservative or non-conservative. The phrase "conservative amino acid substitution" or "conservative variation" refers to the replacement of one amino acid with another amino acid that shares common characteristics. A functional method for defining common properties between amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). By this analysis, groups of amino acids can be defined that preferentially exchange amino acids within a group and are therefore most similar to each other in their effect on the overall structure of the protein (Schulz and Schirmer, ibid).

[0047] Examples of conservative amino acid substitutions include amino acid substitutions within the above subgroups. substitutions such as arginine with lysine and vice versa (so that a positive charge can be maintained), aspartic acid with glutamic acid and vice versa (so that a negative charge can be maintained), threonine with serine (so that a free -OH can be maintained), and asparagine with glutamine (so that a free -NH2 can be maintained).

[0048] "Semi-conservative mutations" include amino acid substitutions within the same group listed above, but not within the same subgroup. For example, substitution of asparagine with aspartic acid, or lysine with asparagine, involves amino acids within the same group, but in different subgroups. "Non-conservative mutations" involve amino acid substitutions between different groups, such as substitution of tryptophan with lysine, or substitution of serine with phenylalanine.

[0049] Furthermore, one or more amino acids may be present in the immunoglobulin heavy chain polypeptide and / or the immunoglobulin light chain polypeptide. Any number of any suitable amino acids may be inserted into the amino acid sequence of an immunoglobulin heavy chain polypeptide and / or light chain polypeptide. In this regard, at least one amino acid (e.g., 2 or more, 5 or more, or 10 or more amino acids), but not more than 20 amino acids (e.g., 18 or less, 15 or less, or 12 or less amino acids) may be inserted into the immunoglobulin heavy chain polypeptide. Preferably, 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) are inserted into the amino acid sequence of an immunoglobulin heavy chain polypeptide and / or light chain polypeptide. In this regard, the amino acid(s) may be inserted at any suitable location into any one of said immunoglobulin heavy chain polypeptides and / or light chain polypeptides. Alternatively, the amino acid(s) are inserted into a CDR (eg, CDR1, CDR2, or CDR3) of an immunoglobulin heavy chain polypeptide and / or light chain polypeptide.

[0050] The isolated immunoglobulin heavy and light chain polypeptides of the present invention are not limited to polypeptides comprising the specific amino acid sequences described herein. Indeed, the immunoglobulin heavy or light chain polypeptides can be any heavy or light chain polypeptides that compete with the immunoglobulin heavy or light chain polypeptides of the present invention for binding to IL-36R. In this regard, for example, the immunoglobulin heavy or light chain polypeptides can be any heavy or light chain polypeptides that bind to the same IL-36R epitope as the epitope recognized by the heavy and light chain polypeptides described herein. Antibody competition can be performed using ELISA, Western blot, or immunohistochemistry methods. This can be assayed using routine peptide competition assays (see, e.g., U.S. Patent Nos. 4,828,981 and 8,568,992; and Braitbard et al., Proteome Sci., 4:12 (2006)).

[0051] The present invention includes one or more of the isolated amino acid sequences of the invention described herein, The present invention provides an IL-36R-binding agent that consists essentially of, or consists of, one or more of the sequences. "IL-36R-binding agent" refers to a molecule that specifically binds to the IL-36R protein, preferably a proteinaceous molecule that specifically binds to the IL-36R protein. Preferably, the IL-36R-binding agent is an antibody or a fragment thereof (e.g., an antigen-binding fragment). The IL-36R-binding agent of the present invention comprises, consists essentially of, or consists of an isolated immunoglobulin heavy chain polypeptide of the present invention and / or an isolated immunoglobulin light chain polypeptide of the present invention. In one embodiment, the IL-36R-binding agent comprises, consists essentially of, or consists of an immunoglobulin heavy chain polypeptide of the present invention or an immunoglobulin light chain polypeptide of the present invention. In another embodiment, the IL-36R-binding agent comprises, consists essentially of, or consists of an immunoglobulin heavy chain polypeptide of the present invention and an immunoglobulin light chain polypeptide of the present invention.

[0052] Any amino acid residue in the immunoglobulin heavy chain polypeptides of the invention and / or the immunoglobulin light chain polypeptides of the invention may be substituted with a different amino acid residue, or deleted or inserted, in any combination, so long as the biological activity of the IL-36R-binding agent is not substantially reduced (e.g., enhanced or improved) as a result of the amino acid substitution, insertion, and / or deletion.

[0053] The "biological activity" of an IL-36R-binding agent includes, for example, binding affinity for a particular IL-36R epitope, neutralization or inhibition of binding of IL-36R to its receptor(s), and / or binding of IL-36R to its receptor(s) in vivo. Neutralization or inhibition of activity (e.g., IC 50 ), pharmacokinetics, and cross-reactivity (e.g., with non-human homologs or orthologs of the IL-36R protein, or with other proteins or tissues). In certain embodiments, the interleukin-36 receptor (IL-36R) binding agents of the invention desirably exhibit one or more of the following biological activities: (a) inhibit the interaction between IL-36R and IL-36α, IL-36β, and / or IL-36γ, (b) inhibit intracellular signaling mediated by IL-36R, and / or (c) cross-react with and inhibit the activity of human and non-human primate (e.g., cynomolgus monkey) IL-36R. Other biological properties or characteristics of antigen-binding agents recognized in the art include, for example, avidity, selectivity, solubility, folding, These properties or characteristics include immunotoxicity, expression, and formulation. Various techniques (ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORE) TM ) or KINEXA TM The activity of the antibody may be observed, measured and / or assessed using a variety of assays, including, but not limited to, in vitro or in vivo neutralization assays, receptor-ligand binding assays, cytokine or growth factor production and / or secretion assays, and signal transduction and immunohistochemistry assays.

[0054] The terms "inhibit" or "neutralize," as used herein with respect to the activity of an IL-36R-binding agent, refer to, for example, the ability to substantially antagonize, prevent, prevent, limit, delay, interrupt, alter, eliminate, stop, or reverse the biological activity of IL-36R or the progression or severity of a disease or condition associated with IL-36R. IL-36R-binding agents of the invention preferably inhibit the activity of IL-36R by at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or more. %, or to a range defined by any two of the foregoing values.

[0055] The IL-36R-binding agent of the present invention can be a whole antibody or an antibody fragment, as described herein. The terms "antibody fragment," "antibody fragment," and "functional fragment of an antibody" are used interchangeably herein and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., Nat. Biotech., 23(9):1126-1129(2005)). The IL-36R-binding agent can be: Any IL-36R-binding antibody fragment may be included. The antibody fragment desirably includes, for example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. Examples of antibody fragments include: (i) a V L , V H , C L and (ii) a Fab fragment, which is a monovalent fragment consisting of the CH1 domain and a disulfide bond in the hinge region. F(ab), a bivalent fragment containing two Fab fragments linked by a cross-link ') 2 fragments, (iii) V of a single arm of an antibody L and V H(iv) Fab' fragments resulting from breaking the disulfide bridges of the F(ab')2 fragment using mild reducing conditions; (v) disulfide-stabilized Fv fragments (dsFv); and (vi) domain antibodies (dAbs), which are antibody single variable region domain (VH or VL) polypeptides that specifically bind to an antigen.

[0056] In embodiments in which the IL-36R-binding agent comprises a fragment of an immunoglobulin heavy or light chain polypeptide, the fragment can be of any size, so long as it binds to IL-36R and preferably inhibits its activity. In this regard, a fragment of an immunoglobulin heavy chain polypeptide desirably contains between about 5 and 18 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of the foregoing values). Similarly, a fragment of an immunoglobulin light chain polypeptide desirably contains between about 5 and 18 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of the foregoing values).

[0057] When the IL-36R-binding agent is an antibody or antibody fragment, the antibody or antibody fragment desirably comprises a heavy chain constant region (F c Preferably, the antibody or antibody fragment comprises a heavy chain constant region based on a wild-type IgG1, IgG2, or IgG4 antibody, or a variant thereof. As such, in some embodiments, when the IL-36R-binding agent is an antibody or antibody fragment, it may be capable of catalyzing antibody-dependent complement-mediated lysis or antibody-dependent complement-mediated lysis, e.g., through interactions with effector molecules and cells. A protein may exhibit one or more effector functions, such as participation in dependent cellular toxicity (eg, activation of the complement system, etc.).

[0058] The IL-36R-binding agent may also be a single-chain antibody fragment. Examples of single-chain antibody fragments include: (i) a single-chain antibody fragment comprising the two domains of an Fv fragment (i.e., V L and V H ) are joined by a synthetic linker that allows them to be synthesized as a single polypeptide chain. Single-chain Fvs (scFvs), which are monovalent molecules consisting of domains (see, e.g., Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16:778 (1998)), and (ii) ) diabodies (dimers of polypeptide chains, each of which is connected by a peptide linker, V L V bonded with H and the peptide linker comprises V H and V L are too short to be paired between different V H -V L on the polypeptide chain Pairing between the complementary domains is promoted to form a dimeric molecule with two functional antigen-binding sites. Antibody fragments include, but are not limited to, antibody fragments (which produce antibodies). Antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent Application Publication No. 2009 / 0093024 A1. do.

[0059] The IL-36R binding agent may also be an intrabody or a fragment thereof. Intrabodies are antibodies that are expressed and function intracellularly. Intrabodies typically lack disulfide bonds and can regulate the expression or activity of target genes through their specific binding activity. Intrabodies are isolated V H and V L Single domains such as domains and scFvs Intrabodies include fragments of intrabodies. Intrabodies may contain intracellular trafficking signals added to the N- or C-terminus of the intrabody to enable expression at high levels in the intracellular compartment where the target protein is located. Upon interaction with the target gene, intrabodies modulate the function of the target protein and / or achieve phenotypic / functional knockout by mechanisms such as promoting target protein degradation or sequestering the target protein in a non-physiological intracellular compartment. Other mechanisms of intrabody-mediated gene inactivation may depend on the epitope targeted by the intrabody (e.g., binding to a catalytic site on the target protein or to an epitope involved in protein-protein, protein-DNA, or protein-RNA interactions).

[0060] The IL-36R-binding agent may also be an antibody conjugate. In this regard, the IL-36R-binding agent may be (1) an antibody, an alternative scaffold, or a fragment thereof; and (2) a conjugate of a protein or non-protein moiety comprising an IL-36R binding agent. For example, the IL-36R-binding agent can be all or part of an antibody conjugated to a peptide, a fluorescent molecule, or a chemotherapeutic agent.

[0061] An IL-36R-binding agent can be or can be derived from a human antibody, a non-human antibody, or a chimeric antibody. A "chimeric" antibody is an antibody or fragment thereof that contains both human and non-human regions. Preferably, the IL-36R-binding agent is a humanized antibody. A "humanized" antibody is a monoclonal antibody comprising a human antibody scaffold and at least one CDR obtained or derived from a non-human antibody. Non-human antibodies include antibodies isolated from any non-human animal, such as, for example, a rodent (e.g., a mouse or a rat). A humanized antibody can comprise one, two, or three CDRs obtained or derived from a non-human antibody. In one embodiment of the present invention, the CDRH3 of the IL-36R-binding agent of the present invention is a mouse monoclonal antibody. The remaining variable and constant regions of the IL-36R-binding agent of the present invention are obtained or derived from a human monoclonal antibody, while the remaining variable and constant regions of the IL-36R-binding agent of the present invention are obtained or derived from a human monoclonal antibody.

[0062] Human, non-human, chimeric, or humanized antibodies can be derived from in vitro sources (e.g., hybridomas or recombinantly produced antibody cell lines) and in vivo sources (e.g., rodent Methods for producing antibodies are known in the art and are described, for example, in Kohler and Milstein, Eur. J. Immunol., 5:511-519. (1976); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); and Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). In certain embodiments, human or chimeric antibodies are used. The body is a system in which one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes. These can be produced using transgenic animals (e.g., mice) in which the endogenous antibody genes have been effectively replaced with human antibody genes. An example of a transgenic mouse in which the endogenous antibody genes have been effectively replaced with human antibody genes is the Medarex HUMAB-MOUSE mouse. TM , Kirin TC MOUSE TM , and Kyowa Kirin KM-MOUSE TM (See, e.g., Lonberg, Nat. Biotechnol., 23(9):1117-25(2005), and Lonberg, Handb. Exp. Pharmacol., 181:69-97(2008)). Humanized antibodies are, for example, produced by grafting non-human CDRs onto a human antibody scaffold. The antibodies can be made using any suitable method known in the art, including by immunoprecipitation (see, e.g., Kashmiri et al., Methods, 36(1):25-34(2005); and Hou et al., J. Biochem., 144(1):115-120(2008)). (2009). In one embodiment, humanized antibodies can be made using methods such as those described in U.S. Patent Application Publication No. 2011 / 0287485 A1.

[0063] In one embodiment, the CDRs (e.g., CDR1, CDR2, or CDR3) or variable regions of the immunoglobulin heavy chain polypeptides and / or immunoglobulin light chain polypeptides described herein are antibody or non-antibody polypeptides, etc., using either protein chemistry or recombinant DNA technology. In this regard, the present invention provides a method for the preparation of a polypeptide comprising at least one of the immunoglobulin heavy and / or light chain polypeptides as described herein. The present invention provides an IL-36R-binding agent comprising one or more CDRs. The IL-36R-binding agent can be any of the IL-36R-binding agents described herein. The antibody may comprise one, two, or three CDRs of an immunoglobulin heavy and / or light chain variable region.

[0064] In a preferred embodiment, the IL-36R-binding agent binds to an IL-36R epitope, thereby inhibiting the binding of IL-36R to any of its ligands (e.g., IL-36α, IL-36β, and IL-36γ). The present invention also provides an isolated or purified IL-36R epitope, which indirectly or allosterically blocks the binding of IL-36R to any ligand of IL-36R.

[0065] The present invention also provides one or more isolated or purified nucleic acids encoding the immunoglobulin heavy chain polypeptides of the invention, the immunoglobulin light chain polypeptides of the invention, and the IL-36R-binding agents of the invention. The acid sequence is provided.

[0066] The term "nucleic acid sequence" is intended to encompass polymers of DNA or RNA, i.e., polynucleotides, which may be single- or double-stranded and may contain non-natural or modified nucleotides. As used herein, the terms "nucleic acid" and "polynucleotide" refer to polymeric forms of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and, thus, include double- and single-stranded DNA, as well as double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides (e.g., but not limited to, methylated and / or capped polynucleotides). Nucleic acids are typically linked via phosphate linkages to form nucleic acid sequences or polynucleotides, although many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, etc.).

[0067] The present invention further includes one or more nucleic acid sequences encoding the immunoglobulin heavy chain polypeptides of the invention, the immunoglobulin light chain polypeptides of the invention, and / or the IL-36R-binding agents of the invention. The present invention provides vectors containing the vectors. The vectors can be, for example, plasmids, episomes, cosmids, viral vectors (e.g., retroviruses or adenoviruses), or phages. Suitable vectors and methods for vector preparation are well known in the art (see, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Cloning). Cholecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994).

[0068] In addition to the nucleic acid sequence encoding the immunoglobulin heavy polypeptide of the invention, the immunoglobulin light chain polypeptide of the invention, and / or the IL-36R-binding agent of the invention, the vector preferably contains components that regulate expression of the coding sequence in a host cell, such as a promoter, an enhancer, a polyadenylation signal, a transcription terminator, a signal peptide (e.g., osteonectin signal peptide), an internal ribosome entry site (IRES), and the like. Exemplary expression control sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0069] Constitutive, inducible and repressible promoters from a variety of different sources Numerous promoters, including those listed above, are well known in the art. Representative sources of promoters include, for example, viral, mammalian, insect, plant, yeast, and bacterial sources, and suitable promoters from these sources are readily available or can be synthetically produced based on sequences publicly available, for example, from depositories such as the ATCC and other commercial or private sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include, for example, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone inducible system (No et al., Proc. Natl. Acad. Sci., 93:3346-3351(1996)), and the T-REX system. TM(Invitrogen, Carlsbad, CA), LACSWITCH TM system (Stratagene, San Diego, CA), and Cre-ERT tamoxifen-inducible replicon binase systems (Indra et al., Nuc. Acid. Res., 27:4324-4327 (1999); Nuc. Acid. Res., 28:e99 (2000); U.S. Patent No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol., 308:123-144 (2005)).

[0070] As used herein, the term "enhancer" refers to a DNA sequence that increases transcription, e.g., of a nucleic acid sequence to which it is operably linked. Enhancers are known to regulate the transcription of a nucleic acid sequence. Enhancers can be located many kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, DNA methylation patterns, or changes in DNA structure. Numerous enhancers from a variety of different sources are well known in the art and are available as or within cloned polynucleotides (e.g., from depositories such as the ATCC and other commercial or private sources). Promoters (e.g., the commonly used CMV promoter) can also be used. Many polynucleotides containing promoters (such as promoters, promoters, promoter sequences ...

[0071] A vector may also contain a "selectable marker gene." As used herein, the term "selectable marker gene" refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected or not selected in the presence of a corresponding selection agent. Suitable selectable marker genes are known in the art and are described, for example, in International Patent Application Publication Nos. WO 1992 / 008796 and WO 1994 / 028143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77:3567-3570 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527-1531 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072-2076 (1981); Colberre-Garapin et al., J. Mol. Biol., 150:1-14 (1981); Santerre et al., Gene, 30:147-156 (1984); Kent et al., Science, 237:901-903(1987);Wigler et al., Cell, 11:223-232(1977);Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026-2034(1962);Lowy et al., Cell, 22:81 7-823 (1980); and U.S. Patent Nos. 5,122,464 and 5,770,359.

[0072] In some embodiments, the vector is an "episomal expression vector" or "episome," which is replicable in a host cell and, under appropriate selective pressure, persists in the host cell as an extrachromosomal segment of DNA (see, e.g., Conese et al., Gene Therapy, 11:1735-1742 (2004)). Exemplary commercially available episomal expression vectors include: Epstein-Barr virus nuclear antigen 1 (EBNA1) and the Epstein-Barr virus (EBV) origin of replication (oriP) are used for episomal plasminogen activators. Examples of vectors include, but are not limited to, the vector pREP4 from Invitrogen (Carlsbad, CA). Non-limiting examples of episomal vectors that use T antigen and the SV40 origin of replication instead of EBNA1 and oriP include pCEP4, pREP7, and pcDNA3.1, and pBK-CMV from Stratagene (La Jolla, CA). Here is an example.

[0073] Other suitable vectors include integrating expression vectors, which are capable of expressing the vector in a host cell. It can be integrated randomly into the DNA of the host cell or by a specific interaction between the expression vector and the host cell chromosome. The vectors may contain recombination sites that allow for recombination. Such integrating expression vectors may utilize endogenous expression regulatory sequences of the host cell chromosome to effect expression of the desired protein. Examples of vectors that integrate in a site-specific manner include, for example, the flp-in system (e.g., pcDNA 1.0) from Invitrogen (Carlsbad, CA). TM 5 / FRT), or the cre-lox system (e.g., Stratagene (La Jolla, CA) Examples of vectors that randomly integrate into a host cell chromosome include components of vectors from Life Technologies, Inc., such as those found in the pExchange-6 Core Vectors. pcDNA3.1 (when transfected in the absence of T antigen) from Millipore (Billerica, MA), UCOE from Millipore (Billerica, MA), and pCI or pFN10A(ACT)FLEXI from Promega (Madison, WI). TM Examples include:

[0074] Viral vectors may also be used. Representative commercially available viral expression vectors include those from Crucell, Examples of vectors that can be used include, but are not limited to, the adenovirus-based Per.C6 system available from Inc. (Leiden, The Netherlands), the lentivirus-based pLP1 from Invitrogen (Carlsbad, CA), and the retroviral vector pFB-ERV plus pCFB-EGSH from Stratagene (La Jolla, CA).

[0075] Nucleic acid sequences encoding the amino acid sequences of the present invention may be present on the same vector (i.e., in cis). A unidirectional promoter can be used to regulate the expression of each nucleic acid sequence. In another embodiment, a combination of bidirectional and unidirectional promoters can be used to regulate the expression of multiple nucleic acid sequences. Alternatively, the nucleic acid sequences encoding the polypeptides of the invention can be provided to the cells on separate vectors (i.e., in trans) and then transfected into the cells. Each nucleic acid sequence in each of the separate vectors can contain the same or different expression control sequences. The separate vectors can be provided to the cell simultaneously or sequentially.

[0076] A vector(s) containing a nucleic acid(s) encoding a polypeptide of the invention can be introduced into a host cell (including any suitable prokaryotic or eukaryotic cell) capable of expressing the polypeptide encoded thereby. Accordingly, the invention provides an isolated cell comprising a vector of the invention. Preferred host cells are those that can be grown easily and reliably, have a reasonably fast growth rate, have a well-characterized expression system, and can be easily and efficiently transformed or transfected.

[0077] Examples of suitable prokaryotic cells include those of the genera Bacillus (e.g., Bacillus subtilis and Bacillus brevis), Escherichia (e.g., E. coli), Pseudomonas, and Streptomyces. Examples of suitable cells include, but are not limited to, cells from the genera Salmonella, Salmonella, and Erwinia. Prokaryotic cells useful for this purpose include various strains of Escherichia coli (e.g., K12, HB101 (ATCC No. 366666) and HB101 (ATCC No. 366666). 3694), DH5α, DH10, MC1061 (ATCC No. 53338), and CC102).

[0078] Preferably, the vector is introduced into a eukaryotic cell. Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include those from the genera Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris.

[0079] Suitable insect cells are described, for example, in Kitts et al., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al., J. Virol., 67:4566-4579 (1993). Preferred insect cells include Sf-9 and HI5 (Invitrogen, Carlsbad, Calif.).

[0080] Preferably, mammalian cells are utilized in the present invention. Numerous suitable mammalian host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, VA). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary (CHO) cells (ATCC No. CCL61), CHO DHFR cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 cell line (ATCC No. CRL1650) and COS-7 cell line (ATCC No. CRL1651), and the CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host cells include primate and rodent cell lines, including transformed cell lines. Cell lines derived from in vitro culture of normal diploid cells, primary tissue, and primary explants are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, and BHK or HaK hamster cell lines (all available from the American Type Culture Collection (ATCC)). Methods for selecting suitable mammalian host cells, as well as methods for transforming, culturing, amplifying, screening, and purifying the cells, are known in the art.

[0081] In one embodiment, the mammalian cell is a human cell. For example, the mammalian cell is a human lymphoid cell line or lymphoid-derived cell line, such as a cell line derived from pre-B lymphocytes. Examples of human lymphoid cell lines include RAMOS (CRL-1596), Daudi (CCL-213), and EB-3. (CCL-85), DT40 (CRL-2111), 18-81 (Jack et al., Proc. Natl. Acad. Sci. USA, 85:1581-1585(1988)), Raji cells (CCL-86), PER.C6 cells (Crucell Holland BV, Leiden, The Netherlands), and derivatives thereof, but are not limited thereto. .

[0082] Nucleic acid sequences encoding the amino acid sequences of the present invention can be introduced into cells by "transfection," "transformation," or "transduction." As used herein, "transfection," "transformation," or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell using physical or chemical methods. Many transfections Infection techniques are known in the art, for example, calcium phosphate DNA co-precipitation (e.g. See Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991); DEAE-dextran; electrophoresis. cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment (Johnston, Nature, 346:776-777 (1990) )); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after propagation of the infectious particles in appropriate packaging cells, many of which are commercially available.

[0083] The present invention provides compositions comprising an effective amount of an immunoglobulin heavy chain polypeptide of the present invention, an immunoglobulin light chain polypeptide of the present invention, an IL-36R-binding agent of the present invention, a nucleic acid sequence of the present invention encoding any of the foregoing, or a vector of the present invention comprising a nucleic acid sequence of the present invention. Preferably, the composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition and comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and an amino acid sequence, IL-36R-binding agent, or vector of the present invention. Any suitable carrier can be used in the context of the present invention, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition will be administered and the particular method used to administer the composition. The composition can optionally be sterile. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier before use. The composition can be made according to conventional techniques, for example, as described in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0084] The present invention further provides a method for treating a disorder responsive to the inhibition or neutralization of IL-36R in a mammal. The method comprises administering the composition to a mammal having a disorder responsive to the inhibition or neutralization of IL-36R, thereby treating the disorder in the mammal. A disorder "responsive to the inhibition of IL-36R" or "responsive to the neutralization of IL-36R" refers to any disease or disorder in which a decrease in IL-36R level or activity has therapeutic benefit in a mammal (preferably a human), or in which inappropriate expression (e.g., overexpression) or increased activity of IL-36R causes or contributes to the pathological effects of the disease or disorder. Disorders responsive to the inhibition of IL-36R include, for example, inflammatory diseases, autoimmune diseases, respiratory diseases, metabolic disorders, and cancer.

[0085] Inflammatory disorders include, for example, allergic inflammation of the skin, lungs, and gastrointestinal tract, atopic dermatitis (also known as atopic eczema), asthma (allergic and non-allergic), epithelial-mediated inflammation, fibrosis (e.g., idiopathic pulmonary fibrosis, scleroderma, renal fibrosis, and scarring), allergic rhinitis, food allergies (e.g., allergies to peanuts, eggs, dairy products, shellfish, tree nuts, etc.), seasonal allergies, and other allergies. do.

[0086] The methods of the present invention can be used to treat autoimmune diseases, such as those described in MacKay IR and Rose NR, eds., The Autoimmune Diseases, Fifth Edition, Academic Press, Waltham, MA (2014). The present invention can be used to treat any type of autoimmune disease (i.e., a disease or disorder caused by an overactivity of the immune system, in which the body attacks and damages its own tissues). Examples of autoimmune diseases that can be treated by the methods of the present invention include multiple sclerosis, asthma, type 1 diabetes, rheumatoid arthritis, scleroderma, Crohn's disease, plaque psoriasis (commonly known as psoriasis), and the like. psoriasis), pustular psoriasis, generalized pustular psoriasis (GPP), palmoplantar pustulosis (PPP), inflammatory bowel disease, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus (SLE), ulcerative colitis, In a preferred embodiment, the method of the present invention is directed to the treatment of pustular psoriasis, generalized pustular psoriasis, palmoplantar pustulosis (PPP), or It is used to treat plaque psoriasis.

[0087] Pustular psoriasis is an uncommon form of psoriasis characterized by white pustules surrounded by red skin. Generalized pustular psoriasis (GPP) is characterized by sudden, recurring episodes of high fever, a generalized rash, and Leukocytosis and elevated serum levels of C-reactive protein, along with generalized pustules, It is a severe disease characterized by the deficiency of interleukin-36 receptor antagonist (interleukin-36Ra) (Marrakchi et al., N. Engl. J. Med., 365(7):620-628 (2011)). GPP is a treatment for patients with or previously suffering from psoriasis vulgaris (PV). however, GPP may occur in patients without a history of PV. (Sugiura et al., J. Invest. Derm., 133: 2514-2521 (2013)) Palmoplantar pustulosis is a chronic inflammatory skin disease characterized by sterile pustules and red, scaly skin on the palms and soles, which significantly impair the quality of life of affected individuals (de Waal, AC and van de Kerkhof, PCM, J. Dermatological Treatment, 22(2): 102-105 (2011)).

[0088] Examples of respiratory diseases that can be treated by the methods of the present invention include, but are not limited to, asthma, cystic fibrosis, emphysema, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome. Examples of metabolic disorders that can be treated by the methods of the present invention include obesity, type 2 diabetes, and bronchial asthma. These include, but are not limited to, diabetes, atherosclerosis, and cardiovascular disease.

[0089] The methods of the present invention may be used to treat any type of cancer known in the art, including, but not limited to, melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gallbladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer, pancreatic cancer, leukemia, lymphoma, and Merkel cell carcinoma (see, e.g., Bhatia et al., Curr. Oncol. Rep., 13(6):488-497(2011)).

[0090] An immune response is induced in a mammal by administering a composition comprising an immunoglobulin heavy chain polypeptide of the present invention, an immunoglobulin light chain polypeptide of the present invention, an IL-36R-binding agent of the present invention, a nucleic acid sequence of the present invention encoding any of the foregoing, or a vector of the present invention comprising a nucleic acid sequence of the present invention. The "immune response" may involve, for example, antibody production and / or activation of immune effector cells (e.g., T cells).

[0091] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures the disease and / or adverse symptoms resulting from the disease. To this end, the methods of the present invention comprise administering a "therapeutically effective amount" of an IL-36R-binding agent. A "therapeutically effective amount" refers to an amount effective, at the dosage and for the period necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the IL-36R-binding agent to elicit a desired response in an individual. For example, a therapeutically effective amount of an IL-36R-binding agent of the present invention is an amount that reduces the biological activity of IL-36R in humans.

[0092] Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., the effect completely or partially prevents a disease or its symptoms. In this regard, the methods of the invention comprise administering a "prophylactically effective amount" of an IL-36R-binding agent. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (e.g., prevention of disease onset).

[0093] Typical doses range, for example, from 1 pg / kg to 20 mg / kg (animal or human body weight). however, doses less than or greater than this exemplary range are within the scope of the present invention. Daily parenteral doses range from about 0.00001 μg / kg to about 20 mg / kg of total body weight (e.g., about 0.001 μg / kg, about 0.1 μg / kg, about 1 μg / kg, about 5 μg / kg, about 10 μg / kg, about 100 μg / kg, about 500 μg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, or a range defined by any two of the foregoing values), preferably from about 0.1 μg / kg to about 10 mg / kg of total body weight (e.g., about 0.5 μg / kg, about 1 μg / kg, about 50 μg / kg, about 150 μg / kg, about 300 μg / kg, about 750 μg / kg, about 1.5 mg / kg, about 5 mg / kg, or a range defined by any two of the foregoing values), more preferably Preferably, about 1 μg / kg to 5 mg / kg (total body weight) (e.g., about 3 μg / kg, about 15 μg / kg, about 75 μg / kg, μg / kg, about 300 μg / kg, about 900 μg / kg, about 2 mg / kg, about 4 mg / kg, or any two of the foregoing values and more preferably about 0.5 to 15 mg / kg (body weight) per day (e.g., about 1 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 6 mg / kg, about 9 mg / kg, about 11 mg / kg, The therapeutic or The effectiveness of the prophylaxis can be monitored by periodic evaluation of the treated patient. Depending on the condition, treatment may be repeated for repeated administrations over several days or longer until a desired suppression of disease symptoms occurs, or treatment may be continued for the life of the patient. However, other administration regimens may be useful and are within the scope of the invention. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0094] A composition comprising an effective amount of an immunoglobulin heavy chain polypeptide of the present invention, an immunoglobulin light chain polypeptide of the present invention, an IL-36R-binding agent of the present invention, a nucleic acid sequence of the present invention encoding any of the foregoing, or a vector of the present invention comprising a nucleic acid sequence of the present invention can be administered to a mammal using standard administration techniques (including oral, intravenous, intraperitoneal, subcutaneous, intrapulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration). The composition is preferably suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, intravaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0095] A composition comprising an effective amount of an immunoglobulin heavy chain polypeptide of the present invention, an immunoglobulin light chain polypeptide of the present invention, an IL-36R-binding agent of the present invention, a nucleic acid sequence of the present invention encoding any of the foregoing, or a vector of the present invention comprising a nucleic acid sequence of the present invention can be administered to a mammal using standard administration techniques (including oral, intravenous, intraperitoneal, subcutaneous, intrapulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration). The composition is preferably suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, intravaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0096] When administered to a mammal (e.g., a human), the biological activity of an IL-36R-binding agent of the present invention can be measured by any suitable method known in the art. For example, biological activity can be assessed by determining the stability of a particular IL-36R-binding agent. In one embodiment of the present invention, an IL-36R-binding agent (e.g., an antibody) has an in vivo half-life of between about 30 minutes and 45 days (e.g., about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 1 day, about 5 days, about 10 days, about 15 days, about 25 days, about 35 days, about 40 days, about 45 days, or a range defined by any two of the foregoing values). In another embodiment, the IL-36R-binding agent is administered for between about 2 hours and 20 days (e.g., about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 2 days, about 3 days, about 7 days, about 12 days, about 14 days, about 17 days, about 19 days, or any of the preceding values). In another embodiment, the IL-36R-binding agent has an in vivo half-life of between about 10 days and about 40 days (e.g., about 10 days, about 13 days, about 16 days, about 18 days, about 20 days, about 23 days, about 26 days, about 29 days, about 30 days, about 33 days, about 37 days, about 38 days, about 39 days, about 40 days, or a range defined by any two of the foregoing values). ) in vivo half-life.

[0097] The stability of the IL-36R binding agents of the present invention is evaluated by the denaturation midpoint (T m ) (the temperature at which 50% of the amino acid sequence is in its native conformation and the other 50% is denatured). Generally, T m The higher the T, the more stable the protein. In one embodiment of the present invention, the IL-36R-binding agent of the present invention has an in vitro denaturation midpoint (T) of about 60 to 100°C. m )of For example, the IL-36R-binding agents of the present invention have an in vitro T of about 65 to 80°C (e.g., 66°C, 68°C, 70°C, 71°C, 75°C, or 79°C), about 80 to 90°C (e.g., about 81°C, 85°C, or 89°C), or about 90 to 100°C (e.g., about 91°C, about 95°C, or about 99°C). m may include:

[0098] The stability of the IL-36R binding agent of the present invention can be evaluated by, for example, measuring the blood half-life, differential scanning calorimetry (DSC ), thermal shift assay, and pulse-chase assay, and any other suitable assay known in the art. Other methods for measuring protein stability in vivo and in vitro that can be used in the context of the present invention are described, for example, in Protein Stability and Folding, B.A. Shirley (ed.), Humana Press, Totowa, New Jersey (1995); Protein Structure, Stability, and Interactions (Methods in Molecular Biology), Shiver J.W. (ed.), Humana Press, New York, NY (2010); and Ignatova, Microb. Cell Fact., 4: 23 (2005).

[0099] The biological activity of a particular IL-36R-binding agent can also be assessed by determining its binding affinity to IL-36R or its epitope. The equilibrium constant for the reversible binding of D The affinity of a binder for a ligand (such as the affinity of an antibody for an epitope) is, for example, about 1 picosecond. from about pM to about 100 micromolar (e.g., about 1 picomolar (pM) to about 100 micromolar (μM) In one embodiment, the IL-36R-binding agent can be 1 nanomolar or less (e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.1 nM, 0.2 ... nM, 0.025 nM, 0.01 nM, 0.001 nM, or a range defined by any two of the foregoing values. )K D In another embodiment, the IL-36R-binding agent can bind to the IL-36R protein at 200 pM or less (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110 pM, 100 pM, 90 pM, 80 pM, 75 pM, 80 pM, 95 pM, 100 pM, 110 pM, 125 pM, 130 pM, 130 pM, 140 pM, 140 pM, 150 pM, 160 pM, 170 pM, 180 pM, 190 pM, 200 pM, 210 pM, 220 pM, 230 pM, 240 pM, 250 pM, 260 pM, 270 pM, 280 pM, 290 pM, 300 pM, 310 pM, 320 pM, 330 pM, 340 pM, 350 pM, 360 pM, 370 pM, 380 pM, 390 pM, 400 pM, 410 pM, 420 pM, 430 pM, 440 pM, 450 pM, 460 pM, 470 pM, 480 pM, 490 pM, 500 pM, 510 pM, 520 pM, 530 pM, 540 pM, 550 pM, 560 pM, 570 pM, 580 pM, 590 pM, 600 pM, 610 pM, 620 pM, 630 pM, 640 pM, pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 1 pM, or K of the range defined by any two of the values ​​listed above D It can bind to IL-36R at the target antigen or Immunoglobulin affinity for an epitope can be measured using any art-recognized assay, including, for example, fluorescence activated cell sorting (FACS), separable beads (e.g., magnetic beads), surface plasmon resonance (SPR), solution phase competition (KINEXA), and the like. TM ), antigen panning, competitive binding assays, and / or or ELISA (see, e.g., Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, NY, 2001).

[0100] The IL-36R-binding agents of the present invention can be administered alone or in combination with other drugs. For example, the IL-36R-binding agents can be administered in combination with other drugs, such as anti-inflammatory drugs (e.g., corticosteroids (e.g., prednisone and fluticasone)), non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin, ibuprofen, etc.), or combinations thereof. profen and naproxen), biologics (e.g., infliximab REMICADE TM ), adalimumab (HUMIRA TM ) or etanercept (ENBREL TM )), methotrexate (MTX), oral retinoids (e.g., acitretin (SORIATANE TM )), and topical steroids) for the treatment or prevention of the diseases disclosed herein.

[0101] In addition to therapeutic uses, the IL-36R-binding agents described herein can be used in diagnostic or research applications. In this regard, the IL-36R-binding agents can be used in methods for diagnosing disorders or diseases in which inappropriate expression (e.g., overexpression) or increased activity of IL-36R causes or contributes to the pathological effects of the disease or disorder. Similarly, the IL-36R-binding agents can be used in assays for monitoring IL-36R protein levels in subjects being tested for diseases or disorders that respond to inhibition of IL-36R. Research applications include, for example, methods utilizing an IL-36R-binding agent and a label to detect IL-36R protein in a sample (e.g., in a human body fluid or in a cell or tissue extract). The IL-36R-binding agents can be used with or without modification (e.g., covalent or non-covalent labeling with a detectable moiety). For example, the detectable moiety can be a radioisotope (e.g., 3 H, 14 C. 32 P, 35 S again teeth 125 I), fluorescent or chemiluminescent compounds (e.g., fluorescein isothiocyanate, rhodamine, or luciferin), enzymes (e.g., alkaline phosphatase, β-galactosidase, The antigen-binding agent ( Any method known in the art for individually conjugating a detectable moiety (e.g., an antibody) to a detectable moiety can be used in the context of the present invention (e.g., Hunter et al., Nature, 194:495-496 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Immunol. Meth., 40:219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30:407-412 (1982).

[0102] IL-36R protein levels can be measured using the IL-36R-binding agents of the present invention by any suitable method known in the art, including, for example, radioimmunoassay (RIA). ) and FACS. The normal or standard expression value of IL-36R can be determined by detecting IL-36R-containing or -containing cells using any suitable technique, for example, under conditions suitable for forming an antigen-antibody complex. This can be established, for example, by combining a sample suspected of containing IL-36R with an IL-36R-specific antibody. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials (e.g., Zola, Monoclonal Antibodies (See A Manual of Techniques, CRC Press, Inc. (1987)). The amount of IL-36R polypeptide expressed is compared with the standard value.

[0103] The IL-36R-binding agent may be provided in a kit (i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay). When the IL-36R-binding agent is labeled with an enzyme, the kit desirably includes substrates and cofactors required by the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). Other additives, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), may also be included in the kit. The relative amounts of the various reagents may be varied to provide concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. The reagents may be provided as dry powders (typically lyophilized) that include excipients that, upon dissolution, provide a reagent solution having the appropriate concentration.

[0104] The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope. [Example]

[0105] Example 1 This example demonstrates that the immunoglobulin heavy chain (HC) and light chain (LC) polypeptides of the present invention can form antibodies in vitro that bind to human IL-36R and block its signaling.

[0106] HEK293T / 17 cells (ATCC CRL-11268) were transfected with human IL-36R (hIL-36R) or cynomolgus IL-36R. (cynoIL-36R) together with the IL-8 promoter (Promega Corp., Madison, WI), and single-cell clones were selected for all subsequent assays.

[0107] HEK293 cells were cultured at 3 × 10 in 10 mL of DMEM + 10% FBS. 6 cells / flask onto a T75 culture flask The plates were plated and incubated overnight at 37°C. The next morning, 24 μl of FUGENE TM HD (Promega Corporation, Madison, WI) in 500 μl of OPTI-MEM TM The DNA encoding IL-36R was added to the medium (Life Technologies, Carlsbad, CA) and incubated at room temperature for 5 minutes. The mixture was incubated for another 25 minutes at room temperature. The allelic variation of cynomolgus IL-36R was examined by Sanger sequencing, and four distinct allelic variants were identified. HEK cell lines expressing each cynomolgus monkey IL-36R allele variant were identified within the population. Both human and cynomolgus monkey IL-8 reporter cell lines were generated endogenously. We used HEK human IL1RAcP expressing this DNA / FUGENE TM The mixture was gently added dropwise to the cells for transfection and incubated overnight at 37°C. 24 hours after transfection, the cells were split and placed in DMEM + 10% FBS containing hygromycin and puromycin for a period of 4 weeks for selection. After 4 weeks, stabilized cells were obtained. Cells were plated at 1 cell / well in 96-well clear-bottom plates (5 plates / cell line). Single cell clones were screened for cell surface expression of IL-36R and expanded, and low passage number (e.g., 1-3) cells were used in the assays described below.

[0108] HEK293 human IL36R / IL8 or HEK293-cynoIL36R / IL8 variant stabilized cell lines were cultured in Accuter Collect the cells in a 96-well clear-bottom plate and incubate at 0.06 x 10 cells / well in 100 µl of DMEM + 10% FBS. 6Cells / well were seeded overnight at 37°C, 5% CO2. The next morning, the plates were inverted in a sink to remove the medium and gently tapped on a paper towel to dry. Diluted antibodies containing various combinations of IL-36Ra and LC polypeptides (see Table 1), IL-36Ra (R&D Systems, Minneapolis, MN), and negative isotype control antibodies were prepared in two-fold serial dilutions to the desired concentrations in DMEM + 10% FBS (Life Technologies, Carlsbad, CA), immediately added to the wells (50 μl / well), and incubated for 20 minutes at 37°C in 5% CO2. I made him bate.

[0109] [Table 1]

[0110] Cells were then stimulated with 50 μl of IL36α, IL36β, or IL36γ ligand (R&D Systems, Minneapolis, MN) and incubated for an additional 24 hours at 37°C, 5% CO2. EC values ​​for each individual cytokine were 50 Luciferase activity was determined empirically prior to the assay. TM Luciferase Assay System (Promega, Cat# E2520, Madison, WI) was used to determine the activity of the luciferase assay substrate:buffer mixture (100 μl) added to each well, incubated at room temperature for 5 minutes, and transferred to a 96-well black-walled, clear-bottom plate. (150 μl). The plate was TM Plate Reader (PerkinElmer, Waltham, WA) The reading was taken on GraphPad PRISM and the luminescence (60 sec delay) was determined. TM Data were analyzed using Software 5 (GraphPad, San Diego, CA).

[0111] The results of the IL-8 luciferase reporter assay for human and cyno IL-36R are shown in Figures 1A to 1F (human IL-36R), Figures 2A to 2C (cyno IL-36R), Figures 10A to 10C (cyno IL-36R), and Figure 10D. The measured potency (IC) of each antibody tested is shown in Table 1. 50 ) are listed in Tables 2 and 3.1 and 3.2.

[0112] [Table 2]

[0113] [Table 3]

[0114] The results of this example demonstrate that the immunoglobulin heavy chain (HC) and light chain (LC) polypeptides of the present invention are We demonstrate that antibodies that bind to human IL-36R and inhibit its signal transduction can be generated in vitro.

[0115] Example 2 This example demonstrates that the immunoglobulin heavy chain (HC) and light chain (LC) polypeptides of the present invention can form antibodies that bind to human IL-36R in vitro.

[0116] DNA samples encoding various immunoglobulin heavy chain (HC) and light chain (LC) polypeptides as described herein were prepared by combining the following: Maxi-prepped DNA (containing 6 μg of HC plasmid and 6 μg of LC plasmid), 1 ml of OPTIMEM TM (Life Technologies, Carlsbad, CA), and 72 μl of FUGENE TMHD Transfection Reagent (Promega, Fitchburg, WI). All reagents were pre-warmed. After thorough mixing and incubation at room temperature for 25 minutes, 1 ml of the reagent / DNA mixture was added to each T225 culture flask containing 8 × 10 6 The transfection was carried out on HEK293-c18 cells (ATCC CRL-10852). 18 hours prior to transfection, cells were plated in T225 culture flasks with 20 ml of DMEM (Life Technologies, Carlsbad, CA) containing 10% FBS (Life Technologies, Carlsbad, CA) per flask and incubated overnight at 37°C in 5% CO2. After transfection, the cells were returned to 37°C in 5% CO2. The next day, the medium in each flask was diluted with 25 ml of 2 The medium was replaced with 93 Freestyle medium (Life Technologies, Carlsbad, CA), and the cells were transferred to an incubator with 8% CO2. Antibody production was allowed to proceed for 7 to 12 days. The supernatant was collected from each flask. The solution was collected, spun down at 3000 rpm for 10 minutes and sterile filtered into a new test tube.

[0117] For antibody purification, approximately 20-30 ml of cell culture supernatant containing the antibody of interest is collected. , 1-2 ml of MAB SELECT SURE TMLX resin (GE Healthcare, Waukesha, WI) was packed and passed through a gravity-flow column pre-equilibrated with PBS buffer (11.9 mM phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4) (Fisher Bioreagents, Waltham, MA). The column was washed with 5 column volumes of PBS buffer. Bound antibody was eluted from the resin with 5–10 column volumes of 0.1 M glycine, pH 3.0. The antibody-containing eluate was concentrated in an Amicon Ultra 10K concentrator (Millipore, Billerica, MA) to an antibody concentration of approximately 0.1–2 mg / mL, and the buffer was exchanged three times with PBS buffer. Antibody concentration was determined using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and purity was assessed by SDS-PAGE analysis. Ta.

[0118] The binding affinities of various purified antibodies, including the immunoglobulin heavy chain (HC) and light chain (LC) polypeptides described herein, were determined using BIACORE TM T200 (Sapidyne Instruments, Boise, Idaho ) assay. TM T200 evaluation software (GE Healthcare, Antibody-antigen binding kinetics and affinity were determined using a CM5 sensor chip (GE Healthcare, Waukesha, WI). The extracellular domain of human IL-36R was immobilized onto a CM5 sensor chip (GE Healthcare, Waukesha, WI) at approximately 100 RU using amine coupling chemistry. Each antibody was reconstituted at various concentrations using HBS-EP+ buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% polysorbate, pH 7.6) (Teknova, Hollister, CA). Each antibody concentration was then injected over the immobilized antigen at a flow rate of 30 μL / min for 2-3 minutes, followed by a 15-minute dissociation period. After each cycle, the surface was regenerated with 60 μL of 3 M MgCl2. Association and Dissociation BIACORE™ was used to report kinetic constants (k and k), on and off rates (k and k, respectively) and affinity (K). TM Global fitting was performed using a 1:1 binding model with mass transport in the T200 evaluation software.

[0119] The binding affinities of various purified antibodies, including the immunoglobulin heavy chain (HC) and light chain (LC) polypeptides described herein, were also evaluated using the KINEXA® 3000 assay (Sapidyne Instruments, Boise, Idaho). The KINEXA® technology measures unbound / free antibody molecules in solution phase after incubation with various concentrations of antigen. Measuring binding events in solution phase using microbeads to maximize surface area avoids mass transport limitations and mobility effects inherent in methods that measure binding to a solid phase. For each experiment, 50 μg of soluble human or cyno IL-36R extracellular domain was used. 50 mg of UltraLink Biosupport beads (Thermo Fisher Scientific, Waltham, MA) were amine-crosslinked. A fixed concentration of antibody (sufficient to generate a signal between 0.8 V and 1.2 V) was incubated with a titrated amount of antigen in sample buffer (1x PBS, pH 7.4, 0.02% NaN3, 0.1% BSA) for a time sufficient to approach or reach equilibrium (incubation time was different for each antibody and dependent on affinity). The antibody-antigen solution was then flowed over the antigen-bound beads at a rate of 0.25 mL / min. Free antibody captured by the beads was then eluted using an ALEXA FLUOR™ chromatograph. TM Detection was performed using 647-conjugated AffiniPure donkey anti-human IgG (H+L) (Jackson ImmunoResearch, West Grove, PA) (500 ng / ml). The KD and / or ABC (active binding concentration) of the antibodies were determined using KINEXA. TM Pro Software The data were obtained from nonlinear regression analysis using a one-site homogeneous binding model in I got a good deal.

[0120] BIACORE TM The KD values ​​obtained by the T200 assay and the KINEXA® 3000 assay are shown in Table 4 and in Figure 3A (KineXA data for humanized 1D9) and Figure 3B (KineXA data for 5D3 APE6194). The results are shown in Figure 3C (KinExA data for humanized 18D4) and Figure 3D (Biacore data for all 18D4).

[0121] [Table 4]

[0122] These data demonstrate that antibodies comprising different combinations of the immunoglobulin HC and LC polypeptides of the invention described herein can bind human IL-36R with high affinity.

[0123] Example 3 This example demonstrates that immunoglobulin heavy chain (HC) and light chain (LC) polypeptides of the present invention can form antibodies in vitro that bind to human IL-36R and inhibit cell signaling and cytokine (e.g., IL-8) release by human primary keratinocyte cells that endogenously express IL-36R.

[0124] The antibodies used in this assay were produced and purified as described above. Normal human epidermal keratinocytes (NHEK) were purchased from Lonza Clonetics (cat# 00192627). Cells were cultured and expanded in a 5% CO2, 37°C incubator using the recommended culture medium (Lonza KBM Gold medium (cat# 00192151) supplemented with Lonza KGM Gold SingleQuot supplement (cat# 0092152)). Passage 2 cells were cultured and expanded in a 5% CO2, 37°C incubator for multiple single-use experiments. The resulting aliquots were frozen in liquid nitrogen.

[0125] Passage 2 cells were thawed and diluted to a density of 100,000 cells / ml in the recommended culture medium above, and 100 μl cells / well were plated into standard flat-bottom 96-well tissue culture plates at a final cell density of 100,000 cells / ml. Cells were plated at a density of 10,000 cells / well. The outer wells were filled with 200 μl / well of phosphate-buffered saline to avoid edge effects. Cells were incubated overnight in 5% CO2 The cells were cultured in an incubator at 37°C to allow adhesion.

[0126] The next day, antibodies were added at concentrations of 10 μg / ml or half-log dilutions ranging from 1 μg / ml to 0 in culture medium. After 30 minutes, recombinant human IL-36 ligand was added to the culture medium at approximately EC 50 Antibody and ligand concentrations were made up at 4x the desired final concentrations and 50 μl was added per well. The final total volume of the wells was 200 μl. Approximately 48 hours later, the plates were centrifuged for 3 minutes and the supernatants were removed and transferred to clean plates and either tested immediately or stored at −80° C. until further analysis.

[0127] Human IL-8 levels in cell supernatants were measured using an R&D Systems DUO-SET TM ELISA kit (cat# DY208) and evaluated by ELISA according to the standard protocol provided by the manufacturer. Graph the data using GraphPad PRISM TM Using the software 50 The value was calculated.

[0128] The results of this assay are shown in Table 5 and Figures 4A-4I.

[0129] [Table 5]

[0130] The results of this example demonstrate that antibodies composed of the combination of HC and LC described herein express IL-36R and are stimulated by cytokines IL-36α, IL-36β, and IL-36γ. We demonstrate that it inhibits the release of inflammatory cytokine (IL-8) from human primary keratinocytes in a dose-dependent manner.

[0131] Example 4 This example demonstrates that the immunoglobulin heavy chain (HC) and light chain (LC) polypeptides of the present invention form antibodies that bind to cynomolgus monkey IL-36R (cyno IL-36R) in vitro, and inhibit IL-36-dependent cell signaling and cytokine production in primary keratinocyte cells that endogenously express IL-36R. (e.g., IL-8) release can be inhibited.

[0132] The antibodies used in this assay were produced and purified as described in Example 3. Cynomolgus monkey epidermal keratinocytes were purchased from CellBiologics (Chicago, IL; cat# MK-6066K). Cells were cultured in the recommended culture medium (CellBiologics Epithelial Medium Kit, cat# M6621 supplemented with CellBiologics Epithelial Cell Medium Supplement, cat# M6621) at 4°C in 5% CO2. The cells were cultured and expanded in an incubator at 37°C. The mixture was frozen in liquid nitrogen in aliquots for 10 min.

[0133] Passage 2 cells were thawed and diluted to a density of 100,000 cells / ml in the above medium, and 100 μl cells / well were plated into standard flat-bottom 96-well tissue culture plates to a final cell density of 10,000 cells / well. The outer wells were filled to avoid edge effects. The wells were filled with 200 μl of PBS to allow cells to adhere. The cells were cultured overnight in a 5% CO2, 37°C incubator.

[0134] The next day, antibodies were added at concentrations of 10 μg / ml or half-log dilutions ranging from 1 μg / ml to 0 in culture medium. After 30 minutes, recombinant cynomolgus IL-36 ligand was added to the culture medium at approximately EC 50 Concentration (each Antibody and ligand concentrations were made up at 4x the desired final concentration and 50 μl was added per well to obtain the final volume of each well. Approximately 48 hours later, the plates were centrifuged for 3 minutes and the supernatant was removed and transferred to a clean plate and either tested immediately or stored at -80°C until further analysis.

[0135] Cynomolgus monkey IL-8 levels in cell supernatants were measured using the eBioscience (San Diego, CA) monkey IL-8 platinum ELISA kit (cat# BMS640 / 3) according to the standard protocol provided by the manufacturer. The data were plotted and analyzed using GraphPad PRISM. TM soft Using the IC 50 The value was calculated.

[0136] The results of this assay are shown in Table 6 and Figures 5A-5F.

[0137] [Table 6]

[0138] The results of this example demonstrate that antibodies composed of the combination of HC and LC described herein express IL-36R and are stimulated by cytokines IL-36α, IL-36β, and IL-36γ. We demonstrate that it inhibits the release of inflammatory cytokines (IL-8) from primary cynomolgus monkey keratinocytes in a dose-dependent manner.

[0139] Example 5 This example shows the IL-36R-expressing antibody composed of the HC and LC described herein. Demonstrated the ability to block human IL-36-mediated IL-8 release from human monocytes in a dose-dependent manner do.

[0140] Leukocyte Reduction System units processed from donor whole blood units were obtained from the San Diego Blood Bank. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density centrifugation (Sigma HISTOPAQUE). TM Monocytes were prepared by standard methods using a human monocyte isolation kit (cat# 10771). The cells were isolated from PBMCs using Kit II (Miltenyi Biotec, San Diego, CA; cat# 130-091-153).

[0141] Monocytes were diluted to a density of 500,000 cells / ml in RPMI 1640 medium containing 10% fetal bovine serum and penicillin / streptomycin, and 100 μl cells / well were added in a standard Cells were plated into flat-bottom 96-well tissue culture plates at a final cell density of 50,000 cells / well. The outer wells were filled with 200 μl / well of PBS to avoid edge effects. The plated cells were allowed to recover for 2–3 hours in a 5% CO2, 37°C incubator.

[0142] After approximately 2-3 hours of incubation, antibodies were added at concentrations of 10 μg / ml or half-log dilutions ranging from 1 μg / ml to 0 μg / ml in the culture medium. After 30 minutes, recombinant human IL-36 ligand was added to the culture medium at approximately EC 50 The antibodies and ligands were added at concentrations (previously determined empirically for each ligand). The end concentration was made 4 times the desired final concentration, and 50 μl was added per well. The final total volume in the wells was 200 μl. Approximately 48 hours later, the plates were centrifuged for 3 minutes and the supernatants were removed and transferred to clean plates and either tested immediately or stored at −80° C. until further analysis.

[0143] Human IL-8 levels in cell supernatants were measured using an R&D Systems DUO-SET TM ELISA kit (cat# DY208) and evaluated by ELISA according to the standard protocol provided by the manufacturer. Graph the data using GraphPad PRISM TM Using the software 50 The value was calculated.

[0144] The results of these experiments are set forth in Table 7 and Figures 6A and 6B.

[0145] [Table 7]

[0146] The results of this example demonstrate that antibodies composed of the combination of HC and LC described herein express IL-36R and are stimulated by cytokines IL-36α, IL-36β, and IL-36γ. We demonstrate that it inhibits the release of inflammatory cytokines (IL-8) from human primary monocytes in a dose-dependent manner.

[0147] Example 6 This example demonstrates that the immunoglobulin heavy chain (HC) and light chain (LC) polypeptides of the invention described herein inhibit IL-36-dependent cytokine release from human primary peripheral blood mononuclear cells. This demonstrates that antibodies can be generated that inhibit the production of IgG.

[0148] Leukocyte Reduction System units processed from donor whole blood units were obtained from the San Diego Blood Bank. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density centrifugation (Sigma HISTOPAQUE). TM Cat# 10771) was prepared by standard methods.

[0149] PBMCs were cultured at 1 × 10 in RPMI 1640 medium containing 10% fetal bovine serum and penicillin / streptomycin. 6 The cells were diluted to a density of 100,000 cells / ml and 100 μl cells / well were plated into a standard flat-bottom 96-well tissue culture plate to a final cell density of 100,000 cells / well. The outer wells were filled with 200 μl / well of PBS to avoid edge effects, and the plated cells were allowed to recover for 2–3 hours in a 5% CO2, 37°C incubator.

[0150] After approximately 2-3 hours of incubation, antibodies were added at concentrations of 10 μg / ml or half-log dilutions ranging from 1 μg / ml to 0 μg / ml in the culture medium. After 30 minutes, recombinant human IL-36 ligand was added to the culture medium at approximately EC 50 The antibodies and ligands were added at concentrations (previously determined empirically for each ligand). The concentration of each solution was made 4 times the desired final concentration, and 50 μl was added to each well. The final total volume of the wells was 200 μl. Approximately 48 hours later, the plates were centrifuged for 3 minutes and the supernatants were removed and transferred to clean plates and either tested immediately or stored at −80° C. until further analysis.

[0151] Human IL-8 levels in cell supernatants were measured using an R&D Systems DUO-SET TM The assay was performed by ELISA using an ELISA kit (cat# DY208) according to the standard protocol provided by the manufacturer. The data was graphed and analyzed using GraphPad PRISM. TM Using the software 50 The value was calculated.

[0152] The results of this assay are shown in Figures 7A-7C and demonstrate that antibodies composed of the HC and LC combinations described herein inhibited the expression of IL-36R and the cytokines IL-36α, IL-36β, and IL-36γ. This study demonstrates that IL-8 inhibits the release of a pro-inflammatory cytokine (IL-8) from human primary peripheral blood mononuclear cells stimulated by IL-8 in a dose-dependent manner.

[0153] Example 7 This example demonstrates that the immunoglobulin heavy chain (HC) and light chain (LC) polypeptides of the invention described herein inhibit IL-36-dependent cytokine release from primary cynomolgus monkey peripheral blood mononuclear cells. We demonstrate that antibodies can be generated that inhibit the release of IgG.

[0154] Peripheral blood mononuclear cells (PBMCs) were obtained from Biotox Sciences (San Diego, CA). Whole blood from monkeys was collected by Ficoll density centrifugation (Sigma HISTOPAQUE) TM cat# 10771) , was prepared by standard methods.

[0155] PBMCs were cultured at 1 × 10 in RPMI 1640 medium containing 10% fetal bovine serum and penicillin / streptomycin. 6 The cells were diluted to a density of 100,000 cells / ml and 100 μl cells / well were plated into a standard flat-bottom 96-well tissue culture plate to a final cell density of 100,000 cells / well. The outer wells were filled with 200 μl / well of PBS to avoid edge effects. The plated cells were allowed to recover for 2-3 hours in a 5% CO2, 37°C incubator.

[0156] After approximately 2-3 hours of incubation, antibodies were added at concentrations of 10 μg / ml or half-log dilutions ranging from 1 μg / ml to 0 μg / ml in the culture medium. After 30 minutes, recombinant cynomolgus monkey IL-36 ligand was added to the culture medium. Approximately EC 50 Antibody and ligand concentrations were made up at 4x the desired final concentrations, and 50 μl was added per well. Approximately 48 hours later, the plates were centrifuged for 3 minutes, after which the supernatant was removed and transferred to a clean plate and either tested immediately or stored at -80°C until further analysis.

[0157] Cynomolgus monkey IL-8 levels in cell supernatants were assessed by ELISA using the eBioscience monkey IL-8 platinum ELISA kit (San Diego, CA; cat# BMS640 / 3) according to the standard protocol provided by the manufacturer. Data were graphed and analyzed using GraphPad PRISM. TM software Using IC 50 The values ​​were calculated. The results of this assay are listed in Table 8.

[0158] [Table 8]

[0159] The results of this example demonstrate that antibodies composed of the combination of HC and LC described herein express IL-36R and are stimulated by cytokines IL-36α, IL-36β, and IL-36γ. We demonstrate that it inhibits the release of inflammatory cytokines (IL-8) from primary cynomolgus monkey peripheral blood mononuclear cells in a dose-dependent manner.

[0160] Example 8 This example demonstrates that the immunoglobulin heavy chain (HC) and light chain (LC) polypeptides of the invention described herein form antibodies that cross-compete for binding to human IL-36R. Demonstrate that this is possible.

[0161] Cross-competitive binding of the target IL-36R by antibodies comprising the various HC and LC polypeptides described herein was measured using BIACORE TMThe assay was performed using a T200 system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). For each assay, a primary antibody was captured on the surface of a chip. Unused complementation sites were then blocked by the addition of a saturating amount of a negative control antibody that does not bind to human IL-36R. After this step, IL-36R was allowed to bind, followed by the addition of a secondary antibody to determine whether the antibodies compete for the same binding site on the monomeric antigen. If the antibodies bind the same epitope, no secondary binding will be observed; if a different binding site on IL-36R is utilized, the secondary antibody will bind to the primary antibody / antigen complex.

[0162] Anti-human IgG (Fc-specific; GE Healthcare, Chalfont St. Giles, United Kingdom) BIACORE using EDC-activated coupling chemistry TM ~8,000RU on the surface of a CM5 chip Anti-IL-36R antibodies (10 μg / mL; 60-second contact time at a flow rate of 10 μL / min) containing various combinations of the HC and LC polypeptides of the present invention described herein were then captured onto the surface of the chip at 25°C, yielding ~500 RU of captured antibody. The chip was then blocked using a non-specific, isotype-matched negative control antibody (APE4909 at 100 μg / mL; contact time: 60 seconds at a flow rate of 10 μL / min). IL-36R diluted (at 1 μM) in running buffer (HBS-EP+, pH 7.6; GE Healthcare, Chalfont St. Giles, United Kingdom) was then flowed over the chip surface (300 seconds at a flow rate of 30 μL / min), followed immediately by the secondary antibody. This allows indirect detection of mass changes on the chip surface. The resulting sensograms generated via surface plasmon resonance (SPR) monitoring were examined to determine cross-competition between the antibodies.

[0163] The results of the competitive binding assay for the anti-IL-36R antibodies of the present invention are shown in Table 9 and Figures 8A and 8B. show.

[0164] [Table 9]

[0165] The results of this example show that antibody APE6155 (5D3) and antibody APE3847 (18D4) compete for binding to the same epitope on human IL-36R, but do not compete with antibody APE5100 for binding to IL-36R. It was demonstrated that neither APE6155 nor APE3847 shares an epitope with APE5100. The competition results were consistent and independent of the order of primary and secondary antibodies against the antigen.

[0166] Example 9 This example demonstrates that the immunoglobulin heavy chain (HC) and light chain (LC) polypeptides of the invention described herein can bind to cells expressing human and cynomolgus IL-36R together with IL-1RAcP.

[0167] We investigated the binding of the antibody to CHO-K cells stably co-expressing human IL-36R and human IL-1RAcP. Allelic variation of cynomolgus monkey IL-36R was examined by Sanger sequencing, and four distinct allelic variants were identified within the cynomolgus monkey population. Antibody binding to CHO-K cells stably co-expressing monkey IL-36R variant 1 and cynomolgus monkey IL-1RAcP was also examined for APE6155 and APE7247. Each antibody was harvested using Accutase, washed, and incubated with CHO cells seeded at 500,000 cells / well. Cells were incubated with antibody concentrations ranging from 33 nM to 16 pM for 30 minutes at 4°C. The cells were incubated and washed three times with FACS staining buffer. The cells were spun, aspirated, and then eluted at 100°C. The cells were incubated with 100 μL of paraformaldehyde for 10 minutes at room temperature. The cells were washed again, aspirated, and stained with 100 μL of anti-human IgG Alexa 647 for 20 minutes at 4°C. The cells were analyzed by FACS. Before analysis on an Array (BD Biosciences), the cells were resuspended in 100 μL of FACS analysis buffer.

[0168] The results of competitive binding assays for the anti-IL-36R antibodies of the present invention are shown in Figures 9A and 9B. Figure 9A shows the binding of the APE06155 and APE07247 antibodies to human IL-36R and CHO cells stably expressing human IL-36R, and Figure 9B shows the binding of the APE06155 and APE07247 antibodies to CHO cells stably expressing cynomolgus monkey variant 1 IL-36R and IL-1RAcP. Figure 1 shows the binding of the same antibody to CHO cells expressing human and cynomolgus monkey IL-36R. Data were fitted using Graphpad Prism software to determine the EC50 values ​​of APE6155 on human and cynomolgus monkey IL-36R-expressing CHO cells. The EC50 values ​​for the binding of backup Ab APE7247 to human and cynomolgus monkey IL-36R-expressing CHO cells were determined to be 1.5 nM and 2.4 nM, respectively, and 2.8 nM and 3.3 nM, respectively. The matched negative control antibody APE00422 showed no binding to either cell line.

[0169] Example 10 This example demonstrates that the immunoglobulin heavy chain (HC) and light chain (LC) polypeptides of the invention described herein exhibit excellent pharmacokinetic characteristics in cynomolgus monkeys. This demonstrates that the compound can be used in vivo with intravenous and subcutaneous bioavailability. Marsh-eating monkeys were administered ANB019 as a single dose intravenous (IV) or subcutaneous (SC) injection. Blood samples were collected from monkeys in the single-dose study 0.5 to 672 hours (4 weeks) post-dose. The obtained serum samples were analyzed by AnaptysBio, Inc. (San Diego, CA) using an in-house ELISA-based method. Pharmacokinetic analysis of ANA020 serum concentration versus time data was performed by AnaptysBio, Inc.

[0170] The serum concentration versus time profile of ANB019 was normal for both IV and SC routes of administration. For IV administration, levels rapidly decreased between Tmax and 24 hours and then declined, consistent with the expected behavior of monoclonal antibodies in non-human primates. Pharmacokinetic parameter estimates from ANB019 serum concentration titers were obtained from non-compartmental analysis and are presented in Table 10. Parameter estimates were obtained from the IgG4 scaffolding expected in monkeys. The pharmacokinetics of ANB019 was consistent with that of conventional monoclonal antibodies. The half-life of ANB019 was ~270 days after IV injection. The estimated bioavailability after SC injection was 60%. It was.

[0171] [Table 10]

[0172] All references cited herein, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each individual reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0173] With respect to the description of the present invention (particularly with respect to the claims that follow), the terms "a" and "an" and the use of "the" and "at least one" and similar referents should be construed to cover both the singular and the plural unless otherwise stated herein or clearly contradicted by context. Use of the term "at least one" following a listing of one or more items (e.g., "at least one of A and B") shall be construed to cover both the singular and the plural unless otherwise stated herein or clearly contradicted by context. Unless otherwise specified, one item (A or B) selected from the items listed, or and any combination of two or more of (A and B). The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise indicated. Recitation of ranges of values ​​herein serves as a shorthand method of referring individually to each separate value falling within that range, unless otherwise stated herein. The present invention is intended to be illustrative and not restrictive, and each individual value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is to be construed as a set forth in the present specification. It is intended only to better illustrate the invention and is not intended to impose limitations on the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0174] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors anticipate that those of skill in the art will employ such variations as appropriate, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. 1. An isolated immunoglobulin light chain polypeptide, comprising: Gln Val Gln Xaa1 Xaa2 Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser Tyr Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Xaa3 Leu Glu Trp Met Gly Trp Ile Tyr Pro Gly Asp Xaa4 Ser Thr Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Xaa5 Asp Xaa6 Ser Ala Xaa7 Thr Ala Tyr Met Glu Leu Xaa8 Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Xaa9 Cys Thr Arg Ser Phe Tyr Thr Met Asp Tyr comprising the amino acid sequence of Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 56), where (a) Xaa1 is leucine (Leu) or phenylalanine (Phe); (b) Xaa2 is valine (Val), methionine (Met), or leucine (Leu); (c) Xaa3 is arginine (Arg) or glycine (Gly); (d) Xaa4 is glycine (Gly), serine (Ser), or alanine (Ala); (e) Xaa5 is arginine (Arg) or alanine (Ala); (f) Xaa6 is threonine (Thr) or lysine (Lys); (g) Xaa7 is serine (Ser) or asparagine (Asn); (h) Xaa8 is serine (Ser) or alanine (Ala), and (i) Xaa9 is tyrosine (Tyr) or phenylalanine (Phe); An isolated immunoglobulin light chain polypeptide.

2. Gln Val Gln Xaa1 Xaa2 Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser Tyr Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Xaa4 Ser Thr Lys Tyr Asn Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Xaa5 Asp Xaa6 Ser Ala Ser Thr Ala Tyr Met Glu Leu Xaa7 Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Xaa8 Cys Thr Arg Ser Phe Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1), wherein (a) Xaa1 is leucine (Leu) or phenylalanine (Phe); (b) Xaa2 is valine (Val), methionine (Met), or leucine (Leu); (c) Xaa3 is arginine (Arg) or glycine (Gly); (d) Xaa4 is glycine (Gly), serine (Ser), or alanine (Ala); (e) Xaa5 is arginine (Arg) or alanine (Ala); (f) Xaa6 is threonine (Thr) or lysine (Lys); (g) Xaa7 is serine (Ser) or alanine (Ala), and (h) Xaa8 is tyrosine (Tyr) or phenylalanine (Phe); 2. The isolated immunoglobulin heavy chain polypeptide of claim 1.

3. SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, Any of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 2. The isolated immunoglobulin heavy chain polypeptide of claim 1, comprising any one of the amino acid sequences of:

4. An isolated immunoglobulin heavy chain polypeptide comprising: Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Xaa1 Met Xaa2 Trp Val Arg Gln Ala Pro Xaa3 Gln Gly Leu Glu Trp Met Gly Met Phe Xaa4 Pro Xaa5 Xaa6 Xaa7 Val Thr Arg Leu Asn Gln Lys Phe Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Thr Ser Met Ile Ile Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 15) wherein (a) Xaa1 is tryptophan (Trp) or tyrosine (Tyr), (b) Xaa2 is histidine (His), asparagine (Asn), or tyrosine (Tyr); (c) Xaa3 is glycine (Gly) or arginine (Arg); (d) Xaa4 is aspartic acid (Asp), glutamic acid (Glu), or histidine (His); (e) Xaa5 is serine (Ser), threonine (Thr), or tyrosine (Tyr); (f) Xaa6 is asparagine (Asn) or glycine (Gly), and (g) Xaa7 is serine (Ser), alanine (Ala), or aspartic acid (Asp); An isolated immunoglobulin heavy chain polypeptide.

5. 5. The isolated immunoglobulin heavy chain polypeptide of claim 4, comprising the amino acid sequence of any one of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:

24.

6. An isolated immunoglobulin light chain polypeptide, comprising: Xaa1 Xaa2 Gln Xaa3 Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Xaa4 Xaa5 Tyr Ser Ile Thr Xaa6 Asp Phe Ala Trp Asn Trp Ile Arg Gln Xaa7 Pro Gly Xaa8 Xaa9 Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Asp Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Xaa10 Xaa11 Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Xaa12 Tyr Xaa13 Cys Ala Ile Arg Gly Pro Tyr Ser Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Xaa14 (SEQ ID NO: 57), wherein (a) Xaa1 is glutamine (Gln) or aspartic acid (Asp); (b) Xaa2 is valine (Val) or leucine (Leu); (c) Xaa3 is leucine (Leu) or phenylalanine (Phe); (d) Xaa4 is threonine (Thr) or serine (Ser); (e) Xaa5 is glycine (Gly) or arginine (Arg); (f) Xaa6 is serine (Ser) or alanine (Ala); (g) Xaa7 is proline (Pro) or phenylalanine (Phe); (h) Xaa8 is lysine (Lys) or asparagine (Asn); (i) Xaa9 is glycine (Gly) or lysine (Lys); (j) Xaa10 is serine (Ser) or threonine (Thr), (k) Xaa11 is valine (Val) or arginine (Arg); (l) Xaa12 is threonine (Thr) or valine (Val); (m) Xaa13 is tyrosine (Tyr) or phenylalanine (Phe), and (n) Xaa14 is alanine (Ala) or absent; An isolated immunoglobulin light chain polypeptide.

7. If the polypeptide is Xaa1 Val Gln Xaa2 Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Asn Trp Ile Arg Gln Xaa4 Pro Gly Xaa5 Xaa6 Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Asp Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Xaa7 Xaa8 Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Xaa9 Cys Ala Ile Arg Gly Pro Tyr Ser Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 25); (a) Xaa1 is glutamine (Gln) or aspartic acid (Asp); (b) Xaa2 is leucine (Leu) or phenylalanine (Phe); (c) Xaa3 is threonine (Thr) or serine (Ser); (d) Xaa4 is proline (Pro) or phenylalanine (Phe); (e) Xaa5 is lysine (Lys) or asparagine (Asn); (f) Xaa6 is glycine (Gly) or lysine (Lys); (g) Xaa7 is serine (Ser) or threonine (Thr); (h) Xaa8 is valine (Val) or arginine (Arg), and (i) Xaa9 is tyrosine (Tyr) or phenylalanine (Phe); 2. The isolated immunoglobulin heavy chain polypeptide of claim 1.

8. Any one amino acid of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, or SEQ ID NO:54 7. The isolated immunoglobulin heavy chain polypeptide of claim 6, comprising the sequence:

9. An isolated immunoglobulin heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:

35.

10. 1. An isolated immunoglobulin light chain polypeptide, comprising: Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Xaa1 Asn Thr Tyr Leu Tyr Trp Xaa2 Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Xaa3 Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys (SEQ ID NO: 36), (a) Xaa1 is glycine (Gly) or alanine (Ala); (b) Xaa2 is phenylalanine (Phe) or tyrosine (Tyr), and (c) Xaa3 is tyrosine (Tyr) or serine (Ser); An isolated immunoglobulin light chain polypeptide.

11. 11. The isolated immunoglobulin light chain polypeptide of claim 10, comprising the amino acid sequence of SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:

39.

12. 1. An isolated immunoglobulin light chain polypeptide, comprising: Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Xaa1 Asn Xaa2 Ile Thr Tyr Phe Tyr Trp Tyr Leu Xaa3 Lys Pro Gly Gln Pro Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys (SEQ ID NO: 40), (a) Xaa1 is serine (Ser) or arginine (Arg); (b) Xaa2 is glycine (Gly) or alanine (Ala), and (c) Xaa3 is glutamine (Gln) or histidine (His); An isolated immunoglobulin light chain polypeptide.

13. 13. The isolated immunoglobulin light chain polypeptide of claim 12, comprising the amino acid sequence of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:

44.

14. 1. An isolated immunoglobulin light chain polypeptide, comprising: Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Xaal Ile Asn Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Xaa2 Leu His Ser Gly Val Pro Ser Arg Phe Ser Xaa3 Ser Gly Ser Gly Xaa4 Asp Xaa5 Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Xaa6 Xaa7 (SEQ ID NO: 58), (a) Xaa1 is aspartic acid (Asp) or tryptophan (Trp); (b) Xaa2 is arginine (Arg) or methionine (Met); (c) Xaa3 is glycine (Gly), serine (Ser), or proline (Pro); (d) Xaa4 is threonine (Thr) or asparagine (Asn); (e) Xaa5 is phenylalanine (Phe) or tyrosine (Tyr); (f) Xaa6 is arginine (Arg) or absent, and (g) Xaa7 is threonine (Thr) or absent; An isolated immunoglobulin light chain polypeptide.

15. The polypeptide is Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Xaa1 Ser Gly Ser Gly Thr It comprises the amino acid sequence of Asp (a) Xaa1 is serine (Ser) or proline (Pro), and (b) Xaa2 is phenylalanine (Phe) or tyrosine (Tyr); 15. The isolated immunoglobulin light chain polypeptide of claim 14.

16. 16. The isolated immunoglobulin light chain polypeptide of claim 15, comprising the amino acid sequence of SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO:

55.

17. An isolated immunoglobulin light chain polypeptide comprising the amino acid sequence of SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:

50.

18. An isolated nucleic acid sequence encoding an immunoglobulin heavy chain polypeptide according to any one of claims 1 to 9.

19. 18. An isolated nucleic acid sequence encoding an immunoglobulin light chain polypeptide according to any one of claims 10 to 17.

20. 20. A vector comprising the isolated nucleic acid sequence of claim 18 or 19.

21. An interleukin-36 receptor (IL-36R) binding agent that exhibits one or more of the following biological activities: (a) inhibiting the interaction between IL-36R and IL-36α, IL-36β, and / or IL-36γ; (b) inhibiting intracellular signaling mediated by IL-36R; (c) Cross-reacting with human IL-36R, cynomolgus monkey IL-36R, and non-human primate IL-36R and its activity It inhibits sex.

22. An interleukin-36 receptor (IL-36R) binding agent comprising an immunoglobulin heavy chain polypeptide according to any one of claims 1 to 9 and / or an immunoglobulin light chain polypeptide according to any one of claims 10 to 17.

23. 23. The IL-36R-binding agent of claim 21 or claim 22, comprising an immunoglobulin heavy chain polypeptide of any one of claims 1 to 9 and an immunoglobulin light chain polypeptide of any one of claims 10 to 17.

24. Claim 21 or claim 22, comprising an immunoglobulin heavy chain polypeptide according to any one of claims 1 to 9 or an immunoglobulin light chain polypeptide according to any one of claims 10 to 17.

1. An IL-36R-binding agent according to claim 1.

25. 25. The IL-36R-binding agent of any one of claims 21 to 24, which is an antibody, an antibody conjugate, or an antigen-binding fragment thereof.

26. F(ab') 2 fragment, Fab' fragment, Fab fragment, Fv fragment, scFv fragment, dsFv fragment, dAb fragment, or single chain binding polypeptide.

23. The IL-36R-binding agent of claim 22, wherein

27. 27. An IL-36R-binding agent that competes for binding to IL-36R with the IL-36R-binding agent of any one of claims 18 to 26.

28. 28. An isolated nucleic acid sequence encoding the IL-36R-binding agent of any one of claims 21 to 27.

29. 29. A vector comprising the isolated nucleic acid sequence of claim 28.

30. 30. An isolated cell comprising the vector of claim 29.

31. (a) the IL-36R-binding agent of any one of claims 21 to 27 or the vector of claim 29 and (b) a pharmaceutically acceptable carrier.

32. 32. A method for treating a disorder responsive to inhibition of IL-36R in a mammal, the method comprising administering to a mammal having a disorder responsive to inhibition of IL-36R an effective amount of the composition of claim 31, thereby treating the disorder in the mammal.

33. 33. The method of claim 32, wherein the disorder is an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, an epithelial-mediated inflammatory disorder, fibrosis, or cancer.

34. 34. The method of claim 33, wherein the disorder is psoriasis vulgaris, pustular psoriasis, generalized pustular psoriasis (GPP), palmoplantar pustulosis (PPP), inflammatory bowel disease, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, COPD, scleroderma, asthma, and ankylosing spondylitis.

35. 35. The method of any one of claims 32 to 34, wherein the half-life of the IL-36R-binding agent in a mammal is between 30 minutes and 45 days.

36. The IL-36R binding agent has a KD between about 1 picomolar (pM) and about 100 micromolar (μM). The method of any one of claims 32 to 35, wherein the antibody binds to IL-36R at

37. 32. Use of the composition of claim 31 for the preparation of a medicament for the treatment of a disorder responsive to the inhibition of IL-36R in a mammal.

38. 38. The use of claim 37, wherein the disorder is an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, an epithelial-mediated inflammatory disorder, fibrosis, or cancer.

39. 38. The use according to claim 37, wherein the disorder is psoriasis vulgaris, pustular psoriasis, generalized pustular psoriasis (GPP), palmoplantar pustulosis (PPP), inflammatory bowel disease, psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, COPD, scleroderma, asthma and ankylosing spondylitis.

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