Antibody composition

Abstract

The present invention relates to a stable liquid aqueous pharmaceutical formulation comprising an anti-interleukin-36 receptor (IL-36R) antibody or antigen-binding antibody fragment; a buffer; a stabilizer; and a surfactant, and to a method of treating a subject with said pharmaceutical formulation.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 187,476, filed May 12, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] Incorporation by Reference of Electronically Submitted Documents The computer-readable nucleotide / amino acid sequence listing, submitted contemporaneously herewith and identified as follows, is incorporated by reference in its entirety herein: 76,894 byte ASCII (text) file entitled "762991_ST25.TXT", dated May 12, 2022. [Background technology]

[0003] Formulations or compositions containing molecules such as antibodies require specific formulations to render the formulation suitable for administration to patients, as well as stability during transportation, storage, and use. Antibody formulations can be adversely affected by denaturation, oxidation, and aggregation, especially when high concentrations of the antibody are desired, which often occurs when the antibody formulation is used for pharmaceutical or therapeutic purposes. There is a need to provide stable liquid, aqueous pharmaceutical formulations of antibodies, particularly IL-36 antibodies, to help meet the medical needs of patients suffering from diseases mediated by interleukin-36 (IL-36) and its receptor. Summary of the Invention

[0004] In one embodiment, the invention provides a stable liquid pharmaceutical formulation comprising an antibody or antigen-binding antibody fragment, a buffer, a stabilizer comprising proline and / or sorbitol, and a non-ionic surfactant. Related compositions and methods of use thereof are also provided. [Brief description of the drawings]

[0005] [Figure 1A]FIG. 1A shows images of the appearance of 1 mL of sample F01 compared to water (leftmost vial) on days 0, 5, 6, 7, and 8 when stored at 45° C. [Figure 1B] FIG. 1B shows images of the appearance of 1 mL sample F02A compared to water (leftmost vial) on days 0, 5, 6, 7, and 8 when stored at 45° C. [Figure 1C] FIG. 1C shows images of the appearance of 1 mL sample F02B compared to water (leftmost vial) on days 0, 5, 6, 7, and 8 when stored at 45° C. [Figure 1D] FIG. 1D shows images of the appearance of 1 mL of sample F02C compared to water (leftmost vial) on days 0, 5, 6, 6, and 8 when stored at 45° C. [Diagram 2] Figure 2 is a plot of turbidity by A350 (AU) versus time (days) for IL-36R formulations F01, F02A, F02B, and F02C stored at 45° C. Lines represent the linear model for each formulation. [Diagram 3] Figure 3 is a plot of purity (% major component) by SEC-HPLC versus time (days) for IL-36R formulations F01, F02A, F02B, and F02C stored at 45° C. Lines represent the linear model for each formulation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Provided herein is a stable liquid aqueous pharmaceutical formulation or composition comprising water; an antibody or antigen-binding antibody fragment; a buffer; a stabilizer comprising proline and / or sorbitol; and a non-ionic surfactant. As used herein, the terms "formulation" and "composition" (e.g., "pharmaceutical formulation" or "pharmaceutical composition") have the same meaning and are used interchangeably.

[0007] Buffers and pH Any suitable buffer may be used, but in some embodiments, the buffer is a histidine buffer. The buffer may be used at a concentration suitable to maintain the desired pH, generally about 5.0 to about 6.5 (e.g., about 5.5 to 6.2 or about 5.8 to 6.0). In some embodiments, the formulation may have a pH of about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5. In some embodiments, the pH is 6.0±0.4, 6.0±0.3, 6.0±0.2, 6.0±0.1, about 6.0, or 6.0.

[0008] In some embodiments, the buffer is a histidine buffer and histidine is present in the formulation at a concentration of about 5-35 mM (e.g., about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, or a range defined by any two of the foregoing values), or about 5-20 mM (e.g., about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, or a range defined by any two of the foregoing values). In some embodiments, the pharmaceutical formulation comprises about 7-25 mM histidine, e.g., about 7-15 mM histidine; or about 10-25 mM histidine, e.g., about 10-15 mM histidine. In some embodiments, the pharmaceutical formulation comprises about 9-11 mM histidine (e.g., about 10 mM histidine) or about 24-26 mM histidine (e.g., about 25 mM histidine).

[0009] Stabilizer In another embodiment, the pharmaceutical formulation further comprises a stabilizer comprising, consisting essentially of, or consisting of proline and / or sorbitol. The stabilizer may have any suitable concentration of proline and / or sorbitol. In some embodiments, the stabilizer comprises, consists essentially of, or consists of proline. In some embodiments, proline is present in the formulation at a concentration of about 100 to about 300 mM (e.g., about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, or a range defined by any two of the foregoing values), such as about 270 mM to about 300 mM (e.g., about 270 mM, about 275 mM, about 280 mM, about 285 mM, about 290 mM, about 295 mM, about 300 mM, or a range defined by any two of the foregoing values), or about 275 to 285 mM. In other embodiments, the stabilizer comprises, consists essentially of, or consists of sorbitol. In some embodiments, sorbitol is present in the formulation at a concentration of about 100 to about 300 mM (e.g., about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, or a range defined by any two of the foregoing values), e.g., about 270 mM to about 300 mM (e.g., about 270 mM, about 275 mM, about 280 mM, about 285 mM, about 290 mM, about 295 mM, about 300 mM, or a range defined by any two of the foregoing values). In yet other embodiments, the stabilizer comprises, consists essentially of, or consists of proline and sorbitol. In some embodiments, the total concentration of sorbitol and proline in the formulation is about 100 to about 300 mM (e.g., about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, or a range defined by any two of the foregoing values), for example, about 270 mM to about 300 mM (e.g., about 270 mM, about 275 mM, about 280 mM, about 285 mM, about 290 mM, about 295 mM, about 300 mM, or a range defined by any two of the foregoing values).For example, the formulation can contain about 50-80 mM sorbitol and about 180-250 mM proline (e.g., about 65-75 mM sorbitol or about 70 mM sorbitol) and about 205-215 mM proline (e.g., about 210 mM proline).

[0010] Surfactants The stable liquid aqueous pharmaceutical formulation also comprises a non-ionic surfactant. In one embodiment, the surfactant is a polysorbate. In a preferred embodiment, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, and mixtures thereof, and is preferably selected from polysorbate 20 or polysorbate 80, or mixtures thereof. In a more preferred embodiment, the surfactant is polysorbate 20.

[0011] In certain embodiments, the surfactant is present at a concentration of about 0.01% to about 0.1% (e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, or a range defined by any two of the foregoing values), such as about 0.01% to about 0.05% or about 0.03% to about 0.07% (e.g., about 0.01%, about 0.015%, about 0.02%, about 0.025%, about 0.03%, about 0.035%, about 0.04%, about 0.045%, about 0.05%, about 0.07%, or a range defined by any two of the foregoing values).

[0012] viscosity The formulation may have any viscosity suitable for the intended use (e.g., parenteral injection, such as subcutaneous injection). In one embodiment, the viscosity of the pharmaceutical formulation is about 15 centipoise ("cps" or "cP") or less. In another embodiment, the viscosity of the pharmaceutical formulation is about 1 cP to about 18 cP (e.g., about 1 cP, about 2 cP, about 3 cP, about 4 cP, about 5 cP, about 6 cP, about 7 cP, about 8 cP, about 9 cP, about 10 cP, about 11 cP, about 12 cP, about 13 cP, about 14 cP, about 15 cP, about 16 cP, about 17 cP, about 18 cP, or a range defined by any two of the foregoing values).

[0013] Osmolarity The formulation may have any suitable osmolarity. In one embodiment, the osmolarity of the pharmaceutical formulation is about 250 mOsm / kg to about 450 mOsm / kg (e.g., about 250 mOsm / kg, about 275 mOsm / kg, about 300 mOsm / kg, about 325 mOsm / kg, about 350 mOsm / kg, about 375 mOsm / kg, about 400 mOsm / kg, about 425 mOsm / kg, about 450 mOsm / kg, or a range defined by any two of the foregoing values). In certain embodiments, the osmolality of the pharmaceutical formulation is about 300 mOsm / kg to about 400 mOsm / kg (e.g., about 300 mOsm / kg, about 310 mOsm / kg, about 320 mOsm / kg, about 330 mOsm / g, about 340 mOsm / kg, about 350 mOsm / kg, about 360 mOsm / kg, about 370 mOsm / kg, about 380 mOsm / kg, about 390 mOsm / kg, about 400 mOsm / kg, or a range defined by any two of the foregoing values).

[0014] In some embodiments, the formulation comprises less than 10 mM (e.g., less than 5 mM, less than 1 mM, or less than 0.5 mM) sodium chloride (NaCl), or is substantially or completely free of NaCl. In some embodiments, the formulation comprises less than 10 mM (e.g., less than 5 mM, less than 1 mM, or less than 0.5 mM) of any tonicity agent, or is substantially or completely free of any tonicity agent. In some embodiments, the formulation consists essentially of or consists of an antibody or antigen-binding antibody fragment, a buffer, a stabilizer, a non-ionic surfactant, and water, as described herein.

[0015] Antibody or antibody fragment The stable liquid, aqueous pharmaceutical formulation of the present invention may comprise any antibody or antigen-binding antibody fragment. In some embodiments, the formulation comprises an anti-IL-36R antibody or antigen-binding antibody fragment, particularly an antibody as described in detail herein.

[0016] In embodiments, the IL-36R antibody is an antibody or antigen-binding antibody fragment that comprises, consists of, or consists essentially of an immunoglobulin heavy chain polypeptide and an immunoglobulin light chain polypeptide, or at least the variable regions thereof (e.g., antigen-binding fragments).

[0017] 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-terminal variable (V H ) region and three C-terminal constant (C H 1. C H 2, and C H 3) region, and each light chain comprises one N-terminal variable (V L ) region and one C-terminal constant (C L) region. The light chain of an antibody can be assigned to one of two different types, either kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain. In a typical immunoglobulin, each light chain is linked to a heavy chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region aligns with the variable region of the heavy chain, and the light chain constant region aligns with the first constant region of the heavy chain. The remaining constant regions of the heavy chains align with each other.

[0018] The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. H Area and V L The regions have the same general structure, and each region contains four framework (FW or FR) regions. As used herein, the term "framework region" refers to a relatively conserved amino acid sequence in the variable region located between the hypervariable or complementarity determining regions (CDRs). There are four framework regions in each variable domain, which are called FR1, FR2, FR3, and FR4. The framework regions form a β-sheet that provides the structural framework of the variable region (see, for example, 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 the antibody, which is responsible for antigen binding. The CDR regions can also be referred to using "H" or "L" in nomenclature to represent the heavy or light chain, respectively, i.e., CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3. The CDRs of a given Ig sequence can be determined by any of several conventional numbering schemes, such as Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo (see, e.g., Kabat, et al., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, NIH (1991); Chothia, et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol., 196:901-917 (1987); Al-Lazikani et al., Standard Conformations for the Canonical Structures of Immunoglobulins, J. Mol. Biol., 273:927-948 (1997); Abhinandan et al., Analysis and Improvements to Kabat and Structurally Correct Numbering of Antibody Variables, 1999). Domains,Mol.Immunol.,45:3832-3839(2008);Lefranc et al.,The IMGT unique numbering for immunoglobulins,T cell Receptors and Ig-like domains,The Immunologist,7:132-136(1999);Lefranc et al., See IMGT unique numbering for immunoglobulin and T cell receptor variable domains and I superfamily V-like domains, Dev. Comp. Immunol., 27:55-77 (2003); and Honegger et al., Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool, J. Mol. Biol. 309:657-670 (2001).

[0020] In some embodiments, the immunoglobulin heavy chain variable region comprises: Gln Val Gln Xaa1 Xaa2 Gln Ser Gly Ala Glu Val 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 Trp Gly Gln Gly Thr Val Thr Val (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); and (e) Xaa5 is glycine (Gly), serine (Ser), 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).In some embodiments, the immunoglobulin heavy chain polypeptide has the amino acid sequence 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 Xaa8 Cys Thr Arg Ser Phe Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser (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).

[0021] The heavy chain polypeptide may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO:56 or SEQ ID NO:1, with any one of the foregoing amino acid substitutions in any suitable combination, or at least the CDRs thereof. In one embodiment, the immunoglobulin heavy chain polypeptide comprises, consists of, or consists essentially of the amino acid sequence of any one of 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, or SEQ ID NO:14, or at least the CDRs thereof.

[0022] In some embodiments, the antibody or antibody fragment of IL-36R comprises a CDR1 (HCDR1) of a heavy chain variable region comprising, consisting of, or consisting essentially of the amino acid sequence of Phe Thr Phe Thr Ser Tyr Asp Ile Asn (SEQ ID NO: 59); (a) Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys Gly (SEQ ID NO: 60); (b) Trp Ile Tyr Pro Gly Asp Ser Ser Thr Lys Tyr Asn Glu Lys Phe Lys Gly (SEQ ID NO: 61); or (c) a CDR2 (HCDR2) of a heavy chain variable region comprising, consisting of, or consisting essentially of the amino acid sequence of Trp Ile Tyr Pro Gly Asp Ala Ser Thr Lys Tyr Asn Glu Lys Phe Lys Gly (SEQ ID NO: 62); and / or an amino acid sequence of Ser Phe Tyr Thr Met Asp The CDR3 (HCDR3) of the heavy chain variable region comprises, consists of, or consists essentially of Tyr (SEQ ID NO: 63).

[0023] In some embodiments, the immunoglobulin heavy chain variable region has the amino acid sequence 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 (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).

[0024] The heavy chain variable region may comprise, consist or consist essentially of the amino acid sequence of SEQ ID NO: 15 or at least the CDRs thereof, with one of the foregoing amino acid substitutions in any suitable combination. In one embodiment, the immunoglobulin heavy chain polypeptide comprises, consists or consists essentially of 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, or at least the CDRs thereof.

[0025] In one embodiment, the antibody or antibody fragment of IL-36 comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of: (a) Tyr Thr Phe Thr Asn Tyr Trp Met His (SEQ ID NO: 64); (b) Tyr Thr Phe Thr Asn Tyr Trp Met Asn (SEQ ID NO: 65); (c) Tyr Thr Phe Thr Asn Tyr Trp Met Tyr (SEQ ID NO: 66); and (d) Tyr Thr Phe Thr Asn Tyr Tyr Met Asn (SEQ ID NO: 67); (a) Met Phe Asp Pro Ser Asn Ser Val Thr Arg Leu Asn Gln Lys Phe Lys Asp (SEQ ID NO: 68); (b) Met Phe Glu Pro Ser Asn Ala Val Thr Arg Leu Asn Gln Lys Phe Lys Asp (SEQ ID NO: 69); (c) Met Phe His Pro Ser Asn Ala Val Thr Arg Leu Asn Gln Lys and (d) an HCDR2 comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of Met Phe His Pro Thr Gly Asp Val Thr Arg Leu Asn Gln Lys Phe Lys Asp (SEQ ID NO: 71); and / or an HCDR3 comprising, consisting of, or consisting essentially of the amino acid sequence Thr Thr Ser Met Ile Ile Gly Gly Phe Ala Tyr (SEQ ID NO: 72).

[0026] In a further embodiment, the immunoglobulin heavy chain variable region comprises: 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 The amino acid sequence of Gly Thr Leu Val Thr Val Ser Ser Xaa14 (SEQ ID NO:57), or at least its CDRs, (wherein 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); Xaa5 is glycine (Gly) or arginine (Arg); 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 phenylalanine (Phe);Xaa14 comprises, consists of, or consists essentially of alanine (Ala) or is absent. In some embodiments, the heavy chain variable region has the amino acid sequence Xaa1 Val Gln Xaa2 Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val 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 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), and (e) Xaa5 is lysine (Lys) or phenylalanine (Phe). (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);

[0027] In some embodiments, the heavy chain variable region may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:25, or at least the CDRs thereof, with one or more of the foregoing amino acid substitutions in any suitable combination. In one embodiment, the immunoglobulin heavy chain variable region comprises, consists of, or consists essentially of 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, or at least the CDRs thereof.

[0028] In additional embodiments, an antibody or antibody fragment of IL-36 may comprise an HCDR1 comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of: (a) Tyr Ser Ile Thr Ser Asp Phe Ala Trp Asn (SEQ ID NO: 73); and (b) Tyr Ser Ile Thr Ala Asp Phe Ala Trp Asn (SEQ ID NO: 74); an HCDR2 comprising, consisting of, or consisting essentially of the amino acid sequence Tyr Ile Ser Tyr Ser Gly Asp Thr Asn Tyr Asn Pro Ser Leu Lys Ser (SEQ ID NO: 75); and / or an HCDR3 comprising, consisting of, or consisting essentially of the amino acid sequence Arg Gly Pro Tyr Ser Phe Thr Tyr (SEQ ID NO: 76).

[0029] In another embodiment, the antibody or antibody fragment of IL-36 comprises an 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, or at least the CDRs thereof.

[0030] In some embodiments, the immunoglobulin heavy chain polypeptide comprises an amino 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 of the aforementioned variable region sequences. As described herein, the "identity" of a nucleic acid or amino acid sequence can be determined by comparing a subject nucleic acid or amino acid sequence to 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 a subject sequence and a reference sequence, divided by the length of the longest sequence (i.e., the length of the subject sequence or the reference sequence, whichever is longer). Many mathematical algorithms for obtaining optimal alignments and calculating the identity between two or more sequences 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., BLAST2.1, BL2SEQ, and newer versions thereof), and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searching).Further, sequence alignment algorithms are described, for example, in 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 This is also disclosed in the UK (1997).

[0031] In addition to the heavy chain variable regions described herein, antibodies or antibody fragments of IL-36R may comprise the heavy chain variable regions of the following amino acid sequence: 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 (b) Xaa2 is phenylalanine (Phe) or tyrosine (Tyr); and (c) Xaa3 is tyrosine (Tyr) or serine (Ser).

[0032] The light chain variable region may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 36, or at least the CDRs thereof, with one or more of the foregoing amino acid substitutions in any suitable combination. In one embodiment, the isolated immunoglobulin light chain variable region comprises, consists of, or consists essentially of the amino acid sequence of any one of SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39, or at least the CDRs thereof.

[0033] In some embodiments, an IL-36R-binding agent comprises a light chain variable region CDR1 (LCDR1) comprising, consisting of, or consisting essentially of an amino acid sequence selected from: (a) Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Tyr (SEQ ID NO: 77); or (b) Arg Ser Ser Lys Ser Leu Leu His Ser Asn Ala Asn Thr Tyr Leu Tyr (SEQ ID NO: 78); a light chain variable region CDR2 (LCDR2) comprising, consisting of, or consisting essentially of the amino acid sequence Arg Met Ser Asn Leu Ala Ser (SEQ ID NO: 79); and a light chain variable region CDR3 (LCDR3) comprising, consisting of, or consisting essentially of the amino acid sequence Met Gln His Leu Glu Tyr Pro Phe Thr (SEQ ID NO: 80).

[0034] In some embodiments, the immunoglobulin light chain variable region has the amino acid sequence 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 (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).

[0035] The light chain variable region may comprise, consist, or consist essentially of the amino acid sequence of SEQ ID NO:40 or at least the CDRs thereof, with any combination of the amino acid substitutions described above. In one embodiment, the immunoglobulin light chain polypeptide comprises, consists, or consists essentially of the amino acid sequence of any one of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:44; or at least the CDRs thereof.

[0036] In some embodiments, the light chain variable region comprises an LCDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of (a) Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Phe Tyr (SEQ ID NO: 81); (b) Arg Ser Ser Lys Ser Leu Leu His Ser Asn Ala Ile Thr Tyr Phe Tyr (SEQ ID NO: 82); or (c) Arg Ser Ser Lys Ser Leu Leu His Arg Asn Ala Ile Thr Tyr Phe Tyr (SEQ ID NO: 83); an LCDR2 comprising, consisting of, or consisting essentially of the amino acid sequence Gln Met Ser Asn Leu Ala Ser (SEQ ID NO: 84); and an LCDR3 comprising, consisting of, or consisting essentially of the amino acid sequence Ala Gln Asn Leu Glu Leu Pro Leu Thr (SEQ ID NO: 85).

[0037] In a further embodiment, the immunoglobulin light chain variable region comprises the amino acid sequence of 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 Xaa1 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 The amino acid sequence of Xaa7 (SEQ ID NO:58) or at least its CDRs (wherein (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)In some embodiments, the immunoglobulin light chain variable region has the amino acid sequence 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) or at least its CDRs, wherein (a) Xaa1 is serine (Ser) or proline (Pro); and (b) Xaa2 is phenylalanine (Phe) or tyrosine (Tyr).

[0038] The light chain variable region may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO:58 or SEQ ID NO:45, or at least the CDRs thereof, with one or more of the foregoing amino acid substitutions in any suitable combination. In one embodiment, the immunoglobulin light chain polypeptide comprises, consists of, or consists essentially of the amino acid sequence of any one of SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:55, or at least the CDRs thereof.

[0039] In some embodiments, the light chain variable region comprises an LCDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of (a) Arg Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn (SEQ ID NO: 86); or (b) Arg Ala Ser Gln Trp Ile Asn Asn Tyr Leu Asn (SEQ ID NO: 87); an LCDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of (a) Tyr Thr Ser Arg Leu His Ser (SEQ ID NO: 88); or (b) Tyr Thr Ser Met Leu His Ser (SEQ ID NO: 89); and an LCDR3 comprising, consisting of, or consisting essentially of the amino acid sequence Gln Gln Gly His Thr Leu Pro Trp Thr (SEQ ID NO: 90).

[0040] In another embodiment, the immunoglobulin light chain variable region comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:48, SEQ ID NO:49, or SEQ ID NO:50, or at least the CDRs thereof.

[0041] In some embodiments, the immunoglobulin light chain variable region comprises an amino 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 of the foregoing immunoglobulin light chain variable region sequences. Nucleic acid or amino acid sequence "identity" can be determined using the methods described herein.

[0042] As described above, an antibody or antibody fragment of IL-36R may comprise any of the heavy and light chain variable region sequences described above or an immunoglobulin heavy and light chain variable region having its CDRs. The CDR sequences may be those described herein or those determined using any of several known methods (e.g., Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo).

[0043] In one embodiment, the IL-36R antibody or antibody fragment has an immunoglobulin heavy chain variable region comprising SEQ ID NO: 15 or SEQ ID NO: 22, or at least the CDR regions thereof; and an immunoglobulin light chain variable region comprising SEQ ID NO: 40 or SEQ ID NO: 44, or at least the CDR sequences thereof, wherein the CDRs are as determined according to any of the various known immunoglobulin numbering schemes, in particular according to Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo. For example, in some embodiments, the antibody or antibody fragment comprises a heavy chain variable region of SEQ ID NO: 22 and a light chain variable region of SEQ ID NO: 44, or at least the CDRs thereof as determined by Kabat. In some embodiments, the antibody or antibody fragment comprises a heavy chain variable region of SEQ ID NO: 22 and a light chain variable region of SEQ ID NO: 44, or at least the CDRs thereof as determined by Chothia. In some embodiments, the antibody or antibody fragment comprises a heavy chain variable region of SEQ ID NO: 22 and a light chain variable region of SEQ ID NO: 44, or at least the CDRs thereof as determined by Martin. In some embodiments, the antibody or antibody fragment comprises a heavy chain variable region of SEQ ID NO: 22 and a light chain variable region of SEQ ID NO: 44, or at least the CDRs thereof as determined by IGMT. In some embodiments, the antibody or antibody fragment comprises a heavy chain variable region of SEQ ID NO: 22 and a light chain variable region of SEQ ID NO: 44, or at least the CDRs thereof as determined by AHo.

[0044] In some embodiments, the antibody or antibody fragment of IL-36R comprises, consists of, or consists essentially of an amino acid sequence selected from the group consisting of: (a) Tyr Thr Phe Thr Asn Tyr Trp Met His (SEQ ID NO: 64); (b) Tyr Thr Phe Thr Asn Tyr Trp Met Asn (SEQ ID NO: 65); (c) Tyr Thr Phe Thr Asn Tyr Trp Met Tyr (SEQ ID NO: 66); and (d) Tyr Thr Phe Thr Asn Tyr Tyr Met Asn (SEQ ID NO: 67); (a) Met Phe Asp Pro Ser Asn Ser Val Thr Arg Leu Asn Gln Lys Phe Lys Asp (SEQ ID NO: 68); (b) Met Phe Glu Pro Ser Asn Ala Val Thr Arg Leu Asn Gln Lys Phe Lys Asp (SEQ ID NO: 69); (c) Met Phe His Pro Ser Asn Ala Val Thr Arg Leu Asn Gln and (d) an HCDR2 comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of Met Phe His Pro Thr Gly Asp Val Thr Arg Leu Asn Gln Lys Phe Lys Asp (SEQ ID NO: 71); and / or an HCDR3 comprising, consisting of, or consisting essentially of the amino acid sequence Thr Thr Ser Met Ile Ile Gly Gly Phe Ala Tyr (SEQ ID NO: 72); and LCDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of Tyr (SEQ ID NO: 83);a light chain variable region comprising an LCDR2 comprising, consisting of, or consisting essentially of the amino acid sequence Gln Met Ser Asn Leu Ala Ser (SEQ ID NO: 84); and an LCDR3 comprising, consisting of, or consisting essentially of the amino acid sequence Ala Gln Asn Leu Glu Leu Pro Leu Thr (SEQ ID NO: 85);

[0045] In certain embodiments, the antibody or antibody fragment of IL-36R comprises a heavy chain variable region comprising an HCDR1 comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of Tyr Thr Phe Thr Asn Tyr Trp Met Asn (SEQ ID NO: 65); an HCDR2 comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of Met Phe His Pro Thr Gly Asp Val Thr Arg Leu Asn Gln Lys Phe Lys Asp (SEQ ID NO: 71); and / or an HCDR3 comprising, consisting of, or consisting essentially of the amino acid sequence Thr Thr Ser Met Ile Ile Gly Gly Phe Ala Tyr (SEQ ID NO: 72); and an LCDR1 comprising, consisting of, or consisting essentially of the amino acid sequence Arg Ser Ser Lys Ser Leu Leu His Arg Asn Ala Ile Thr Tyr Phe Tyr (SEQ ID NO: 83); and a LCDR3 comprising, consisting of, or consisting essentially of the amino acid sequence Ala Gln Asn Leu Glu Leu Pro Leu Thr (SEQ ID NO: 85).

[0046] Furthermore, an antibody or antibody fragment of IL-36R may comprise immunoglobulin heavy and light chain variable regions that have a specified percent identity to the heavy and light chain variable region sequences, e.g., are 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). In some embodiments, the diversity in sequence occurs outside the CDRs (as determined by any known method, including Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo), such that heavy and light chain sequences that have a specified sequence identity to the specified sequences described herein retain the CDRs of such sequences. In embodiments, the antibody or antibody fragment of IL-36R comprises an immunoglobulin heavy chain variable region 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 SEQ ID NO:15 or SEQ ID NO:22, and optionally, the sequence retains the CDRs of SEQ ID NO:15 or SEQ ID NO:22; the antibody or antibody fragment comprises an immunoglobulin heavy chain variable region 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 SEQ ID NO:15 or SEQ ID NO:22, and optionally, the sequence retains the CDRs of SEQ ID NO:15 or SEQ ID NO:22; and optionally, the sequence retains the CDRs of SEQ ID NO:40 or SEQ ID NO:44, wherein the CDRs are as determined according to any of the various known immunoglobulin numbering schemes, in particular according to Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo.In certain embodiments, the antibody or antibody fragment of IL-36R comprises an immunoglobulin heavy chain variable region 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 SEQ ID NO:22, optionally wherein the sequence retains the CDRs of SEQ ID NO:22; the antibody or antibody fragment comprises an immunoglobulin heavy chain variable region 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 SEQ ID NO:44, optionally wherein the CDRs retain the CDRs of SEQ ID NO:44, according to any of a variety of known immunoglobulin numbering schemes, in particular according to Kabat, Chothia, Martin (Enhanced Chothia), IGMT, or AHo.

[0047] Diversity in sequence identity can be achieved by the addition, substitution, or deletion of one or more amino acid residues. Amino acid "replacement" or "substitution" refers to the replacement of one amino acid at a given position or residue with another amino acid at the same position or residue in a polypeptide sequence. Amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative, depending on whether the replacement is with an amino acid residue that has similar properties to the replaced residue. A functional method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analysis, groups of amino acids can be defined as those amino acids within the group that are preferentially exchanged with each other and are therefore most similar to each other in their effect on the overall protein structure (Schulz and Schirmer, supra).

[0048] Amino acids may be broadly classified as "aromatic" or "aliphatic". 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).

[0049] The aliphatic amino acids can be subdivided into four subgroups: the "large aliphatic non-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; and the "small residue subgroup" consists of glycine and alanine. The group of charged / polar amino acids can be subdivided into three subgroups: the "positively charged subgroup" consisting of lysine and arginine, the "negatively charged subgroup" consisting of glutamic acid and aspartic acid, and the "polar subgroup" consisting of asparagine and glutamine.

[0050] The aromatic amino acids can be subdivided into two subgroups: the "nitrogen ring subgroup", consisting of histidine and tryptophan, and the "phenyl subgroup", consisting of phenylalanine and tyrosine.

[0051] Examples of conservative amino acid substitutions include substitutions of amino acids within the above subgroups, such as substitutions of arginine with lysine (or vice versa) so that a positive charge can be maintained, substitutions of aspartic acid with glutamic acid (or vice versa) so that a negative charge can be maintained, substitutions of threonine with serine so that a free -OH can be maintained, and substitutions of asparagine with glutamine so that a free -NH2 can be maintained. "Semi-conservative mutations" include amino acid substitutions of amino acids within the same group listed herein but not within the same subgroup. For example, substitutions of asparagine with aspartic acid or lysine with asparagine involve amino acids within the same group but from different subgroups. "Non-conservative mutations" include amino acid substitutions between different groups, such as substitutions of tryptophan with lysine or serine with phenylalanine, etc.

[0052] When an immunoglobulin light or heavy chain variable region "consists essentially of" any of the foregoing heavy or light chain variable region amino acid sequences, it may contain additional elements that do not significantly affect the polypeptide, such as those described herein. When an immunoglobulin light or heavy chain variable region "consists of," the polypeptide does not include any additional elements.

[0053] An IL-36R antibody or antibody fragment can be a binding agent that competes with an IL-36R binding agent comprising an immunoglobulin heavy or light chain polypeptide described herein for binding to IL-36R, e.g., binds to the same epitope or an overlapping epitope. Antibody competition can be assayed using routine peptide competition assays utilizing ELISA, Western blot, or immunohistochemical methods (e.g., U.S. Pat. Nos. 4,828,981 and 8,568,992; and Braitbard et al., Proteome Sci., 4:12 (2006)).

[0054] The amount of antibody or antigen-binding fragment thereof contained within the pharmaceutical formulation of the present invention can vary depending on the particular properties desired for the formulation, as well as the particular context and purpose for which the formulation is intended to be used. In certain embodiments, the pharmaceutical formulation has a concentration of about 75 mg / mL to about 175 mg / mL (e.g., about 75 mg / mL, about 85 mg / mL, about 95 mg / mL, about 105 mg / mL, about 115 mg / mL, about 125 mg / mL, about 135 mg / mL, about 145 mg / mL, about 155 mg / mL, about 165 mg / mL, about 175 mg / mL, or a range defined by any two of the foregoing values), preferably about 75 mg / mL to about 150 mg / mL (e.g., about 75 mg / mL, about 80 mg / mL, about 165 mg / mL, about 175 mg / mL, or a range defined by any two of the foregoing values). , about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 105 mg / mL, about 110 mg / mL, about 115 mg / mL, about 120 mg / mL, about 125 mg / mL, about 130 mg / mL, about 135 mg / mL, about 140 mg / mL, about 145 mg / mL, about 150 mg / mL, or a range defined by any two of the aforementioned values), more preferably about 75 mg / mL to about 125 mg / mL.

[0055] Treatment method Provided herein is a method of treating a subject in need of an anti-IL-36R antibody or antigen-binding antibody fragment, comprising administering an effective amount of a pharmaceutical formulation described herein. The subject may be any subject in need of an anti-IL-36R antibody or antigen-binding antibody fragment, including any subject having a disorder condition responsive to inhibition or neutralization of IL-36R. A disorder "responsive to IL-36R inhibition" or "responsive to IL-36R neutralization" refers to any disease or disorder in which a reduction in the level or activity of IL-36R has a 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 IL-36R inhibition include, for example, inflammatory diseases, autoimmune diseases, respiratory diseases, metabolic disorders, and cancer.

[0056] 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, hidradenitis suppurativa, acne, fibrosis (e.g., idiopathic pulmonary fibrosis, scleroderma, renal fibrosis, and scarring), allergic rhinitis, food allergies (e.g., allergies to peanuts, eggs, dairy products, shellfish, nuts, etc.), seasonal allergies, and other allergies.

[0057] The method of the present invention can be used to treat any type of autoimmune disease (i.e., a disease or disorder caused by an overactive immune system in which the body attacks and damages its own tissues), such as those described in, for example, MacKay IR and Rose NR, eds., The Autoimmune Diseases, Fifth Edition, Academic Press, Waltham, MA (2014). Examples of autoimmune diseases that can be treated by the method of the present invention include, but are not limited to, multiple sclerosis, asthma, type 1 diabetes, rheumatoid arthritis, scleroderma, Crohn's disease, plaque psoriasis (commonly referred to as 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, ankylosing spondylitis, ichthyosis, and skin toxicity. In preferred embodiments, the methods of the invention are used to treat pustular psoriasis, generalized pustular psoriasis, palmoplantar pustulosis (PPP), or plaque psoriasis.

[0058] Pustular psoriasis is a rare form of psoriasis characterized by white pustules surrounded by reddened skin. Generalized pustular psoriasis (GPP) is a life-threatening disease characterized by sudden recurrent episodes of high fever, generalized rash, and disseminated pustules accompanied by leukocytosis and elevated serum levels of C-reactive protein, which may be caused by a deficiency of interleukin-36 receptor antagonist (interleukin-36Ra) (Marrakchi et al., N. Engl. J. Med., 365(7):620-628(2011)). GPP often develops in patients who have or have a history of psoriasis vulgaris (PV), but can also develop 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 or soles that significantly impairs the quality of life of affected individuals (de Waal, AC and van de Kerkhof, PCM, J. Dermatological Treatment, 22(2):102-105 (2011)).

[0059] Examples of respiratory diseases that can be treated by the method 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 method of the present invention include, but are not limited to, obesity, type 2 diabetes, atherosclerosis and cardiovascular disease.

[0060] The methods of the invention can 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, gastric 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)).

[0061] Administration of the pharmaceutical formulations described herein induces an immune response in a mammal. An "immune response" can involve, for example, antibody production and / or activation of immune effector cells (e.g., T cells).

[0062] 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., treatment reduces the severity of one or more adverse symptoms in a patient in need of an anti-IL-36R antibody, including, but not limited to, removal of scaling, inhibition of erythema, pruritus, and reduction of inflammation.

[0063] To this end, the methods of the present invention involve administering a "therapeutically effective amount" of the pharmaceutical formulations described herein.

[0064] A "therapeutically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve a desired pharmacological and / or physiological effect. A therapeutically effective amount may vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the pharmaceutical formulation to elicit a desired response in the individual. For example, a therapeutically effective amount of a pharmaceutical formulation described herein is an amount that reduces the biological activity of any one of the IL-36 cytokines and / or IL-36R, such that a therapeutically effective amount of a pharmaceutical formulation described herein is an amount that reduces the biological activity of IL-36R in a subject. In another example, a therapeutically effective amount of a pharmaceutical formulation described herein is an amount that reduces an adverse symptom in a subject.

[0065] In some embodiments, the pharmacological and / or physiological effect may be prophylactic, i.e., the effect completely or partially prevents a disease, disorder, or condition characterized by increased IL-36 biological activity, or a symptom thereof. In this regard, the method of the present invention includes administering a "prophylactically effective amount" of the pharmaceutical formulation. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. In some embodiments, the subject may be a subject with a genetic predisposition to a disease, disorder, or condition characterized by increased IL-36 biological activity. EXAMPLES

[0066] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0067] Example 1 In this example, sample preparation of four different formulations of IL-36R antibody formulations is described.

[0068] Starting with approximately 200 mL of UF / DF rinse (Lot C6903), anti-IL-36R antibody (heavy chain variable region SEQ ID NO: 22 and light chain variable region SEQ ID NO: 44) was buffer exchanged by UF / DF into formulations F02A, F02B, F02C (listed below in Table 1). 2 Diafiltration was performed with a Sartorius vivaflow TFF cassette. Complete buffer exchange of approximately 7 diavolumes (approximately 150 mL / DV) was achieved. The solution was then concentrated to approximately 30-35 mL so that the protein concentration was >100 mg / mL and diluted with UF / DF rinses as necessary to achieve a target concentration of 100 mg / mL. Samples of F01 were also prepared by concentrating the UF / DF rinses to a target concentration of 100 mg / mL. For all samples, 1 mL of each solution was placed into rubber-stoppered glass drug product (DP) vials, sealed with parafilm, and stored at 2-8 °C until placed at 45 °C as described in the next example.

[0069] Example 2 This example shows the relative protein concentration, pH osmolality, and viscosity of the four formulations.

[0070] Validation testing of the formulations was initiated at AnaptysBio (San Diego, CA). Protein concentration (mg / mL), pH, osmolality (mOsm / kg), and viscosity (cP) were measured for the four different formulations.

[0071] pH measurements were performed using an Oakton PC700 pH meter with a Mettler Toledo inLab microprobe, 3mol / L KCl. Instrument control and data acquisition was done on the meter body. Once the pH had stabilized, the pH value was recorded manually from the meter display. The pH procedure was as follows: 200μL of each input solution was pipetted into a 1.5mL Eppendorf tube for pH measurement. Once the meter had stabilized, the pH measurement was recorded.

[0072] Osmolality analysis was performed using a Precision Systems Osmette III-5010 automated osmometer. Instrument control and data acquisition was performed on the meter body. Once the instrument had stabilized, osmolality values ​​were recorded manually from the meter display. The osmolality procedure was as follows: 10 μL of each input solution was pipetted in triplicate into the instrument for measurement. Once the device had stabilized, osmolality measurements were recorded.

[0073] Viscosity analysis was performed using a RheoSense HVROC-L (microVISC) viscometer. Instrument control and data acquisition were performed using microVISC software. For all measurements, the instrument was placed in a microVISC temperature-controlled chamber set at 25 °C. The procedure for viscosity measurements was as follows: 400 μL of each sample formulated at 100 mg / mL was drawn into a microVISC disposable pipette for automatic injection by the instrument for each measurement. Sample injections were performed in at least triplicate when possible. Cleaning was performed by injecting 1% Aquet detergent solution between each sample set. Viscosity standards were also checked to ensure the integrity of the viscosity data collected. Viscosity data acquired by the microVISC software was exported to Microsoft excel. The results are shown in Table 1.

[0074] [Table 1]

[0075] The measured osmolality for all formulations was higher than expected. This is likely due to the Denon effect, which is commonly observed when using a centrifuge to concentrate protein solutions. The osmolality standards used to ensure proper performance of the osmometer were all within acceptable limits. Differences in measured viscosity (cP) were also observed among the four formulations, with F02C being closest to F01 and F02B being the highest.

[0076] Example 3 This experiment demonstrates the improved stability of the F02A formulation.

[0077] IL-36R antibodies in four different formulations (i.e., F01, F02A, F02B, F02C as described in Table 1) were incubated at 45° C. for up to 8 days. 1 mL samples were assessed for color, clarity, and appearance against appropriate standards in glass DP vials. Sample images were taken in a light box under illumination against a black background. FIG. 1A shows images of the appearance of F01 compared to water at days 0, 5, 6, 7, and 8. FIG. 1B shows images of the appearance of F02A compared to water at days 0, 5, 6, 7, and 8. FIG. 1C shows images of the appearance of F02B compared to water at days 0, 5, 6, 7, and 8. FIG. 1D shows images of the appearance of F02C compared to water at days 0, 5, 6, 7, and 8. In comparison, F02A, F02B, and F02C were substantially less opaque than F01. The opacity observed for F02C was similar to F01. F02A and F02B were notably less opaque than F02C. After stressing at 45°C for 8 days, the level of opacity did not appear to increase for any of the formulations.

[0078] After storing at 45°C for 8 days, the turbidity of each sample was measured. 350 Turbidity was measured by. Turbidity analysis was performed using a Thermo Scientific Nanodrop 2000c spectrophotometer. Instrument control and data acquisition was performed using nanodrop software. The turbidity procedure was as follows: 2 μL of each formulated sample and control at 100 mg / mL was applied to each measurement nanodrop using a UV-VIS application with a 10 mm path length recording the absorbance of the sample at 350 nm. All measurements were performed at least in triplicate. NTU turbidity standards were also checked to reference the sample turbidity data collected. The turbidity data acquired by the nanodrop software was exported to Microsoft excel. Table 2 shows the A 350 FIG. 2 shows the results of turbidity analysis of IL-36R formulations stored at 45° C. 350 Table 3 shows the bivariate plot of turbidity by time (days).350 ) is shown.

[0079] [Table 2]

[0080] [Table 3]

[0081] All formulations showed a trend towards increasing turbidity as indicated by the p-values ​​reported in Table 3. To further evaluate the turbidity data, a common slope test was used to compare the turbidity change among the four formulations. Analysis was performed using a crossover design in the Fit Model platform of JMP® to determine whether there was a statistically significant difference between the slopes of each formulation. The test demonstrated that there was a statistically significant difference in the change in turbidity over time among all formulations as indicated by an F-ratio greater than 4 and a p-value of <0.0001. F01 is inferior to F02A-C as it has almost twice the slope. The results of the common slope test are shown in Table 4.

[0082] [Table 4]

[0083] The purity of samples stored at 45° C. for up to 8 days was measured by SEC-HPLC (% Principal Components). Sample purity by SEC-HPLC was measured using a Tosoh Tskgel G3000SWxl 5 μm column. After dilution to 10 mg / mL with formulation buffer (F01, F02A, F02B, F02C), samples were analyzed with an injection volume of 10 μL, a runtime of 20 min, a flow rate of 1.0 mL / min, and a column temperature of 25° C. Samples were analyzed singly. Table 5 shows the results of the purity measurements. Figure 3 shows a bivariate plot of the SEC-HPLC (% Principal Components) of IL-36R formulations stored at 45° C. Table 6 shows the results of a goodness-of-fit analysis of the results. All formulations showed a trend towards decreased turbidity, as indicated by the p-values ​​reported in Table 6.

[0084] [Table 5]

[0085] [Table 6]

[0086] A common slope test was used to compare the purity change among the four formulations. The results of the common slope test are shown in Table 7. Analysis was performed using a crossover design in the Fit Model platform of JMP® to determine whether there was a statistically significant difference between the slopes of each formulation. The test demonstrated that there was a statistically significant difference in the purity change over time among all formulations, as indicated by an F-ratio greater than 4 and a p-value of 0.003. As shown in Table 7, F01 and F02C are inferior to F02A and F02B, as indicated by the nearly two-fold slope. The similar purity loss observed for F02C is reasonable given that the composition of this formulation is similar to F01.

[0087] [Table 7]

[0088] This experiment shows that F02A and F02B have improved stability over time in terms of turbidity and purity when the formulations are exposed to 45° C. for 8 days.

[0089] All references cited in this specification, including publications, patent applications, and patents, are hereby incorporated by reference as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0090] Use of the terms "a" and "an" and "the" and "at least one" and similar referents in connection with the description of the present invention (particularly in connection with the claims that follow) should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. Use of the term "at least one" following a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B), unless otherwise specified herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise specified herein or clearly contradicted by context. The recitation of ranges of values ​​herein is intended to serve merely as a shorthand for referring individually to each separate value within the range, and each separate value is incorporated into the specification as if it were individually recited herein, unless otherwise specified 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., "etc.") provided herein is intended merely to further elucidate the invention, and does not pose a limitation on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0091] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors expect that such variations will be utilized by those of skill in the art, and the inventors intend that the invention 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. A stable aqueous pharmaceutical formulation comprising: (a) water; (b) a histidine buffer; (c) a stabilizer comprising proline and / or sorbitol; (d) a non-ionic surfactant; and (e) an antibody or antigen-binding antibody fragment.

2. the stabilizer comprises proline, Optionally, about 100-300 mM proline, or containing about 270-300 mM proline; 2. The pharmaceutical formulation of claim 1.

3. the stabilizer comprises sorbitol; Optionally, about 100-300 mM sorbitol, or containing about 270-300 mM sorbitol; 2. The pharmaceutical formulation of claim 1.

4. the stabilizer comprises proline and sorbitol; Optionally, About 100-300 mM of a combination of proline and sorbitol, or about 270-300 mM of a combination of proline and sorbitol; or 10. The pharmaceutical formulation of claim 1 comprising about 50-80 mM sorbitol and 180-250 mM proline.

5. 10. The pharmaceutical formulation of claim 1, comprising about 5-35 mM histidine, or about 10-25 mM histidine.

6. 2. The pharmaceutical formulation of claim 1, wherein the pH is about 5.0 to 6.5, or about 5.5 to 6.2, or about 5.8 to 6.

0.

7. the non-ionic surfactant is polysorbate or polysorbate-20, and optionally About 0.01 to 0.1% by weight of polysorbate-20, or 10. The pharmaceutical formulation of claim 1, comprising about 0.01 to 0.05% by weight of polysorbate-20.

8. 10. The pharmaceutical formulation of claim 1, comprising about 75-175 mg / mL of the antibody or antigen-binding antibody fragment, or about 75-125 mg / mL of the antibody or antigen-binding antibody fragment.

9. 10. The pharmaceutical formulation of claim 1 having a viscosity of less than 15 cps.

10. 2. The pharmaceutical formulation of claim 1, comprising less than 10 mM NaCl, and optionally substantially or completely free of NaCl.

11. The formulation, (a) about 75-150 mg / mL of antibody or antigen-binding antibody fragment; (b) about 9-11 mM histidine; (c) about 275-285 mM proline; and (d) about 0.01% to 0.05% polysorbate-20 and optionally the formulation comprises a pH of about 5.8-6.0 and a viscosity of less than 15 cps; and optionally the formulation comprises less than 10 mM NaCl or is substantially or completely free of NaCl; or, The formulation comprises: (a) about 75-150 mg / mL of antibody or antigen-binding antibody fragment; (b) about 9-11 mM histidine; (c) about 205-215 mM proline; (d) about 0.01% to 0.05% polysorbate-20; and (e) about 65-75 mM sorbitol and optionally the formulation comprises a pH of about 5.8-6.0 and a viscosity of less than 15 cps; and optionally the formulation comprises less than 10 mM NaCl or is substantially or completely free of NaCl; or, The formulation comprises: (a) about 75-150 mg / mL of antibody or antigen-binding antibody fragment; (b) about 24-26 mM histidine; (c) about 275-285 mM proline; and (d) about 0.03% to 0.07% polysorbate-20 and optionally having a pH of about 5.8-6.0 and a viscosity of less than 15 cps, and optionally containing less than 10 mM NaCl or being substantially or completely free of NaCl; or, The formulation comprises: (a) about 75-150 mg / mL of antibody or antigen-binding antibody fragment; (b) about 10 mM histidine; (c) about 280 mM proline; and (d) about 0.03% polysorbate-20 and optionally the formulation comprises a pH of about 5.8-6.0 and a viscosity of less than 15 cps; and optionally the formulation comprises less than 10 mM NaCl or is substantially or completely free of NaCl; or, The formulation comprises: (a) about 75-150 mg / mL of antibody or antigen-binding antibody fragment; (b) about 10 mM histidine; (c) about 210 mM proline; (d) about 0.03% polysorbate-20; and (e) about 70 mM sorbitol and optionally the formulation comprises a pH of about 5.8-6.0 and a viscosity of less than 15 cps; and optionally the formulation comprises less than 10 mM NaCl or is substantially or completely free of NaCl; or, The formulation comprises: (a) about 75-150 mg / mL of antibody or antigen-binding antibody fragment; (b) about 25 mM histidine; (c) about 280 mM proline; and (d) about 0.05% polysorbate-20 2. The pharmaceutical formulation of claim 1, comprising:

12. the antibody is an anti-IL-36 antibody, and optionally (a)(i) a light chain variable region comprising: a complementarity determining region (CDR) 1 domain (CDRL1) comprising the amino acid sequence of SEQ ID NO: 83; a CDRL2 domain comprising the amino acid sequence of SEQ ID NO: 84; and a CDRL3 domain comprising the amino acid sequence of SEQ ID NO: 85; and (ii) a heavy chain variable region comprising: a CDRH1 domain comprising the amino acid sequence of SEQ ID NO: 65; a CDRH2 domain comprising the amino acid sequence of SEQ ID NO: 71; and a CDRH3 domain comprising the amino acid sequence of SEQ ID NO: 72; or (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:44 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

22.

13. A method for treating a patient in need of an anti-IL-36R antibody, comprising administering an effective amount of a pharmaceutical formulation according to any one of claims 1 to 12, Optionally, the method is a method for treating an autoimmune, inflammatory, respiratory, or metabolic disease or disorder, or cancer.

14. 13. The pharmaceutical formulation according to any one of claims 1 to 12, for treating a disease or disorder responsive to inhibition or neutralization of IL-36R, optionally wherein said disease or disorder is an autoimmune, inflammatory, respiratory, or metabolic disease or disorder, or cancer.

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