Novel anti-IL-36R antibody

Abstract

The present disclosure provides anti-IL-36R antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding them, pharmaceutical compositions comprising them, and uses thereof.

Description

[Technical field]

[0001]

[0001] The present disclosure relates generally to novel anti-IL-36R antibodies. [Background technology]

[0002]

[0002] Interleukin-36 receptor (IL-36R) is expressed on the cell surface and belongs to the IL1R family. There are three agonists of IL-36R, namely, IL-36α, IL-36β, and IL-36γ, and two antagonists, namely, IL-36Ra (IL36RN) and IL-38. The ligands of IL-36R are mainly derived from keratinocytes, epithelial cells, T / B lymphocytes, monocytes, dendritic cells, macrophages, etc. IL-36R is usually expressed in keratinocytes, fibroblasts, dendritic cells, endothelial cells, monocytes, macrophages, Langerhans cells, CD4+ T cells, etc., and functions by signal transduction through the NFκB or MARK pathway. The important functions of IL-36R in cells are to induce proinflammatory cytokines and chemokines, and to promote cell activation and proliferation. Summary of the Invention [Problem to be solved by the invention]

[0003]

[0003] However, IL-36R-related skin diseases have a high unmet medical need, and therefore there remains a need for novel anti-IL-36R antibodies. [Means for solving the problem]

[0004]

[0004] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or two or more antibodies.

[0005] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof capable of specifically binding to human IL-36R, the antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, wherein HCDR1 is selected from the group consisting of DYYX, ... 1 X 2 (SEQ ID NO: 191), SEQ ID NO: 19, 33, 48, 64, 79, 94, 109, 124 or 139, and HCDR2 is LIRNKAAGYTIYYX 3 X 4 X 5 VKG (SEQ ID NO: 192), comprising the amino acid sequence of SEQ ID NO: 20, 34, 49, 65, 80, 95, 110, 125 or 140, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 5, 21, 35, 50, 66, 81, 96, 111, 126 or 141; 1 is M or L, and X 2 is N, H, S or R, and X 3 is S or A, and X 4 is A or D, and X 5 is S or P.

[0006] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain complementarity determining region 1 (LCDR1), a LCDR2 and a LCDR3, wherein LCDR1 is selected from the group consisting of RASX 18 NINIWLS (SEQ ID NO: 193), LCDR2 comprises the amino acid sequence of SEQ ID NO: 22, 36, 51, 67, 82, 97, 112, 127 or 142, LCDR3 comprises the amino acid sequence of SEQ ID NO: 7, 23, 37, 52, 68, 83, 98, 113, 128 or 113, and LCDR4 comprises the amino acid sequence of X 19 QSQSYPLT (SEQ ID NO: 194), comprising the amino acid sequence of SEQ ID NO: 24, 38, 53, 69, 84, 99, 114, 129 or 143, wherein X 18 is Q or R, and X 19 is Q or L.

[0007] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and / or a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein (a) HCDR1 is DYYX 1 X 2 (SEQ ID NO: 191), HCDR2 is LIRNKAAGYTIYYX 3 X 4 X 5 VKG (SEQ ID NO: 192), HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR1 comprises RASX 18 NINIWLS (SEQ ID NO: 193), LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises X 19 QSQSYPLT (SEQ ID NO: 194) 1 is M or L, and X 2 is N, H, S or R, and X 3 is S or A, and X 4 is A or D, and X 5 is S or P, and X 18 is Q or R, and X 19is Q or L; (b) HCDR1 comprises the amino acid sequence of SEQ ID NO:19, HCDR2 comprises the amino acid sequence of SEQ ID NO:20, HCDR3 comprises the amino acid sequence of SEQ ID NO:21, LCDR1 comprises the amino acid sequence of SEQ ID NO:22, LCDR2 comprises the amino acid sequence of SEQ ID NO:23, and LCDR3 comprises the amino acid sequence of SEQ ID NO:24; (c) HCDR1 comprises the amino acid sequence of SEQ ID NO:33, HCDR2 comprises the amino acid sequence of SEQ ID NO:34, HCDR3 comprises the amino acid sequence of SEQ ID NO:35, LCDR1 comprises the amino acid sequence of SEQ ID NO:36, LCDR2 comprises the amino acid sequence of SEQ ID NO:37, and LCDR3 comprises the amino acid sequence of SEQ ID NO:38; (d) HCDR1 comprises the amino acid sequence of SEQ ID NO:48, HCDR2 comprises the amino acid sequence of SEQ ID NO:49, HCDR3 comprises the amino acid sequence of SEQ ID NO:50, LCDR1 comprises the amino acid sequence of SEQ ID NO:51, LCDR2 comprises the amino acid sequence of SEQ ID NO:52, and LCDR3 comprises the amino acid sequence of SEQ ID NO:53. (e) HCDR1 comprises the amino acid sequence of SEQ ID NO:64, HCDR2 comprises the amino acid sequence of SEQ ID NO:65, HCDR3 comprises the amino acid sequence of SEQ ID NO:66, LCDR1 comprises the amino acid sequence of SEQ ID NO:67, LCDR2 comprises the amino acid sequence of SEQ ID NO:68, and LCDR3 comprises the amino acid sequence of SEQ ID NO:69; (f) HCDR1 comprises the amino acid sequence of SEQ ID NO:79, HCDR2 comprises the amino acid sequence of SEQ ID NO:80, HCDR3 comprises the amino acid sequence of SEQ ID NO:81, LCDR1 comprises the amino acid sequence of SEQ ID NO:82, LCDR2 comprises the amino acid sequence of SEQ ID NO:83, and LCDR3 comprises the amino acid sequence of SEQ ID NO:84; (g) HCDR1 comprises the amino acid sequence of SEQ ID NO:94, HCDR2 comprises the amino acid sequence of SEQ ID NO:95, HCDR3 comprises the amino acid sequence of SEQ ID NO:96, LCDR1 comprises the amino acid sequence of SEQ ID NO:97, LCDR2 comprises the amino acid sequence of SEQ ID NO:98, and LCDR3 comprises the amino acid sequence of SEQ ID NO:99;(h) HCDR1 comprises the amino acid sequence of SEQ ID NO: 109, HCDR2 comprises the amino acid sequence of SEQ ID NO: 110, HCDR3 comprises the amino acid sequence of SEQ ID NO: 111, LCDR1 comprises the amino acid sequence of SEQ ID NO: 112, LCDR2 comprises the amino acid sequence of SEQ ID NO: 113, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 114; (i) HCDR1 comprises the amino acid sequence of SEQ ID NO: 124, HCDR2 comprises the amino acid sequence of SEQ ID NO: 125, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 126, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 127; CDR1 comprises the amino acid sequence of SEQ ID NO: 127, LCDR2 comprises the amino acid sequence of SEQ ID NO: 128, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 129; or (j) HCDR1 comprises the amino acid sequence of SEQ ID NO: 139, HCDR2 comprises the amino acid sequence of SEQ ID NO: 140, HCDR3 comprises the amino acid sequence of SEQ ID NO: 141, LCDR1 comprises the amino acid sequence of SEQ ID NO: 142, LCDR2 comprises the amino acid sequence of SEQ ID NO: 113, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 143;

[0008]

[0008] In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, 180, 184 or 188, HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, 151, 164 or 173, HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, LCDR1 comprises the amino acid sequence of SEQ ID NO: 6 or 152, LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 8 or 153.

[0009] In some embodiments, (a) HCDR1 comprises the amino acid sequence of SEQ ID NO:3, HCDR2 comprises the amino acid sequence of SEQ ID NO:4, HCDR3 comprises the amino acid sequence of SEQ ID NO:5, LCDR1 comprises the amino acid sequence of SEQ ID NO:6, LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO:8; or (b) HCDR1 comprises the amino acid sequence of SEQ ID NO:3, HCDR2 comprises the amino acid sequence of SEQ ID NO:151, HCDR3 comprises the amino acid sequence of SEQ ID NO:5, and LCDR1 comprises the amino acid sequence of SEQ ID NO:152. (c) HCDR1 comprises the amino acid sequence of SEQ ID NO:3, HCDR2 comprises the amino acid sequence of SEQ ID NO:164, and HCDR3 comprises the amino acid sequence of SEQ ID NO:5, LCDR1 comprises the amino acid sequence of SEQ ID NO:6, LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO:153; or (d) HCDR1 comprises the amino acid sequence of SEQ ID NO:3, HCDR2 comprises the amino acid sequence of SEQ ID NO:173, and HCDR (e) HCDR1 comprises the amino acid sequence of SEQ ID NO:3, HCDR2 comprises the amino acid sequence of SEQ ID NO:151, HCDR3 comprises the amino acid sequence of SEQ ID NO:5, LCDR1 comprises the amino acid sequence of SEQ ID NO:6, LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO:153; or (f) HCDR1 comprises the amino acid sequence of SEQ ID NO:18 0, HCDR2 comprises the amino acid sequence of SEQ ID NO: 151, HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, LCDR1 comprises the amino acid sequence of SEQ ID NO: 152, LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153, or (g) HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, HCDR2 comprises the amino acid sequence of SEQ ID NO: 151, HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, LCDR1 comprises the amino acid sequence of SEQ ID NO: 152, and LCDR2 comprises the amino acid sequence of SEQ ID NO: 7,and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153; or (h) HCDR1 comprises the amino acid sequence of SEQ ID NO: 184, HCDR2 comprises the amino acid sequence of SEQ ID NO: 151, HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, LCDR1 comprises the amino acid sequence of SEQ ID NO: 152, LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153; or (i) HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, HCDR2 comprises the amino acid sequence of SEQ ID NO: 151, HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, LCDR1 comprises the amino acid sequence of SEQ ID NO: 152, LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 15 3, (j) HCDR1 comprises the amino acid sequence of SEQ ID NO: 188, HCDR2 comprises the amino acid sequence of SEQ ID NO: 151, HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, LCDR1 comprises the amino acid sequence of SEQ ID NO: 152, LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153, or (k) HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, HCDR2 comprises the amino acid sequence of SEQ ID NO: 151, HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, LCDR1 comprises the amino acid sequence of SEQ ID NO: 152, LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises one or more of heavy chain framework region 1 (HFR1), HFR2, HFR3, and HFR4, and / or one or more of light chain framework region 1 (LFR1), LFR2, LFR3, and LFR4, wherein HFR1 is selected from the group consisting of X, ... 6 VQLX7ESGGGLVKPGGSLRLSCAASGX 8 X 9 FX 10 (SEQ ID NO: 195) or a homologous sequence having at least 85% sequence identity thereto, and HFR2 comprises WX 11 RQAPGKGLEWVX 12(SEQ ID NO: 196) or a homologous sequence having at least 85% sequence identity thereto, and HFR3 is RFTISRDX 13 X 14 KSX 15 LYLQMNSLX 16 X 17 EDTAVYYCVR (SEQ ID NO: 197) or a homologous sequence having at least 85% sequence identity thereto, HFR4 comprises the amino acid sequence of SEQ ID NO: 157 or a homologous sequence having at least 85% sequence identity thereto, and LFR1 comprises X 20 IVMTQSPX 21 X 22 X 23 SX 24 SX 25 GX 26 RX 27 TX 28 X 29 C (SEQ ID NO: 198) or a homologous sequence having at least 85% sequence identity thereto, and LFR2 is WYQQKPGX 30 APX 31 LFIY (SEQ ID NO: 199) or a homologous sequence having at least 85% sequence identity thereto, and LFR3 is GVPX 32 RFSGSGSGTX 33 FTLTISSLQX 34 EDFAX 35 YYC (SEQ ID NO: 200) or a homologous sequence having at least 85% sequence identity thereto; LFR4 comprises the amino acid sequence of SEQ ID NO: 161 or a homologous sequence having at least 85% sequence identity thereto; X 6 is Q or E, and X 7 is Q or V, and X 8 is F or Y, and X 9 is A, D or N, and X 10 is T or G, and X 11 is I or V, and X 12 is S or A, and X 13 is N or D, and X 14 is A or S, and X 15 is S or T, and X 16 is R or K, and X17 is A or T, and X 20 is D or E, and X 21 is S or A, and X 22 is S or T, and X 23 is L or V, and X 24 is A or V, and X 25 is V or P, and X 26 is D or E, and X 27 is V or A, and X 28 is I or L, and X 29 is T or S, and X 30 is Q or K, and X 31 is K or R, and X 32 is S or A, and X 33 is D or E, and X 34 is S or P, and X 35 is T or V.

[0011] In some embodiments, HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 9, 25, 39, 54, 70, 85, 100, 115, 130, 144, 154, 165, 174, 178, 181, 186, 190, and 195, and HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 10, 26, 40, 55, 71, 86, 101, 116, 131, 145, 155, 166, 175, and 196, HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 11, 27, 41, 56, 72, 87, 56, 117, 132, 146, 156, 167, 167, 156, 156, 156, 156, 156, 156, 156, and 197, HFR4 comprises a sequence selected from the group consisting of SEQ ID NOs: 12 , 42, 57, 73, 102, 118, and 157, LFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 13, 28, 43, 58, 74, 88, 103, 119, 133, 147, 158, 168, and 198, LFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 14, 29, 44, 59, 75, 89, 104, 120, 134, 159, 169, and 199, LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 30, 45, 60, 76, 90, 105, 121, 135, 148, 160, 170, and 200, and LFR4 comprises a sequence selected from the group consisting of SEQ ID NOs: 16, 61, 91, 106, 136, 161.

[0012]

[0012] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof provided herein comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 17, 31, 46, 62, 77, 92, 107, 122, 137, 149, 162, 171, 176, 179, 182, 183, 185, 187, and 189, as well as homologous sequences thereof having at least 80% sequence identity and retaining specific binding affinity for human IL-36R.

[0013]

[0013] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof provided herein comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 18, 32, 47, 63, 78, 93, 108, 123, 138, 150, 163, 172, and 177, as well as homologous sequences thereof having at least 80% sequence identity and retaining specific binding affinity for human IL-36R.

[0014] In some embodiments, in the antibody or antigen-binding fragment thereof provided herein, the heavy chain variable region comprises the sequence of SEQ ID NO:1 and the light chain variable region comprises the sequence of SEQ ID NO:2, or the heavy chain variable region comprises the sequence of SEQ ID NO:17 and the light chain variable region comprises the sequence of SEQ ID NO:18, or the heavy chain variable region comprises the sequence of SEQ ID NO:31 and the light chain variable region comprises the sequence of SEQ ID NO:32, or the heavy chain variable region comprises the sequence of SEQ ID NO:46 and the light chain variable region comprises the sequence of SEQ ID NO:47, or the heavy chain variable region comprises the sequence of SEQ ID NO:62. or the heavy chain variable region comprises the sequence of SEQ ID NO: 77 and the light chain variable region comprises the sequence of SEQ ID NO: 78; or the heavy chain variable region comprises the sequence of SEQ ID NO: 92 and the light chain variable region comprises the sequence of SEQ ID NO: 93; or the heavy chain variable region comprises the sequence of SEQ ID NO: 107 and the light chain variable region comprises the sequence of SEQ ID NO: 108; or the heavy chain variable region comprises the sequence of SEQ ID NO: 122 and the light chain variable region comprises the sequence of SEQ ID NO: 123; or the heavy chain variable region comprises the sequence of SEQ ID NO: 137 and the light chain variable region comprises the sequence of SEQ ID NO: or the heavy chain variable region comprises the sequence of SEQ ID NO: 149 and the light chain variable region comprises the sequence of SEQ ID NO: 150; or the heavy chain variable region comprises the sequence of SEQ ID NO: 162 and the light chain variable region comprises the sequence of SEQ ID NO: 163; or the heavy chain variable region comprises the sequence of SEQ ID NO: 171 and the light chain variable region comprises the sequence of SEQ ID NO: 172; or the heavy chain variable region comprises the sequence of SEQ ID NO: 176 and the light chain variable region comprises the sequence of SEQ ID NO: 177; or the heavy chain variable region comprises the sequence of SEQ ID NO: 179 and the light chain variable region comprises the sequence of SEQ ID NO: 150. or the heavy chain variable region comprises the sequence of SEQ ID NO: 182 and the light chain variable region comprises the sequence of SEQ ID NO: 150, or the heavy chain variable region comprises the sequence of SEQ ID NO: 183 and the light chain variable region comprises the sequence of SEQ ID NO: 150, or the heavy chain variable region comprises the sequence of SEQ ID NO: 185 and the light chain variable region comprises the sequence of SEQ ID NO: 150, or the heavy chain variable region comprises the sequence of SEQ ID NO: 187 and the light chain variable region comprises the sequence of SEQ ID NO: 150, or the heavy chain variable region comprises the sequence of SEQ ID NO: 189 and the light chain variable region comprises the sequence of SEQ ID NO: 150.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises one or more amino acid residue substitutions or modifications and still retains specific binding affinity for human IL-36R. In some embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences of the heavy chain variable region or the light chain variable region and / or in one or more of the non-CDR sequences. In some embodiments, at least one of the substitutions is a conservative substitution.

[0016] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise an Fc region, optionally an Fc region of a human immunoglobulin (Ig), or optionally an Fc region of a human IgG. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgM. In some embodiments, the Fc region derived from human IgG4 comprises the following mutations: M252Y / S254T / T256E (YTE). In some embodiments, the Fc region derived from human IgG4 comprises the following mutations: T307Q / N434A (QA).

[0017] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are humanized. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are monoclonal antibodies, bispecific antibodies, multispecific antibodies, recombinant antibodies, chimeric antibodies, labeled antibodies, bivalent antibodies, anti-idiotypic antibodies, or fusion proteins.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof provided herein is a diabody, Fab, Fab', F(ab') 2 , Fd, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) 2, bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabody (ds diabody), single chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single chain domain antibody, nanobody, domain antibody, or bivalent domain antibody.

[0019] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a) a K of 2E-08M or less (preferably, 1E-08M or less, e.g., 9E-09M or less, 8E-09M or less, 7E-09M or less, 6E-09M or less, 5E-09M or less, 4E-09M or less, 3E-09M or less, 2E-09M or less, or 1E-10M or less) as measured by a biolayer interferometry (BLI) assay. d and b) has a binding affinity for human IL-36R of 1E-08M or less (e.g., 9E-09M or less, 8E-09M or less, 7E-09M or less, 6E-09M or less, 5E-09M or less, 4E-09M or less, 3E-09M or less, 2E-09M or less, or 1E-10M or less) as measured by a surface plasmon resonance (SPR) assay. d and c) an EC50 of 0.1 μg / mL or less (e.g., 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, 0.01 μg / mL, or 0.005 μg / mL) as measured by an enzyme-linked immunosorbent assay (ELISA) assay. 50 and d) an EC50 of 0.5 μg / mL or less (e.g., 0.4 μg / mL, 0.3 μg / mL, 0.2 μg / mL, 0.19 μg / mL, 0.18 μg / mL, 0.17 μg / mL, 0.16 μg / mL, 0.15 μg / mL, or 0.1 μg / mL) as measured by a fluorescence-activated cell sorting (FACS) assay. 50 and having binding affinity for membrane human IL-36R.

[0020]

[0020] In some embodiments, the antibody or antigen-binding fragment thereof provided herein can block IL-36R signaling induced by an IL-36R agonist (preferably, IL-36α) as measured by an IL-36R reporter assay.

[0021]

[0021] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have one or more properties selected from the group consisting of: a) being capable of inhibiting IL-36R agonist-induced IL-8 release in cells; b) having the ability to inhibit IL-36R agonist-induced IL-6 release in cells; and d) having the ability to inhibit IL-36R agonist-induced TNF-a release in cells, wherein the IL-36R agonist includes IL-36α, IL-36β, and / or IL-36γ.

[0022]

[0022] In another aspect, the present disclosure provides an anti-IL-36R antibody or antigen-binding fragment thereof that does not compete with an antibody or antigen-binding fragment thereof in the art for binding to human IL-36R. In some embodiments, the antibody or antigen-binding fragment thereof does not compete with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO:205 and a light chain variable region comprising the sequence of SEQ ID NO:206 for binding to human IL-36R. In some embodiments, the antibody or antigen-binding fragment thereof does not compete with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO:209 and a light chain variable region comprising the sequence of SEQ ID NO:210 for binding to human IL-36R.

[0023] In some embodiments, the disclosed anti-IL-36R antibodies or antigen-binding fragments thereof bind to human IL-36R with high specificity. In some embodiments, the disclosed anti-IL-36R antibodies or antigen-binding fragments thereof do not bind to cynomolgus monkey IL-36R or mouse IL-36R. In some embodiments, the disclosed anti-IL-36R antibodies or antigen-binding fragments thereof do not bind to human IL-1R1.

[0024]

[0024] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are bispecific. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can specifically bind to a second antigen other than IL-36R or a second epitope on IL-36R. In some embodiments, the second antigen other than IL-36R is selected from the group consisting of IL-17, IL-23, TNF, IL-12, and IL-1.

[0025] In another aspect, the present disclosure provides an antibody-drug conjugate comprising an anti-IL-36R antibody or antigen-binding fragment thereof disclosed herein linked to one or more conjugate moieties. In some embodiments, the conjugate moieties may comprise a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or any combination thereof.

[0026]

[0026] In some embodiments, the conjugate portion comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, or other anti-cancer drug.

[0027] In some embodiments, the conjugate moiety comprises a drug or therapeutic agent.

[0028] In another aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and a TCR signaling domain, wherein the antigen-binding domain specifically binds to IL-36R and comprises an antigen-binding fragment of an anti-IL-36R antibody provided herein. In some embodiments, the antigen-binding fragment further comprises a costimulatory domain. In some embodiments, the antigen-binding domain of the CAR specifically binds to human IL-36R and comprises an antigen-binding fragment of the present disclosure.

[0028]

[0029] In some embodiments, the antigen-binding domain of the CAR specifically binds human IL-36R and comprises a heavy chain variable region comprising the HCDR1, HCDR2 and HCDR3 sequences provided herein, and / or a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences provided herein. 1 X 2 (SEQ ID NO: 191), HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, 33, 48, 64, 79, 94, 109, 124, or 139, LIRNKAAGYTIYYX 3 X 4 X 5 VKG (SEQ ID NO: 192), HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, 34, 49, 65, 80, 95, 110, 125, or 140, HCDR3 (X 1 is M or L, and X 2 is N, H, S or R, and X 3 is S or A, and X 4 is A or D, and X 5 is S or P), and / or RASX 18 NINIWLS (SEQ ID NO: 193), LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, 36, 51, 67, 82, 97, 112, 127, or 142, LCDR2 comprising the amino acid sequence of SEQ ID NO: 7, 23, 37, 52, 68, 83, 98, 113, 128, or 113, X 19 QSQSYPLT (SEQ ID NO: 194), LCDR3(X) comprising the amino acid sequence of SEQ ID NO: 24, 38, 53, 69, 84, 99, 114, 129, or 143 18 is Q or R, and X 19is Q or L. In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, 180, 184 or 188, HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, 151, 164 or 173, HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, LCDR1 comprises the amino acid sequence of SEQ ID NO: 6 or 152, LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 8 or 153.

[0029]

[0030] In some embodiments, the antigen-binding domain of the CAR specifically binds human IL-36R and comprises a VH / VL pair selected from the group consisting of SEQ ID NOs: 1 / 2, 17 / 18, 31 / 32, 46 / 47, 62 / 63, 77 / 78, 92 / 93, 107 / 108, 122 / 123, and 137 / 138. In some embodiments, the antigen-binding domain of the CAR specifically binds human IL-36R and comprises a VH / VL pair selected from the group consisting of SEQ ID NOs: 162 / 163, 171 / 172, 176 / 177, 179 / 150, 182 / 150, 149 / 150, 183 / 150, 185 / 150, 187 / 150, and 189 / 150.

[0030]

[0031] In another aspect, the present disclosure provides a nucleic acid sequence encoding the chimeric antigen receptor (CAR) of the present application. In another aspect, the present disclosure provides a cell comprising the nucleic acid sequence of the present disclosure. In another aspect, the present disclosure provides a cell genetically modified to express the CAR of the present disclosure. In another aspect, the present disclosure provides a vector comprising the nucleic acid sequence of the present disclosure.

[0031]

[0032] In another aspect, the disclosure provides a method for stimulating a T cell-mediated immune response against an IL-36R-expressing cell or tissue in a mammal, comprising administering to the mammal an effective amount of a cell genetically modified to express a CAR of the disclosure.

[0032]

[0033] In another aspect, the present disclosure provides a method of treating a mammal having an IL-36R-related disease or condition, comprising administering to the mammal an effective amount of a cell of the present disclosure, thereby treating the mammal. In some embodiments, the cell is an autologous T cell. In some embodiments, the mammal is a human subject. In some embodiments, the mammal has been identified as having an IL-36R positive cell or a cell in which IL-36R signaling is dysregulated.

[0033]

[0034] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, a polynucleotide encoding the antibody or antigen-binding fragment thereof, an antibody-drug conjugate, a CAR, or a recombinant immune cell of the present disclosure, and one or more pharma- ceutically acceptable carriers.

[0034]

[0035] In another aspect, the disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof of the disclosure.

[0036] In another aspect, the disclosure provides a vector comprising an isolated polynucleotide of the disclosure.

[0035]

[0037] In another aspect, the disclosure provides a host cell comprising a vector of the disclosure.

[0038] In another aspect, the disclosure provides a kit comprising an antibody or antigen-binding fragment thereof and / or a pharmaceutical composition of the disclosure and a second therapeutic agent.

[0036]

[0039] In another aspect, the present disclosure provides a method of expressing an antibody or antigen-binding fragment thereof of the present disclosure, comprising culturing a host cell of the present disclosure under conditions in which a vector of the present disclosure is expressed.

[0037]

[0040] In another aspect, the disclosure provides a method of treating, preventing, or ameliorating a disease or disorder responsive to IL-36R inhibition, comprising administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, a polynucleotide encoding the antibody or antigen-binding fragment thereof, an antibody-drug conjugate, a recombinant immune cell, and / or a pharmaceutical composition of the disclosure.

[0038]

[0041] In some embodiments, the disease or disorder is associated with dysregulation of IL-36R-mediated signaling. In some embodiments, dysregulation of IL-36R-mediated signaling comprises dysregulation of IL-36 (e.g., IL-36α, IL-36β, or IL-36γ), an IL-36R antagonist, and / or IL-38. In some embodiments, dysregulation of IL-36R-mediated signaling comprises overactivation of IL-36 (e.g., IL-36α, IL-36β, or IL-36γ), or oversuppression of an IL-36R antagonist, or oversuppression of IL-38, compared to control levels (e.g., levels in a healthy subject).

[0039]

[0042] In some embodiments, the disease or disorder is an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, or cancer.

[0043] In some embodiments, the inflammatory disease is selected from the group consisting of allergic inflammation of the skin, lungs, and digestive 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. In some embodiments, the inflammatory disease is selected from the group consisting of allergic inflammation of the skin, lungs, and digestive tract, atopic dermatitis (also known as atopic eczema), asthma (allergic and non-allergic), and epithelial-mediated inflammation.

[0040]

[0044] In some embodiments, the autoimmune disease is selected from the group consisting of multiple sclerosis, asthma, type 1 diabetes mellitus, rheumatoid arthritis, scleroderma, Crohn's disease, plaque psoriasis (commonly referred to as psoriasis), hidradenitis suppurativa, generalized pustular psoriasis (GPP), palmoplantar pustulosis (PPP), inflammatory bowel disease, psoriatic arthritis, systemic lupus erythematosus (SLE), ulcerative colitis, ankylosing spondylitis, atopic dermatitis, and acne vulgaris. In some embodiments, the method is useful for treating pustular psoriasis, generalized pustular psoriasis, palmoplantar pustulosis (PPP), plaque psoriasis, atopic dermatitis, and acne vulgaris. In some embodiments, the autoimmune disease is selected from the group consisting of psoriasis vulgaris (commonly referred to as psoriasis), hidradenitis suppurativa, generalized pustular psoriasis (GPP), palmoplantar pustulosis (PPP), inflammatory bowel disease, atopic dermatitis, acne vulgaris, and Beh-CHETS disease (also referred to as Behcet's syndrome).

[0041]

[0045] In some embodiments, the respiratory disease is selected from the group consisting of asthma, cystic fibrosis, emphysema, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome.

[0046] In some embodiments, the metabolic disease is selected from the group consisting of obesity, type 2 diabetes, atherosclerosis, and cardiovascular disease.

[0042]

[0047] In some embodiments, the cancer may be 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.

[0043]

[0048] In some embodiments, the disease or disorder is selected from the group consisting of psoriasis, pustular psoriasis, hidradenitis suppurativa, ichthyosis, inflammatory bowel disease (IBD), atopic dermatitis, acne vulgaris, and Behcet's disease.

[0044]

[0049] In another aspect, the present disclosure provides a method of modulating the activity of IL-36R in an IL-36R-positive cell, the method comprising exposing the IL-36R-positive cell to an antibody or antigen-binding fragment thereof of the present disclosure and / or a pharmaceutical composition of the present disclosure.

[0045]

[0050] In another aspect, the present disclosure provides a method of detecting the presence or amount of IL-36R in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof of the present disclosure and determining the presence or amount of IL-36R in the sample.

[0046]

[0051] In another aspect, the present disclosure provides a method of diagnosing an IL-36R-related disease, disorder, or condition in a subject, the method comprising the steps of: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof of the present disclosure and / or a pharmaceutical composition of the present disclosure; b) determining the presence or amount of IL-36R in the sample; and c) correlating the presence or amount of IL-36R with the presence or status of the IL-36R-related disease, disorder, or condition in the subject.

[0047]

[0052] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the sequence of SEQ ID NO: 3, an HCDR2 comprising the sequence of SEQ ID NO: 151, an HCDR3 comprising the sequence of SEQ ID NO: 5, an LCDR1 comprising the sequence of SEQ ID NO: 152, an LCDR2 comprising the sequence of SEQ ID NO: 7, and an LCDR3 comprising the sequence of SEQ ID NO: 153.

[0048]

[0053] In another aspect, the present disclosure provides the use of an antibody or antigen-binding fragment thereof of the present disclosure and / or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating, preventing, or ameliorating a disease or disorder responsive to IL-36R inhibition in a subject.

[0049]

[0054] In another aspect, the present disclosure provides the use of the antibody or antigen-binding fragment thereof and / or the pharmaceutical composition of the present disclosure in the manufacture of a diagnostic reagent for diagnosing a disease or disorder responsive to IL-36R inhibition in a subject.In another aspect, the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof and / or the pharmaceutical composition of the present disclosure, which is useful for detecting IL-36R.In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the sequence of SEQ ID NO:3, an HCDR2 comprising the sequence of SEQ ID NO:151, an HCDR3 comprising the sequence of SEQ ID NO:5, an LCDR1 comprising the sequence of SEQ ID NO:152, an LCDR2 comprising the sequence of SEQ ID NO:7, and an LCDR3 comprising the sequence of SEQ ID NO:153. [Brief description of the drawings]

[0050] [Figure 1]

[0055] FIG. 1 shows a binding affinity study of IL-36R antibodies. [Diagram 2]

[0056] FIG. 2 shows that the antibodies block IL-36R signaling (IL-36R reporter assay). [Diagram 3]

[0057] FIG. 3 shows the blocking activity of humanized IL-36R antibody 5F7. [Figure 4]

[0058] FIG. 4 shows the blocking activity of 5F7 after affinity maturation. [Figure 5-1]

[0059] FIG. 5 shows the binding kinetics curves of antibodies to hIL-36R (tested by SPR). [Figure 5-2] FIG. 5 shows the binding kinetics curves of antibodies to hIL-36R (tested by SPR). [Figure 6]

[0060] FIG. 6 shows binding of 5F7-2a8 to hIL-36R-his in ELISA. [Figure 7]

[0061] FIG. 7 shows binding of antibodies to membrane hIL-36R. [Figure 8]

[0062] FIG. 8 shows binding of antibodies to membrane cynomolgus IL-36R. [Figure 9]

[0063] FIG. 9 shows binding of antibodies to membrane mouse IL-36R. [Figure 10]

[0064] FIG. 10 shows a binding test to hIL1R1. [Figure 11]

[0065] FIG. 11 shows that 5F7-2a8 blocks IL-36α / β / γ-induced IL-8 release in A431 cells. [Figure 12]

[0066] FIG. 12 shows that 5F7-2a8 blocks IL-36α-induced IL-8 release in HDF cells. [Figure 13]

[0067] FIG. 13 shows that 5F7-2a8 blocks IL-36α-induced IL-8 / IL-6 / TNF-α release in HEK cells. [Figure 14]

[0068] FIG. 14 shows that 5F7-2a8 blocks IL-36β-induced IL-8 / IL-6 / TNF-α release in HEK cells. [Figure 15]

[0069] FIG. 15 shows that 5F7-2a8 blocks IL-36γ-induced IL-8 / IL-6 / TNF-α release in HEK cells. [Figure 16]

[0070] FIG. 16 shows the binding affinity of antibodies to hFcRn. [Figure 17]

[0071] FIG. 17 shows the results of a pharmacokinetic study of Fc variants of 5F7-2a8 in cynomolgus monkeys. [Figure 18]

[0072] FIG. 18 shows the blocking activity of IL36R antibodies tested by IL36R reporter assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051]

[0073] Detailed Description of the Invention

[0074] The following description of the present disclosure is intended only to describe various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is clear to those skilled in the art that various equivalents, modifications and alterations can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments should be included in this specification. All references cited in this specification, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0052]

[0075] definition

[0076] The term "antibody" as used herein includes any immunoglobulin, monoclonal, polyclonal, polyvalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A natural intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consisting of a variable region (VH) and a first, second, third, and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively). Mammalian light chains are classified as lambda or kappa, each light chain consisting of a variable region (VL) and a constant region. Antibodies have a "Y" shape, with the axis of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonds. Each arm of the Y comprises the variable region and first constant region of one heavy chain bound to the variable and constant regions of one light chain. The variable regions of the light and heavy chains are involved in antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2 and LCDR3, heavy chain CDRs including HCDR1, HCDR2 and HCDR3).The boundaries of the CDRs of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the rules of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3):651-63(1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901(1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83(1989); Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)). The three CDRs are inserted between adjacent sections known as framework regions (FRs) (light chain FRs including LFR1, LFR2, LFR3 and LFR4, and heavy chain FRs including HFR1, HFR2, HFR3 and HFR4), which are more highly conserved than the CDRs and form a scaffold supporting the highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to a class based on the amino acid sequence of the constant region of their heavy chains.The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain).

[0053]

[0077] In certain embodiments, the antibodies provided herein include any antigen-binding fragment thereof. As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody that contains one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain an intact native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab') 2 , Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bispecific antibodies, multispecific antibodies, camelized single chain domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies. Antigen-binding fragments can bind to the same antigen that the parent antibody binds.

[0054]

[0078] "Fab," with respect to an antibody, means the portion of an antibody that consists of a single light chain (both variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0055]

[0079] "Fab'" refers to a Fab fragment that includes part of the hinge region.

[0080] "F(ab') 2 " means a Fab' dimer.

[0081] "Fc" refers to the portion of an antibody (e.g., of an IgG, IgA, or IgD isotype) that consists of the second and third constant domains of a first heavy chain linked via disulfide bonds to the second and third constant domains of a second heavy chain. For antibodies of the IgM and IgE isotypes, the Fc further comprises a fourth constant domain. The Fc portion of an antibody is involved in various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0056]

[0082] "Fv" refers to the minimum fragment of an antibody that contains a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0083] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked together directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)).

[0057]

[0084] By "single chain Fv-Fc antibody" or "scFv-Fc" is meant an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0085] "Camelized single domain antibodies", "heavy chain antibodies", or "HCAbs" are antibodies that consist of two V HIt refers to antibodies that contain heavy chains and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2):25-38(1999); Muyldermans S., J Biotechnol. Jun; 74(4):277-302(2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies are originally derived from the Camelidae family (camels, dromedaries, and llamas). Camelized antibodies have no light chains but have bona fide antigen-binding capacity (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428): 446-8 (1993); Nguyen VK. et al. Immunogenetics. Apr; 54(1): 39-47 (2002); Nguyen VK. et al. Immunology. May; 109(1): 93-101 (2003)). The variable domain of heavy chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)).

[0058]

[0086] "Nanobody" refers to an antibody fragment consisting of a VHH domain and two constant domains, CH2 and CH3, of a heavy chain antibody.

[0087] A "diabody" or "dAb" refers to a small antibody fragment with two antigen-binding sites, arranged in the same polypeptide chain as the V L V linked to domain H Domain Included (V H -V L or V L -V H) (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. Jul 15; 90(14):6444-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes).

[0059]

[0088] "Domain antibody" refers to an antibody fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more V H The domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody. H The domains may target the same or different antigens.

[0060]

[0089] As used herein, the term "valency" refers to the presence of a specific number of antigen-binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen-binding fragment that has only one single antigen-binding site, and the term "multivalent" refers to an antibody or antigen-binding fragment that has multiple antigen-binding sites. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, in an antigen-binding molecule. In some embodiments, an antibody or antigen-binding fragment thereof is bivalent.

[0061]

[0090] As used herein, a "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes, which may be on the same antigen or on two different antigens.

[0062]

[0091] In certain embodiments, an "scFv dimer" is a dimer of another V H -V L Dimerized with the V moiety (dimerized via a peptide linker) H -V L and a bivalent diabody or bispecific scFv (BsFv) comprising one part V H V in other parts L In another embodiment, an "scFv dimer" is a combination of a V (linked by a peptide linker) and a V (linked by a peptide linker) to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). L1 -V H2 (also linked by a peptide linker) V H1 -V L2 and a bispecific diabody comprising V H1 and V L1 In cooperation with V H2 and V L2 cooperate, with each cooperated pair having a different antigen specificity.

[0063]

[0092] "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, "(dsFv) 2 " or "(dsFv-dsFv')" refers to three peptide chains, i.e., two Vs linked by a peptide linker (e.g., a long flexible linker) and connected via disulfide bridges. L Two V's attached to the part H In some embodiments, dsFv-dsFv' are bispecific, in which the respective disulfide-paired heavy and light chains have different antigen specificities.

[0064]

[0093] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of a heavy and / or light chain derived from one species and the remainder of the heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody may contain a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0065]

[0094] As used herein, the term "humanized" means that the antibody or antigen-binding fragment contains CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, a constant region derived from a human.

[0066]

[0095] As used herein, the term "affinity" refers to the strength of the non-covalent interactions between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.

[0096] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen. Specific binding is, for example, the K D value, i.e., the ratio of the dissociation rate to the association rate (k off / k on The binding affinity of the ligand can be characterized by its binding affinity, which is expressed by K D may be determined using any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). -6 M or less (for example, 5×10 -7 M or less, 2×10 -7 M or less, 10 -7 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 10 -8 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10-9 M or less, or 10 -9 M or less) K D The value may indicate specific binding between the antibody or antigen-binding fragment thereof and IL-36R (eg, human IL-36R).

[0067]

[0097] As used herein, the ability to "compete for binding to human IL-36R" refers to the ability of a first antibody or antigen-binding fragment to inhibit the binding interaction between human IL-36R and a second anti-IL-36R antibody to any detectable extent. In certain embodiments, an antibody or antigen-binding fragment that competes for binding to human IL-36R inhibits the binding interaction between human IL-36R and a second anti-human IL-36R antibody by at least 85%, or at least 90%. In certain embodiments, the inhibition may be greater than 95%, or greater than 99%.

[0068]

[0098] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitopes in the antigen. Epitopes may be linear or conformational (i.e., involving separated amino acid residues). For example, an antibody or antigen-binding fragment may be considered to bind to the same / closely related epitope as the reference antibody if it blocks at least 85%, or at least 90%, or at least 95% of the binding of the reference antibody to the antigen.

[0069]

[0099] As used herein, the term "amino acid" refers to an amine (-NH 2 ) and a carboxyl (-COOH) functional group. In this disclosure, the names of amino acids may also be represented as standard one-letter or three-letter codes, which are summarized below.

[0070] [Table 1]

[0071]

[0100] "Conservative substitution" in relation to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitution can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between amino acid residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between amino acid residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between amino acid residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitution usually does not cause significant changes in the conformational structure of a protein, and therefore can retain the biological activity of the protein.

[0072]

[0101] As used herein, the term "homologous" refers to a nucleic acid sequence (or its complementary strand) or amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence when optimally aligned.

[0073]

[0102] "Percent sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps to maximize the number of identical amino acids (or nucleic acids). In other words, the percent sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) with respect to the reference sequence being compared by the total number of amino acid residues (or bases) in either the candidate or reference sequence, whichever is shorter. Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for calculating percent amino acid (or nucleic acid) sequence identity can be accomplished using publicly available tools such as, for example, BLASTN, BLASTp (available from the US National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available from the European Bioinformatics Institute website, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. One skilled in the art may use the default parameters provided by the tool or may customize appropriate parameters for the alignment, for example by selecting a suitable algorithm.

[0074]

[0103] As used herein, "effector function" refers to the biological activity attributable to the binding of the Fc region of an antibody to its effector, such as the C1 complex and Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC), mediated by the interaction of the antibody with C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity (ADCC), mediated by the binding of the Fc region of an antibody to an Fc receptor on an effector cell, and phagocytosis. Effector function can be assessed using various assays, such as Fc receptor binding assays, C1q binding assays, and cytolysis assays.

[0075]

[0104] An "isolated" material is altered by the hand of man from its natural state. If an "isolated" composition or material occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not "isolated," but the same polynucleotide or polypeptide is "isolated" if it is sufficiently separated from the coexisting materials in its natural state and exists in a substantially pure state. An "isolated nucleic acid sequence" refers to a sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that has a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (ion exchange chromatography or reverse phase HPLC).

[0076]

[0105] As used herein, the term "vector" refers to a vehicle into which a genetic element is operably inserted and expression of the genetic element occurs, thereby producing a protein, RNA, or DNA encoded by the genetic element, or replicating the genetic element. A vector may be used to transform, transduce, or transfect a host cell to cause expression of the genetic element it carries into the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector may contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, a vector may contain an origin of replication. A vector may contain substances that aid in the entry of the vector into a cell, including, but not limited to, viral particles, liposomes, or protein coatings. A vector may be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) comprising a nucleic acid sequence provided herein encoding an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0077]

[0106] As used herein, the phrase "host cell" means a cell into which an exogenous polynucleotide and / or vector can be introduced or has been introduced.

[0107] The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" or "subject" are used interchangeably herein.

[0078]

[0108] As used herein, "treating" a disease, disorder, or condition or "treatment" thereof includes preventing or alleviating the disease, disorder, or condition, slowing the onset or rate of development of the disease, disorder, or condition, reducing the risk of developing the disease, disorder, or condition, preventing or delaying the onset of symptoms associated with the disease, disorder, or condition, reducing or terminating symptoms associated with the disease, disorder, or condition, completely or partially ameliorating the disease, disorder, or condition, curing the disease, disorder, or condition, or some combination thereof.

[0079]

[0109] The terms "diagnosis", "diagnose" or "diagnosing" refer to the identification of a pathological condition, disease or condition, such as the identification of an IL-36R-associated disease, or the identification of a subject having an IL-36R-associated disease that may benefit from a particular treatment regimen. In some embodiments, diagnosis includes the identification of an abnormal amount or activity of IL-36R. In some embodiments, diagnosis refers to the identification of cancer or an autoimmune disease in a subject.

[0080]

[0110] As used herein, the term "biological sample" or "sample" refers to a biological composition comprising cells and / or other molecular entities obtained or derived from a subject of interest and characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological characteristics. Biological samples include, but are not limited to, cells, tissues, organs, and / or biological fluids of a subject obtained by any method known to those of skill in the art. In some embodiments, the biological sample is a fluid sample. In some embodiments, the fluid sample is whole blood, plasma, serum, mucus (including nasal drip and sputum), peritoneal fluid, pleural fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis, abdominal fluid, peritoneal fluid, or pericardial fluid. In some embodiments, the biological sample is tissue or cells obtained from the heart, liver, spleen, lungs, kidneys, skin, or blood vessels of a subject.

[0081]

[0111] As used herein, "IL-36R" refers to the interleukin-36 receptor, a cell surface receptor and member of the IL1R family known to be involved in inflammatory responses triggered in skin and other epithelial tissues. IL-36R is a heterodimeric protein composed of the receptor subunit IL-1Rrp2 (also known as IL-1RL2, interleukin-1 receptor-like 2 or interleukin-1 receptor-related protein 2) and the co-receptor subunit interleukin-1 receptor IL-1RAcP, an auxiliary protein shared with IL-1R and IL-33R. IL-36R recognizes three different agonists, IL-36α, IL-36β, and IL-36γ (also known as IL-1F6, IL-1F8, and IL-1F9), which signal through the IL-36R / IL-1RAcP receptor to activate NF-κB and MAPKs, such as p38 and JNK, to promote inflammatory responses and induce the expression of inflammatory cytokines. There are also two receptor antagonists, IL-36Ra and IL-38, which bind to the IL-36 receptor and reduce the expression of proinflammatory cytokines.

[0082]

[0112] In certain embodiments, the IL-36R is human IL-36R, which comprises human IL-1Rrp2 and human IL-1RAcP. In some embodiments, the human IL-1Rrp2 sequence is available under Genbank accession number NP_003845.2. In some embodiments, the amino acid sequence of human IL-1Rrp2 is as set forth in SEQ ID NO:211. In some embodiments, the human IL-1RAcP sequence is available under Genbank accession number NP_002173.1. In some embodiments, the amino acid sequence of human IL-1RAcP is as set forth in SEQ ID NO:212.

[0083]

[0113] Amino acid sequence of human IL-1Rrp2 (SEQ ID NO:211):

[0114] MWSLLLCGLSIALPLSVTADGCKDIFMKNEILSASQPFAFNCTFPPITSGEVSVTWYKNSSKIPVSKIIQSRIHQDETWILFLPMEWGDSGVYQCVIKGRDSCHRIHVNLTVFEKHWCDTSIGGLPNLSDEYKQILHLGKDDS LTCHLHFPKSCVLGPIKWYKDCNEIKGERFTVLETRLLVSNVSAEDRGNYACQAILTHSGKQYEVLNGITVSITERAGYGGSVPKIIYPKNHSIEVQLGTTLIVDCNVTDTKDNTNLRCWRVNNTLVDDYYDESKRIREGVETH VSFREHNLYTVNITFLEVKMEDYGLPFMCHAGVSTAYIILQLPAPDFRAYLIGGLIALVAVAVSVVYIYNIFKIDIVLWYRSAFHSTETIVDGKLYDAYVLYPKPHKESQRHAVDALVLNILPEVLERQCGYKLFIFGRDEFPG QAVANVIDENVKLCRRLIVIVVPESLGFGLLKNLSEEQIAVYSALIQDGMKVILIELEKIEDYTVMPESIQYIKQKHGAIRWHGDFTEQSQCMKTKFWKTVRYHMPPRRCRPFPPVQLLQHTPCYRTAGPELGSRRKKCTLTTG

[0115] Amino acid sequence of human IL-1RAcP (SEQ ID NO:212):

[0116] MTLLWCVVSLYFYGILQSDASERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLKFNYSTAHSAGLTLIWYWTRQDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTGNYTCMLRNTTYCSKVAFPLEVVQKDSCFNSPM KLPVHKLYIEYGIQRITCPNVDGYFPSSVKPTITWYMGCYKIQNFNNVIPEGMNLSFLIALISNNGNYTCVVTYPENGRTFHLTRTLTVKVVGSPKNAVPPVIHSPNDHVVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTID GKKPDDITIDVTINESISHSRTEDETRTQILSIKKVTSEDLKRSYVCHARSAKGEVAKAAKVKQKVPAPRYTVELACGFGATVLLVVILIVVYHVYWLEMVLFYRAHFGTDETILDGKEYDIYVSYARNAEEEEFVLLTLRG VLENEFGYKLCIFDRDSLPGGIVTDETLSFIQKSRRLLVVLSPNYVLQGTQALLELKAGLENMASRGNINVILVQYKAVKETKVKELKRAKTVLTVIKWKGEKSKYPQGRFWKQLQVAMPVKKSPRRSSSDEQGLSYSSLKNV

[0117] The term "anti-IL-36R antibody" refers to an antibody capable of specifically binding to IL-36R (e.g., human IL-36R). The term "anti-human IL-36R antibody" refers to an antibody capable of specifically binding to human IL-36R.

[0084]

[0118] As used herein, an "IL-36R-associated" disease, disorder, or condition refers to any disease or condition caused, exacerbated, or otherwise associated with increased or decreased expression or activity of IL-36R. In some embodiments, the IL-36R-associated disease, disorder, or condition is an immune-related disorder, such as, for example, an autoimmune disease. In some embodiments, the IL-36R-associated disease, disorder, or condition is a disorder associated with excessive cell proliferation, such as, for example, cancer. In certain embodiments, the IL-36R-associated disease or condition is characterized by expression or overexpression of the IL-36R gene. In certain embodiments, the IL-36R-associated disease or condition is characterized by overexpression of IL-36R and / or dysregulation of IL-36R-mediated signaling.

[0085]

[0119] The term "pharmaceutical acceptable" indicates that the specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation and physiologically compatible with the recipient thereof.

[0086]

[0120] The term "IL-36R positive cells" as used herein refers to cells that exhibit abnormal IL-36R expression levels compared to control cells. The abnormal expression levels may be up-regulated or down-regulated compared to the levels of control cells, and may be associated with dysregulation of IL-36R-mediated signal transduction. The control cells may be normal or healthy counterpart cells, which may or may not express IL-36R. If the control cells express IL-36R, the abnormal expression levels of IL-36R positive cells may be up-regulated or down-regulated. If the control cells do not express IL-36R, the abnormal expression levels of IL-36R positive cells may be up-regulated.

[0087]

[0121] Anti-IL-36R antibody

[0122] The present disclosure provides anti-IL-36R antibodies and antigen-binding fragments thereof. The anti-IL-36R antibodies and antigen-binding fragments provided herein can specifically bind to IL-36R.

[0088]

[0123] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein exhibit a 10 -7 M or less, 8×10 -8 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 8×10 -9 M or less, 5×10 -9 M or less, 2×10 -9 M or less, 10 -9 M or less, 8×10 -10 M or less, 7×10 -10 M or less, or 6 x 10 -10 K below M D values, see, e.g., Murphy, M. et al., Current protocols in protein science, Chapter 19, unit 19.14, 2006. D Values ​​are measured by the method described in Example 6 of the present disclosure.

[0089]

[0124] The binding of the antibodies or antigen-binding fragments thereof provided herein to human IL-36R is measured using a "50% effective concentration" (EC 50 ) value. 50 The values ​​represent the concentration of antibody at which 50% of its maximal binding is observed. EC 50 Values ​​can be measured by binding assays known in the art, for example, direct or indirect binding assays such as enzyme-linked immunosorbent assay (ELISA), flow cytometry assays, and other binding assays. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have an EC of 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, 0.09 nM or less, 0.08 nM or less, 0.07 nM or less, 0.06 nM or less, or 0.05 nM or less by enzyme-linked immunosorbent assay (ELISA) assay.50 In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein specifically bind to human IL-36R at an EC of 0.1 μg / mL or less, 0.09 μg / mL or less, 0.08 μg / mL or less, 0.07 μg / mL or less, 0.06 μg / mL or less, 0.05 μg / mL or less, 0.04 μg / mL or less, 0.03 μg / mL or less, 0.02 μg / mL or less, 0.01 μg / mL or less, or 0.005 μg / mL or less by ELISA assay. 50 (i.e., 50% binding concentration). In certain embodiments, 50 Values ​​are measured by the method described in Example 7 of this disclosure.

[0090]

[0125] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein specifically bind to membrane human IL-36R as measured by a fluorescence-activated cell sorting (FACS) assay. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have an EC of 0.5 μg / mL or less, 0.4 μg / mL or less, 0.3 μg / mL or less, 0.2 μg / mL or less, 0.19 μg / mL or less, 0.18 μg / mL or less, 0.17 μg / mL or less, 0.16 μg / mL or less, 0.15 μg / mL or less, or 0.1 μg / mL or less as measured by a FACS assay. 50 In certain embodiments, the EC 50 Values ​​are measured by the method described in Example 8 of the present disclosure.

[0091]

[0126] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein specifically bind to human IL-36R as measured by biolayer interferometry (BLI). In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have a K of 2E-08M or less (preferably 1E-08M or less, e.g., 9E-09M or less, 8E-09M or less, 7E-09M or less, 6E-09M or less, 5E-09M or less, 4E-09M or less, 3E-09M or less, 2E-09M or less, or 1E-10M or less) as measured by BLI. d and has binding affinity for recombinant human IL-36R protein. In certain embodiments, d Values ​​are measured by the method described in Example 2 of the present disclosure.

[0092]

[0127] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein exhibit no detectable binding to either cynomolgus monkey IL-36R or mouse IL-36R, or exhibit binding to either cynomolgus monkey or mouse IL-36R at a level comparable to that of a negative control antibody under comparable assay conditions. In addition, the negative control antibody can be any antibody known not to bind to either cynomolgus monkey IL-36R or mouse IL-36R.

[0093]

[0128] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein can block IL-36R signaling induced by an IL-36R agonist (e.g., IL-36α, IL-36β, and / or IL-36γ, preferably IL-36α), as measured by an IL-36R reporter assay. In certain embodiments, the IL-36R reporter assay is as described in Example 3 of the present disclosure.

[0094]

[0129] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are capable of inhibiting IL-36R agonist-induced IL-6 release in cells, where the IL-36R agonist comprises IL-36α, IL-36β, and / or IL-36γ. In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are capable of inhibiting IL-36R agonist-induced TNF-a release in cells, where the IL-36R agonist comprises IL-36α, IL-36β, and / or IL-36γ.

[0095]

[0130] Exemplary Anti-IL-36R Antibodies

[0131] In certain embodiments, the present disclosure provides anti-IL-36R antibodies (e.g., anti-human IL-36R antibodies) and antigen-binding fragments thereof, including DYYX 1 X 2 (SEQ ID NO: 191), LIRNKAAGYTIYYX 3 X 4 X 5 VKG (SEQ ID NO: 192), SEQ ID NOs: 19, 33, 48, 64, 79, 94, 109, 124, 139, 20, 34, 49, 65, 80, 95, 110, 125, 140, 5, 21, 35, 50, 66, 81, 96, 111, 126, and 141; 1 is M or L, and X 2 is N, H, S or R, and X 3 is S or A, and X 4 is A or D, and X 5 is S or P. In certain embodiments, the present disclosure further encompasses antibodies and antigen-binding fragments thereof having no more than one, no more than two, or no more than three amino acid residue substitutions relative to any of the sequences herein.

[0096]

[0132] In certain embodiments, the present disclosure provides anti-IL-36R antibodies (e.g., anti-human IL-36R antibodies) and antigen-binding fragments thereof, comprising RASX18 NINIWLS (SEQ ID NO: 193), X 19 QSQSYPLT (SEQ ID NO: 194), SEQ ID NOs: 22, 36, 51, 67, 82, 97, 112, 127, 142, 7, 23, 37, 52, 68, 83, 98, 113, 128, 113, 24, 38, 53, 69, 84, 99, 114, 129, and 143; 18 is Q or R and X 19 is Q or L. In certain embodiments, the present disclosure further encompasses antibodies and antigen-binding fragments thereof having no more than one, no more than two, or no more than three amino acid residue substitutions relative to any of the sequences herein.

[0097]

[0133] As used herein, the antibody "5F7" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:1 and a light chain variable region having the sequence of SEQ ID NO:2.

[0134] As used herein, the antibody "9A6" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:17 and a light chain variable region having the sequence of SEQ ID NO:18.

[0098]

[0135] As used herein, the antibody "9C12" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:31 and a light chain variable region having the sequence of SEQ ID NO:32.

[0099]

[0136] As used herein, the antibody "10D12" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:46 and a light chain variable region having the sequence of SEQ ID NO:47.

[0100]

[0137] As used herein, the antibody "10F6" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:62 and a light chain variable region having the sequence of SEQ ID NO:63.

[0101]

[0138] As used herein, the antibody "9H11" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:77 and a light chain variable region having the sequence of SEQ ID NO:78.

[0102]

[0139] As used herein, the antibody "1C11" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:92 and a light chain variable region having the sequence of SEQ ID NO:93.

[0103]

[0140] As used herein, the antibody "1A21" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:107 and a light chain variable region having the sequence of SEQ ID NO:108.

[0104]

[0141] As used herein, the antibody "1J3" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:122 and a light chain variable region having the sequence of SEQ ID NO:123.

[0105]

[0142] As used herein, the antibody "1J22" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:137 and a light chain variable region having the sequence of SEQ ID NO:138.

[0106]

[0143] In certain embodiments, the disclosure provides anti-IL-36R antibodies and antigen-binding fragments thereof that comprise one or more (e.g., one, two, three, four, five, or six) CDR sequences of antibodies 5F7, 9A6, 9C12, 10D12, 10F6, 9H11, 1C11, 1A21, 1J3, or 1J22.

[0107]

[0144] In certain embodiments, the present disclosure provides an HCDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 3, 19, 33, 48, 64, 79, 94, 109, 124, and 139; an HCDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 20, 34, 49, 65, 80, 95, 110, 125, and 140; and an HCDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 5, 21, 35, 50, 66, 81, 96, 111, 126, and 141; and / or The present invention provides anti-IL-36R antibodies and antigen-binding fragments thereof, comprising an LCDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 6, 22, 36, 51, 67, 82, 97, 112, 127, and 142; an LCDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 7, 23, 37, 52, 68, 83, 98, 113, and 128; and an LCDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 8, 24, 38, 53, 69, 84, 99, 114, 129, and 143.

[0108]

[0145] In certain embodiments, the present disclosure provides anti-IL-36R antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO:3, an HCDR2 comprising the sequence of SEQ ID NO:4, an HCDR3 comprising the sequence of SEQ ID NO:5, and / or an LCDR1 comprising the sequence of SEQ ID NO:6, an LCDR2 comprising the sequence of SEQ ID NO:7, and an LCDR3 comprising the sequence of SEQ ID NO:8.

[0109]

[0146] In certain embodiments, the present disclosure provides anti-IL-36R antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 19, an HCDR2 comprising the sequence of SEQ ID NO: 20, an HCDR3 comprising the sequence of SEQ ID NO: 21, and / or an LCDR1 comprising the sequence of SEQ ID NO: 22, an LCDR2 comprising the sequence of SEQ ID NO: 23, and an LCDR3 comprising the sequence of SEQ ID NO: 24.

[0110]

[0147] In certain embodiments, the present disclosure provides anti-IL-36R antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 33, an HCDR2 comprising the sequence of SEQ ID NO: 34, an HCDR3 comprising the sequence of SEQ ID NO: 35, and / or an LCDR1 comprising the sequence of SEQ ID NO: 36, an LCDR2 comprising the sequence of SEQ ID NO: 37, and an LCDR3 comprising the sequence of SEQ ID NO: 38.

[0111]

[0148] In certain embodiments, the present disclosure provides anti-IL-36R antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO:48, an HCDR2 comprising the sequence of SEQ ID NO:49, an HCDR3 comprising the sequence of SEQ ID NO:50, and / or an LCDR1 comprising the sequence of SEQ ID NO:51, an LCDR2 comprising the sequence of SEQ ID NO:52, and an LCDR3 comprising the sequence of SEQ ID NO:53.

[0112]

[0149] In certain embodiments, the present disclosure provides anti-IL-36R antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 64, an HCDR2 comprising the sequence of SEQ ID NO: 65, an HCDR3 comprising the sequence of SEQ ID NO: 66, and / or an LCDR1 comprising the sequence of SEQ ID NO: 67, an LCDR2 comprising the sequence of SEQ ID NO: 68, and an LCDR3 comprising the sequence of SEQ ID NO: 69.

[0113]

[0150] In certain embodiments, the present disclosure provides anti-IL-36R antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO:79, an HCDR2 comprising the sequence of SEQ ID NO:80, an HCDR3 comprising the sequence of SEQ ID NO:81, and / or an LCDR1 comprising the sequence of SEQ ID NO:82, an LCDR2 comprising the sequence of SEQ ID NO:83, and an LCDR3 comprising the sequence of SEQ ID NO:84.

[0114]

[0151] In certain embodiments, the present disclosure provides anti-IL-36R antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO:94, an HCDR2 comprising the sequence of SEQ ID NO:95, an HCDR3 comprising the sequence of SEQ ID NO:96, and / or an LCDR1 comprising the sequence of SEQ ID NO:97, an LCDR2 comprising the sequence of SEQ ID NO:98, and an LCDR3 comprising the sequence of SEQ ID NO:99.

[0115]

[0152] In certain embodiments, the present disclosure provides anti-IL-36R antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 109, an HCDR2 comprising the sequence of SEQ ID NO: 110, an HCDR3 comprising the sequence of SEQ ID NO: 111, and / or an LCDR1 comprising the sequence of SEQ ID NO: 112, an LCDR2 comprising the sequence of SEQ ID NO: 113, and an LCDR3 comprising the sequence of SEQ ID NO: 114.

[0116]

[0153] In certain embodiments, the present disclosure provides anti-IL-36R antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 124, an HCDR2 comprising the sequence of SEQ ID NO: 125, an HCDR3 comprising the sequence of SEQ ID NO: 126, and / or an LCDR1 comprising the sequence of SEQ ID NO: 127, an LCDR2 comprising the sequence of SEQ ID NO: 128, and an LCDR3 comprising the sequence of SEQ ID NO: 129.

[0117]

[0154] In certain embodiments, the present disclosure provides anti-IL-36R antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 139, an HCDR2 comprising the sequence of SEQ ID NO: 140, an HCDR3 comprising the sequence of SEQ ID NO: 141, and / or an LCDR1 comprising the sequence of SEQ ID NO: 142, an LCDR2 comprising the sequence of SEQ ID NO: 113, and an LCDR3 comprising the sequence of SEQ ID NO: 143.

[0118]

[0155] Table 1 below shows the CDR amino acid sequences of antibodies 5F7, 9A6, 9C12, 10D12, 10F6, 9H11, 1C11, 1A21, 1J3, and 1J22. The boundaries of the CDRs were defined or identified according to the Kabat rules. Table 2 below shows the heavy and light chain variable region amino acid sequences of antibodies 5F7, 9A6, 9C12, 10D12, 10F6, 9H11, 1C11, 1A21, 1J3, and 1J22.

[0119]

[0156]

[0120] [Table 2-1]

[0121] [Table 2-2]

[0122]

[0157]

[0123] [Table 3-1]

[0124] [Table 3-2]

[0125]

[0158] Given that antibodies 5F7, 9A6, 9C12, 10D12, 10F6, 9H11, 1C11, 1A21, 1J3, and 1J22 can each bind to IL-36R and antigen-binding specificity is provided primarily by the CDR1, CDR2, and CDR3 regions, the HCDR1, HCDR2 and HCDR3 sequences, and LCDR1, LCDR2 and LCDR3 sequences of antibodies 5F7, 9A6, 9C12, 10D12, 10F6, 9H11, 1C11, 1A21, 1J3, and 1J22 can be "mixed and matched" (i.e., CDRs of different antibodies can be mixed and matched, but each antibody must include HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3) to create anti-IL-36R binding molecules of the disclosure. Such "mixed and matched" antibodies can be tested for IL-36R binding using the binding assays described above and in the Examples. Preferably, when VH CDR sequences are mixed and matched, the HCDR1, HCDR2 and / or HCDR3 sequences from a particular VH sequence are replaced with structurally similar CDR sequences. Similarly, when VL CDR sequences are mixed and matched, the LCDR1, LCDR2 and / or LCDR3 sequences from a particular VL sequence are preferably replaced with structurally similar CDR sequences. It will be readily apparent to one of ordinary skill in the art that novel VH and VL sequences can be created by replacing one or more VH and / or VL CDR region sequences for monoclonal antibodies 5F7, 9A6, 9C12, 10D12, 10F6, 9H11, 1C11, 1A21, 1J3, and 1J22 with structurally similar sequences from the CDR sequences disclosed herein.

[0126]

[0159] It is known that CDR is involved in antigen binding.However, it has been found that not all six CDRs are essential or invariant.In other words, it is possible to replace, change or modify one or more CDRs in anti-IL-36R antibody 5F7, 9A6, 9C12, 10D12, 10F6, 9H11, 1C11, 1A21, 1J3 and 1J22 while substantially retaining specific binding affinity to IL-36R.

[0127]

[0160] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof are capable of specifically binding to IL-36R. The CDR sequences provided in Table 1 above are derived from mouse antibodies, but they can be grafted into any suitable FR sequence of any suitable species, such as mouse, human, rat, rabbit, among others, using suitable methods known in the art, such as recombinant techniques.

[0128]

[0161] In certain embodiments, the antibody and antigen-binding fragment provided herein are humanized. Humanized antibody or antigen-binding fragment is desirable for its reduced immunogenicity in humans. Humanized antibody is chimeric in its variable region because non-human CDR sequence is grafted to human or substantially human FR sequence. Humanization of antibody or antigen-binding fragment can be essentially performed by replacing non-human (such as mouse) CDR gene with corresponding human CDR gene in human immunoglobulin gene (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0129]

[0162] To this end, suitable human heavy and light chain variable domains can be selected using methods known in the art. In an illustrative example, a "best-fit" approach can be used, in which the variable domain sequence of a non-human (e.g., rodent) antibody is screened or BLASTed against known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as a human scaffold for grafting the non-human CDR sequences (see, e.g., Sims et al., (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mot. Biol. 196:901). Alternatively, frameworks derived from the consensus sequence of all human antibodies may be used for grafting of non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0130]

[0163] Table 3 below shows the CDR amino acid sequences of the humanized antibodies of antibody 5F7, designated 5F7-hu-2, 5F7-hu-3, 5F7-hu-5, 5F7-2a1, 5F7-2a6, 5F7-2a8, 5F7-2c4, 5F7-2d3, 5F7-2g10, and 5F7-2h1. The boundaries of the CDRs are defined or specified according to the Kabat convention. Table 4 below shows the heavy and light chain variable region amino acid sequences of the humanized antibodies 5F7-hu-2, 5F7-hu-3, 5F7-hu-5, 5F7-2a1, 5F7-2a6, 5F7-2a8, 5F7-2c4, 5F7-2d3, 5F7-2g10, and 5F7-2h1. Table 5 below shows the FR amino acid sequences of the eight humanized antibodies 5F7-hu-2, 5F7-hu-3, 5F7-hu-5, 5F7-2a1, 5F7-2a6, 5F7-2a8, 5F7-2c4, 5F7-2d3, 5F7-2g10, and 5F7-2h1.

[0131]

[0164]

[0132] [Table 4-1]

[0133] [Table 4-2]

[0134]

[0165]

[0135] [Table 5-1]

[0136] [Table 5-2]

[0137]

[0166]

[0138] [Table 6-1]

[0139] [Table 6-2]

[0140] [Table 6-3]

[0141]

[0167] In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein consists of substantially all human sequences, except for the CDR sequences, which are non-human. In some embodiments, the variable region FR and, if present, the constant region are entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences can be from different human immunoglobulin genes, e.g., the FR sequences are from one human antibody and the constant region is from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises human heavy chain HFR1-4 and / or light chain LFR1-4.

[0142]

[0168] In some embodiments, the FR region derived from a human may comprise the same amino acid sequence as the original human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are replaced with the corresponding residue from the parent non-human antibody. This is desirable in certain embodiments to allow the humanized antibody or fragment thereof to closely approximate the structure of the non-human parent antibody, thereby optimizing the binding properties (e.g., increasing binding affinity). In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein comprises the substitution of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues in each of the human FR sequences, or the substitution of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues in all of the FR sequences of the heavy or light chain variable domains. In some embodiments, such changes in amino acid residues may be present only in the heavy chain FR regions, only in the light chain FR regions, or in both chains. In certain embodiments, one or more amino acids of the human FR sequences are randomly mutated to increase binding affinity. In certain embodiments, one or more amino acids of the human FR sequences are backmutated to the corresponding amino acid of the parent non-human antibody to increase binding affinity.

[0143]

[0169] In certain embodiments, the present disclosure also provides humanized anti-IL-36R antibodies and antigen-binding fragments thereof, comprising: 6 VQLX7ESGGGLVKPGGSLRLSCAASGX 8 X 9 FX 10 Heavy chain HFR1, WX comprising the sequence of (SEQ ID NO: 195) or a homologous sequence having at least 80% sequence identity thereto 11 RQAPGKGLEWVX 12 Heavy chain HFR2, RFTISRDX comprising the sequence of (SEQ ID NO: 196) or a homologous sequence having at least 80% sequence identity thereto 13 X 14 KSX 15 LYLQMNSLX 16 X 17and a heavy chain HFR3 comprising the sequence of SEQ ID NO: 197, or a homologous sequence having at least 80% sequence identity thereto, and a heavy chain HFR4 comprising the sequence of SEQ ID NO: 157, or a homologous sequence having at least 80% sequence identity thereto; 6 is Q or E and X 7 is Q or V, and X 8 is F or Y, and X 9 is A, D or N, and X 10 is T or G, and X 11 is I or V, and X 12 is S or A, and X 13 is N or D, and X 14 is A or S, and X 15 is S or T, and X 16 is R or K, and X 17 is A or T, and antigen-binding fragments thereof.

[0144]

[0170] In certain embodiments, the present disclosure provides humanized anti-IL-36R antibodies and antigen-binding fragments thereof, comprising: 20 IVMTQSPX 21 X 22 X 23 SX 24 SX 25 GX 26 RX 27 TX 28 X 29 Light chain LFR1, WYQQKPGX, comprising the sequence of C (SEQ ID NO: 198) or a homologous sequence having at least 80% sequence identity thereto 30 APX 31 Light chain LFR2, GVPX, comprising the sequence LFIY (SEQ ID NO: 199) or a homologous sequence having at least 80% sequence identity thereto 32 RFSGSGSGTX 33 FTLTISSLQX 34 EDFAX 35 A light chain LFR3 having a sequence of SEQ ID NO: 161 or a homologous sequence having at least 80% sequence identity thereto, and a light chain LFR4 having a sequence of SEQ ID NO: 161 or a homologous sequence having at least 80% sequence identity thereto,20 is D or E, and X 21 is S or A, and X 22 is S or T, and X 23 is L or V, and X 24 is A or V, and X 25 is V or P, and X 26 is D or E, and X 27 is V or A, and X 28 is I or L, and X 29 is T or S, and X 30 is Q or K, and X 31 is K or R, and X 32 is S or A, and X 33 is D or E, and X 34 is S or P, and X 35 is T or V, and antigen-binding fragments thereof.

[0145]

[0171] In certain embodiments, the present disclosure also provides humanized anti-IL-36R antibodies and antigen-binding fragments thereof, comprising heavy chain HFR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 165, 174, 178, 181, 154, 178, 186, 178, and 190, heavy chain HFR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 166, 175, and 155, heavy chain HFR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 167 and 156, and heavy chain HFR4 comprising the sequence of SEQ ID NO: 157, and / or light chain LFR1 comprising a sequence from the group consisting of SEQ ID NOs: 168 and 158, light chain LFR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 169 and 159, light chain LFR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 170 and 160, and light chain LFR4 comprising the sequence of SEQ ID NO: 161.

[0146]

[0172] In certain embodiments, the disclosure also provides humanized anti-IL-36R antibodies and antigen-binding fragments thereof comprising HFR1, HFR2, HFR3 and / or HFR4 sequences contained in a heavy chain variable region selected from the group consisting of 5F7-hu-2-VH (SEQ ID NO: 162), 5F7-hu-3-VH (SEQ ID NO: 171), 5F7-hu-5-VH (SEQ ID NO: 176), 5F7-2a1-VH (SEQ ID NO: 179), 5F7-2a6-VH (SEQ ID NO: 182), 5F7-2a8-VH (SEQ ID NO: 149), 5F7-2c4-VH (SEQ ID NO: 183), 5F7-2d3-VH (SEQ ID NO: 185), 5F7-2g10-VH (SEQ ID NO: 187), and 5F7-2h1-VH (SEQ ID NO: 189).

[0147]

[0173] In certain embodiments, the present disclosure also provides humanized anti-IL-36R antibodies and antigen-binding fragments thereof comprising LFR1, LFR2, LFR3 and / or LFR4 sequences contained in a light chain variable region selected from the group consisting of 5F7-hu-2-VL (SEQ ID NO: 163), 5F7-hu-3-VL (SEQ ID NO: 172), 5F7-hu-5-VL (SEQ ID NO: 177), 5F7-2a1-VL / 5F7-2a6-VL / 5F7-2a8-VL / 5F7-2c4-VL / 5F7-2d3-VL / 5F7-2g10-VL / 5F7-2h1-VL (SEQ ID NO: 150).

[0148]

[0174] In certain embodiments, humanized anti-IL-36R antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO:162, SEQ ID NO:171, SEQ ID NO:176, SEQ ID NO:179, SEQ ID NO:182, SEQ ID NO:149, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, and SEQ ID NO:189, and / or a light chain variable domain sequence selected from the group consisting of SEQ ID NO:163, SEQ ID NO:172, SEQ ID NO:177, and SEQ ID NO:150.

[0149]

[0175] The present disclosure also provides 1) "5F7-hu-2", which comprises the heavy chain variable region of 5F7-hu-2-VH (SEQ ID NO: 162) and the light chain variable region of 5F7-hu-2-VL (SEQ ID NO: 163); 2) "5F7-hu-3", which comprises the heavy chain variable region of 5F7-hu-3-VH (SEQ ID NO: 171) and the light chain variable region of 5F7-hu-3-VL (SEQ ID NO: 172); 3) "5F7-hu-5", comprising the heavy chain variable region of 5F7-hu-5-VH (SEQ ID NO: 176) and the light chain variable region of 5F7-hu-5-VL (SEQ ID NO: 177); 4) "5F7-2a1", comprising the heavy chain variable region of 5F7-2a1-VH (SEQ ID NO: 179) and the light chain variable region of 5F7-2a1-VL (SEQ ID NO: 150); 5) "5F7-2a6", comprising the heavy chain variable region of 5F7-2a6-VH (SEQ ID NO: 182) and the light chain variable region of 5F7-2a6-VL (SEQ ID NO: 150); 6) "5F7-2a8", comprising the heavy chain variable region of 5F7-2a8-VH (SEQ ID NO: 149) and the light chain variable region of 5F7-2a8-VL (SEQ ID NO: 150); 7) "5F7-2c4" comprising the heavy chain variable region of 5F7-2c4-VH (SEQ ID NO: 183) and the light chain variable region of 5F7-2c4-VL (SEQ ID NO: 150); 8) "5F7-2d3", comprising the heavy chain variable region of 5F7-2d3-VH (SEQ ID NO: 185) and the light chain variable region of 5F7-2d3-VL (SEQ ID NO: 150); 9) "5F7-2g10" comprising the heavy chain variable region of 5F7-2g10-VH (SEQ ID NO: 187) and the light chain variable region of 5F7-2g10-VL (SEQ ID NO: 150); 10) "5F7-2h1" comprising the heavy chain variable region of 5F7-2h1-VH (SEQ ID NO: 189) and the light chain variable region of 5F7-2h1-VL (SEQ ID NO: 150) Exemplary humanized antibodies of 5F7 are also provided, including:

[0150]

[0176] These exemplary humanized anti-IL-36R antibodies retained specific binding ability or affinity for IL-36R and were at least as good or better in that respect than the parental murine antibody 5F7, e.g., data provided in Examples 4-16.

[0151]

[0177] In some embodiments, the anti-IL-36R antibodies and antigen-binding fragments provided herein comprise all or a portion of the heavy chain variable domain and / or all or a portion of the light chain variable domain. In one embodiment, the anti-IL-36R antibodies or antigen-binding fragments provided herein are single-domain antibodies consisting of all or a portion of the heavy chain variable domain provided herein. Further information on such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0152]

[0178] In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin (Ig) constant region, which optionally further comprises a heavy and / or light chain constant region. In certain embodiments, the heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region (or optionally a CH2-CH3-CH4 region). In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein comprise a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In certain embodiments, the light chain constant region comprises a Cκ or Cλ. The constant region of the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein may be identical to the sequence of the wild-type constant region or may differ in one or more mutations.

[0153]

[0179] In certain embodiments, the heavy chain constant region comprises an Fc region. The Fc region is known to mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of antibodies. The Fc regions of different Ig isotypes have different abilities to induce effector functions. For example, it is recognized that the Fc regions of IgG1 and IgG3 induce both ADCC and CDC more effectively than the Fc regions of IgG2 and IgG4. In certain embodiments, the anti-IL-36R antibodies and antigen-binding fragments thereof provided herein comprise an Fc region of an IgG1 or IgG3 isotype capable of inducing ADCC or CDC, or comprise a constant region of an IgG4 or IgG2 isotype with reduced or depleted effector function. In certain embodiments, the anti-IL-36R antibodies and antigen-binding fragments thereof provided herein comprise a wild-type human IgG4 Fc region or other wild-type human IgG4 allele. In certain embodiments, the anti-IL-36R antibodies and antigen-binding fragments thereof provided herein comprise a human IgG4 Fc region comprising an S228P mutation. In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 203. In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein comprise a human IgG4 Fc region comprising the following mutations: M252Y / S254T / T256E (YTE). In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein comprise a human IgG4 Fc region comprising the following mutations: T307Q / N434A (QA). In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein comprise a human IgG4 Fc region comprising the following mutations: S228P and M252Y / S254T / T256E (YTE). In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 201.In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein comprise a human IgG4 Fc region comprising the S228P mutation and the T307Q / N434A (QA) mutation. In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 204. In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein comprise a human IgG4 Fc region comprising the S228P mutation, the M252Y / S254T / T256E (YTE) mutation, and the T307Q / N434A (QA) mutation.

[0154]

[0180] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have sufficient specific binding affinity for IL-36R to provide for diagnostic and / or therapeutic purposes.

[0155]

[0181] The antibodies or antigen-binding fragments thereof provided herein can be monoclonal, polyclonal, humanized, chimeric, recombinant, bispecific, multispecific, labeled, bivalent, anti-idiotypic, or fusion proteins. Recombinant antibodies are antibodies prepared using recombinant methods in vitro, rather than in an animal.

[0156]

[0182] In certain embodiments, the present disclosure provides an anti-IL-36R antibody or antigen-binding fragment thereof that does not compete for binding to human IL-36R with an antibody selected from the group consisting of: a) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 205 and a light chain variable region comprising the sequence of SEQ ID NO: 206; b) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 209 and a light chain variable region comprising the sequence of SEQ ID NO: 210, wherein the antibody or antigen-binding fragment thereof is not that of BI655130 or REGN14.

[0157]

[0183] As used herein, "BI655130" refers to an antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:205 and a light chain variable region having the amino acid sequence of SEQ ID NO:206.

[0158]

[0184] As used herein, "REGN14" refers to an antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:209 and a light chain variable region having the amino acid sequence of SEQ ID NO:210.

[0159]

[0185] Antibody variants

[0186] The antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.

[0160]

[0187] In certain embodiments, the antibody variants comprise one or more of the CDR sequences provided in Tables 1 and 3 above, one or more of the non-CDR sequences of the heavy or light chain variable regions provided in Tables 2 and 4 above, and / or one or more modifications or substitutions in the constant region (e.g., Fc region). Such variants retain the binding specificity of their parent antibodies to IL-36R, but have one or more desirable properties imparted by the modifications or substitutions. For example, the antibody variants may have improved antigen binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or eliminated effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., one or more introduced cysteine ​​residues).

[0161]

[0188] Methods known in the art, such as "alanine scanning mutagenesis," may be used to screen parent antibody sequences to identify suitable or preferred residues for modification or substitution (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies are produced and screened for the desired properties. If substitution at a particular amino acid position demonstrates a desired change in function, that position can be identified as a potential residue for modification or substitution. Potential residues may be further evaluated by substituting different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0162]

[0189] Affinity variants

[0190] The affinity variants of the antibodies may include modifications or substitutions in one or more CDR sequences provided in Tables 1 and 3 above, one or more FR sequences provided in Table 5 above, or the heavy or light chain variable region sequences provided in Tables 2 and 4 above. Since it is known in the art that a CDR region is adjacent to two FR regions in the variable region, the FR sequences can be easily identified by those skilled in the art based on the CDR sequences in Tables 1 and 3 above and the variable region sequences in Tables 2 and 4 above. The affinity variants retain the specific binding affinity of the parent antibody to IL-36R, or even have improved IL-36R specific binding affinity over the parent antibody. In certain embodiments, at least one (or all) of the substitutions in the CDR sequences, FR sequences, or variable region sequences include conservative substitutions.

[0163]

[0191] Those skilled in the art will understand that one or more amino acid residues may be substituted in the CDR sequences provided in Tables 1 and 3 above, and the variable region sequences provided in Tables 2 and 4 above, and the resulting antibody or antigen-binding fragment may still retain or even have improved binding affinity or capacity to IL-36R. To achieve this goal, various methods known in the art may be used. For example, phage display technology may be used to generate and express a library of antibody variants (such as Fab or scFv variants), which are then screened for binding affinity to human IL-36R. As another example, computer software may be used to virtually mimic the binding of an antibody to human IL-36R, and amino acid residues on the antibody that form the binding interface may be identified. Such residues may be avoided in substitutions to prevent a reduction in binding affinity, or may be targeted for substitution to provide stronger binding.

[0164]

[0192] In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein comprises one or more amino acid residue substitutions in one or more of the CDR sequences and / or in one or more of the FR sequences. In certain embodiments, the affinity variant comprises a total of 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less substitutions in the CDR sequences and / or in the FR sequences.

[0165]

[0193] In certain embodiments, an anti-IL-36R antibody or antigen-binding fragment thereof comprises one, two, or three CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the CDRs listed in Tables 1 and 3 above, yet retains specific binding affinity for IL-36R at similar or even higher levels than its parent antibody.

[0166]

[0194] In certain embodiments, an anti-IL-36R antibody or antigen-binding fragment thereof comprises one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the variable region sequences listed in Tables 2 and 4 above, and retains specific binding affinity for IL-36R at a similar or even higher level than its parent antibody. In some embodiments, a total of 1-10 amino acids are substituted, inserted, or deleted in the variable region sequences listed in Tables 2 and 4 above. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (e.g., within the FRs).

[0167]

[0195] Glycosylation variants

[0196] The anti-IL-36R antibodies or antigen-binding fragments thereof provided herein also encompass glycosylation variants obtained by increasing or decreasing the degree of glycosylation of the antibodies or antigen-binding fragments thereof.

[0168]

[0197] The antibody or antigen-binding fragment thereof may contain one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of an antibody is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, e.g., an asparagine residue in a tripeptide sequence, such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by modifying the amino acid sequence such that one of the above tripeptide sequences (for N-linked glycosylation sites) or a serine or threonine residue (for O-linked glycosylation sites) present in the sequence is substituted. New glycosylation sites can likewise be created by introducing such a tripeptide sequence or a serine or threonine residue.

[0169]

[0198] In certain embodiments, the anti-IL-36R antibodies and antigen-binding fragments provided herein contain a mutation at N297 (eg, N297A, N297Q, or N297G) to remove a glycosylation site.

[0170]

[0199] Cysteine ​​engineered variants

[0200] The anti-IL-36R antibodies or antigen-binding fragments thereof provided herein also include cysteine ​​engineered variants that contain one or more introduced free cysteine ​​amino acid residues.

[0171]

[0201] A free cysteine ​​residue is a residue that is not part of a disulfide bridge. Cysteine ​​engineered variants are useful for conjugating, for example, with cytotoxic and / or imaging compounds, labels, or especially radioisotopes, at the site of engineered cysteine, for example, by maleimide or haloacetyl. Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine ​​residues are known in the art, see, for example, WO2006 / 034488.

[0172]

[0202] Fc variants

[0203] The anti-IL-36R antibodies or antigen-binding fragments thereof provided herein also encompass Fc variants comprising modification or substitution of one or more amino acid residues in the Fc region and / or hinge region to provide altered effector functions, such as, for example, ADCC and CDC. Methods for modifying ADCC activity by antibody engineering have been described in the art, see, for example, Shields RL.et al., J Biol Chem.2001.276(9):6591-604; Idusogie EE.et al., J Immunol.2000.164(8):4178-84; Steurer W.et al., J Immunol.1995,155(3):1165- 74; Idusogie EE.et al., J Immunol.2001,166(4):2571-5; Lazar GA.et al., PNAS,2006,103(11):4005-4010; Ryan MC.et al., Mol.Cancer Ther.,2007,6:3009-3018; Richards JO,.et al., Mol Cancer See Ther. 2008, 7(8):2517-27; Shields R Let al., J. Biol. Chem, 2002, 277:26733-26740; Shinkawa T. et al., J. Biol. Chem, 2003, 278:3466-3473.

[0173]

[0204] The CDC activity of the antibodies or antigen-binding fragments provided herein can also be modified, for example, by improving or diminishing C1q binding and / or CDC (see, e.g., WO99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351 for other examples of Fc region variants). One or more amino acids selected from amino acid residues 329, 331, and 322 of the Fc region can be replaced with different amino acid residues to modify C1q binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC) (see, e.g., U.S. Patent No. 6,194,551 by Idusogie et al.). One or more amino acid substitutions can also be introduced to modify the ability of the antibody to fix complement (see, PCT Publication WO 94 / 29351 by Bodmer et al.).

[0174]

[0205] In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein have reduced effector function and comprise one or more amino acid substitutions in IgG1 at positions selected from the group consisting of 234, 235, 237, and 238, 268, 297, 309, 330, and 331. In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein are of the IgG1 isotype and comprise one or more amino acid substitutions selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A, L234F, L235E, P331S, and any combination thereof. In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein are of the IgG2 isotype and contain one or more amino acid substitutions selected from the group consisting of H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A, or any combination thereof (e.g., H268Q / V309L / A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S). In certain embodiments, the anti-IL-36R antibody or antigen-binding fragment thereof provided herein is of IgG4 isotype and contains one or more amino acid substitutions selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A, M252Y / S254T / T256E, T307Q / N434, and any combination thereof. In certain embodiments, the anti-IL-36R antibody or antigen-binding fragment thereof provided herein is of IgG2 / IgG4 cross-isotype. Examples of IgG2 / IgG4 cross-isotypes are described in Rother RP et al., Nat Biotechnol 25:1256-1264(2007).

[0175]

[0206] In certain embodiments, the anti-IL-36R antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and contain the following amino acid substitutions: M252Y / S254T / T256 (YTE). In certain embodiments, the anti-IL-36R antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and contain the following amino acid substitutions: T307Q / N434A (QA).

[0176]

[0207] In certain embodiments, the anti-IL-36R antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and include one or more amino acid substitutions, for example at position 228. In certain embodiments, the anti-IL-36R antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and include an S228P mutation in the Fc region. In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments provided herein include a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 203. In certain embodiments, the anti-IL-36R antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and include an S228P mutation and an M252Y / S254T / T256 (YTE) mutation amino acid substitution. In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments provided herein include a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 201. In certain embodiments, the anti-IL-36R antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and contain the amino acid substitutions of S228P and T307Q / N434A (QA) mutations. In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments provided herein comprise a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO:204.

[0177]

[0208] In certain embodiments, the anti-IL-36R antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions that improve pH-dependent binding to neonatal Fc receptor (FcRn). Such variants can have an extended pharmacokinetic half-life because they bind to FcRn at acidic pH, allowing it to escape degradation in lysosomes and then be translocated and released extracellularly. Methods for engineering antibodies or antigen-binding fragments thereof to improve binding affinity to FcRn are known in the art, see, e.g., Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277(2010); and Hinton, P. et al., J. Immunology, 176:346-356(2006).

[0178]

[0209] In certain embodiments, the anti-IL-36R antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions that facilitate and / or promote heterodimerization at the interface of the Fc region. These modifications include the introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, where the protuberance is located within the cavity to promote interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating antibodies with these modifications are known in the art, for example, as described in U.S. Pat. No. 5,731,168.

[0179]

[0210] antigen-binding fragment

[0211] Anti-IL-36R antigen-binding fragments are also provided herein. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-IL-36R antibodies provided herein, including, for example, the exemplary antibodies whose CDRs are shown in Tables 1 and 3 above and whose variable sequences are shown in Tables 2 and 4 above, as well as various variants thereof (affinity variants, glycosylation variants, Fc variants, cysteine ​​engineered variants, etc.).

[0180]

[0212] In certain embodiments, the anti-IL-36R antigen-binding fragments provided herein include diabodies, Fab, Fab', F(ab') 2 , Fd, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single-chain domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies.

[0181]

[0213] A variety of techniques are available for the production of such antigen-binding fragments. Illustrative methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression in host cells such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., for ScFv), and F(ab') 2 The chemical coupling of two Fab'-SH fragments to form the fragment (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

[0182]

[0214] In certain embodiments, the antigen-binding fragment is an scFv. The generation of scFvs is described, for example, in WO 93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458. ScFvs can be fused to effector proteins at the amino or carboxyl terminus to provide fusion proteins (see, for example, Antibody Engineering, edited by Borrebaeck).

[0183]

[0215] In certain embodiments, the anti-IL-36R antibodies or antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. Any molecule with more than two valencies is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0184]

[0216] A bivalent molecule is monospecific if both binding sites are specific for binding to the same antigen or epitope. In certain embodiments, this provides stronger binding to antigen or epitope than its monovalent counterpart. Similarly, multivalent molecules can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, the first valency of the binding site and the second valency of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have the same specificity but different sequence).

[0185]

[0217] Bivalent molecules can also be bispecific if the two binding sites are specific for different antigens or epitopes. This also applies to multivalent molecules. For example, a trivalent molecule can be bispecific if the two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0186]

[0218] bispecific antibody

[0219] In certain embodiments, the anti-IL-36R antibody or antigen-binding fragment thereof is bispecific, hi certain embodiments, the antibody or antigen-binding fragment thereof is further linked to a second functional moiety having a different binding specificity than the IL-36R antibody or antigen-binding fragment thereof.

[0187]

[0220] In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to a second antigen other than IL-36R or a second epitope on IL-36R. In some embodiments, the second antigen other than IL-36R is selected from the group consisting of IL-17, IL-23, TNF, IL-12, and IL-1.

[0188]

[0221] Conjugates

[0222] In some embodiments, the anti-IL-36R antibody or antigen-binding fragment thereof further comprises one or more conjugate moieties. The conjugate moieties can be linked to the antibody or antigen-binding fragment thereof. The conjugate moieties are moieties that can bind to the antibody or antigen-binding fragment thereof. It is contemplated that various conjugate moieties can be linked to the antibody or antigen-binding fragment thereof provided herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate moieties can be linked to the antibody or antigen-binding fragment thereof by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among others. In some embodiments, the antibody or antigen-binding fragment thereof can be linked to one or more conjugates via a linker.

[0189]

[0223] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein may be engineered to contain specific sites in addition to the epitope-binding moiety that are available for attachment to one or more conjugate moieties. For example, such sites may contain one or more reactive amino acid residues, such as cysteine ​​or histidine residues, that facilitate covalent linkage to the conjugate moiety.

[0190]

[0224] In some embodiments, the antibody moiety is linked to the conjugate moiety through a chemical bond or linker, hi some embodiments, the antibody moiety and the conjugate moiety are linked using a variety of well-known bifunctional reagents and chemistries suitable for conjugation to proteins. Such reagents include, but are not limited to, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HQ), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds bis-(p-azidobenzoyl)-hexane-diamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0191]

[0225] In certain embodiments, the antibody or antigen-binding fragment thereof may be indirectly linked to the conjugate moiety or through another conjugate moiety.For example, the antibody or antigen-binding fragment thereof provided herein may be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin.In some embodiments, the conjugate moiety comprises a clearance modifier (e.g., a polymer such as PEG that extends half-life), a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a detectable label (e.g., a luminescent label, a fluorescent label, an enzyme substrate label), a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, a purification moiety, or other therapeutic agent or drug.

[0192]

[0226] Therapeutic agents or drugs useful as conjugate moieties can be those useful for treating psoriasis, for example, GPP (generalized pustular psoriasis), PPP (palmoplantar pustulosis), HS (hidradenitis suppurativa), and the like.

[0193]

[0227] In some embodiments, the conjugate moiety comprises a hormone-based immunosuppressant. In some embodiments, the conjugate moiety comprises a glucocorticoid or steroid. Non-limiting exemplary glucocorticoids or steroids include budesonide, flunisolide, triamcinolone acetonide, fluticasone propionate, beclomethasone dipropionate, and ciclesonide.

[0194]

[0228] In some embodiments, the conjugate moiety comprises a therapeutic agent or drug for treating psoriasis. In some embodiments, the conjugate moiety can be a non-biological drug, such as methotrexate, cyclosporine, fumaric acid esters (FAE), hydroxycarbamide, fumarate salts (e.g., dimethyl fumarate), retinoids (synthetic forms of vitamin A), glucocorticoids, or steroids. In some embodiments, the conjugate moiety can be a biologic that blocks immune processes involved in psoriasis, targeting specific aspects of the immune system that contribute to psoriasis. In some embodiments, the biologic is a monoclonal antibody or antigen-binding fragment thereof, such as anti-IL17, anti-IL12 / 23, anti-IL23, and / or anti-TNF alpha. Examples of such monoclonal antibodies include, but are not limited to, infliximab, adalimumab, golimumab, certolizumab pegol, ixekizumab, ustekinumab, guselkumab, efalizumab, alefacept, secukinumab, and brodalumab.

[0195]

[0229] In some embodiments, the conjugate moiety comprises a therapeutic agent or drug for treating GPP (generalized pustular psoriasis). In some embodiments, the conjugate moiety comprises a therapeutic agent or drug, such as a glucocorticoid or steroid, etanercept, PUVA, hydroxyurea, dapsone, cyclosporine A, adalimumab, etretinate, isotretinoin, or acitretin.

[0196]

[0230] In some embodiments, the conjugate moiety comprises a therapeutic agent or drug for treating PPP (palmoplantar pustulosis). In some embodiments, the conjugate moiety comprises a therapeutic agent or drug, such as a retinoid, cyclosporine, tetracycline, colchicine, Tripterygium wilfordii, Tripterygium hypoglaucum hutch, or an anti-interleukin 23 monoclonal antibody (e.g., guselkumab) or an antigen-binding fragment thereof.

[0197]

[0231] In some embodiments, the conjugate moiety comprises a therapeutic agent or drug for treating HS (hidradenitis suppurativa). In some embodiments, the conjugate moiety comprises a therapeutic agent or drug, such as a corticosteroid, an antibiotic, an antiandrogen, or an anti-inflammatory agent. Examples of antibiotics include, but are not limited to, rifampicin, clindamycin, tetracycline, and minocycline. Examples of antiandrogens include, but are not limited to, spironolactone, flutamide, cyproterone acetate, ethinyl estradiol, finasteride, dutasteride, and metformin. Examples of anti-inflammatory agents include, but are not limited to, TNF inhibitors, such as infliximab, etanercept, and adalimumab.

[0198]

[0232] In some embodiments, the conjugate moiety comprises an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordi protein, dianthin protein, Phytolacca americana protein, Momordica chalantia inhibitor, curcin, crotin, Sapoonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0199]

[0233] A "toxin" can be any agent that is detrimental to or damages or kills a cell. Examples of toxins include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethate, 5-fluorouracil decarbazine), 5-fluorouracil decarbazine, ... These include, without limitation, cisplatin, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), antimitotic agents (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin binders.

[0200]

[0234] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioisotopes, luminescent labels, chromogenic moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection. A variety of radioisotopes are available for the production of such radioconjugates. Examples include: 211 At, 131 I, 125I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Radioisotopes include Pb, and Lu. In some embodiments, the conjugate moiety may include a radioisotope for scintigraphic detection, or a spin label for NMR detection or MRI. Suitable radioisotopes or spin labels include: 123 I, 131 I, 111 In, 13 C. 19 F, 15 N, 17 Examples include various isotopes of O, Gd, Mn, and Fe.

[0201]

[0235] In certain embodiments, the conjugate moiety may be a clearance modifier that helps to extend the half-life of the antibody. Illustrative examples include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, and others. The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody varies, and when two or more polymers are attached, they may be the same or different molecules.

[0202]

[0236] In certain embodiments, the conjugated moiety can be a purification moiety, such as a magnetic bead.

[0237] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are used as the basis for a conjugate.

[0203]

[0238] Polynucleotides and Recombinant Methods

[0239] The present disclosure provides an isolated polynucleotide encoding an anti-IL-36R antibody or antigen-binding fragment thereof provided herein. As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as encompasses the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0204]

[0240] DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody). The encoding DNA may also be obtained by synthetic methods.

[0205]

[0241] The isolated polynucleotide encoding the anti-IL-36R antibody or antigen-binding fragment thereof can be inserted into a vector for further cloning (amplification of the DNA) or expression using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0206]

[0242] The present disclosure provides a vector comprising the isolated polynucleotide provided herein. In certain embodiments, the polynucleotide provided herein encodes an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40), lambda phage, and M13 phage, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, p GEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, p Includes LexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0207]

[0243] A vector containing a polynucleotide sequence encoding an antibody or an antigen-binding fragment thereof can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or DNA expression in a vector herein are prokaryotic cells, yeast, or higher eukaryotic cells as described above. Suitable prokaryotic cells for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Escherichia, Enterobacteriaceae, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0208]

[0244] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for anti-IL-36R antibody-encoding vectors. Among lower eukaryotic host microorganisms, Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used. However, Schizosaccharomyces pombe is capable of inhibiting Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus, Yarrowia (EP 402,226), Pichia pastoris (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa, Schwanniomyces Many other genera, species, and strains are commonly available and useful herein, such as Schwanniomyces, e.g., A. occidentalis, and filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, e.g., A. nidulans and A. niger.

[0209]

[0245] Suitable host cells for the expression of the glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses may be used as viruses herein in accordance with the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

[0210]

[0246] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become routine procedure. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and human hepatocellular carcinoma line (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293, and their derivatives.

[0211]

[0247] The host cells are transformed with the above expression or cloning vectors for the production of anti-IL-36R antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In another embodiment, the antibodies may be produced by homologous recombination as known in the art. In certain embodiments, the host cells are capable of producing the antibodies or antigen-binding fragments thereof provided herein.

[0212]

[0248] The present disclosure also provides a method for expressing the antibody or antigen-binding fragment thereof provided herein, comprising culturing a host cell provided herein under conditions in which the vector of the present disclosure is expressed. The host cells used to produce the antibody or antigen-binding fragment thereof provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing the host cells. Additionally, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Pat. Re. 30,985 may be used as a culture medium for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those of skill in the art may also be included at appropriate concentrations. Temperature, pH, and other culture conditions will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0213]

[0249] When recombinant techniques are used, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate debris of host cells or lysed fragments are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, for example, a Pellicon ultrafiltration unit from Amicon or Millipore. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and an antibiotic may be included to prevent the growth of adventitious contaminants.

[0214]

[0250] The anti-IL-36R antibody or antigen-binding fragment thereof prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0215]

[0251] In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0216]

[0252] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5 to 4.5, preferably performed at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0217]

[0253] Pharmaceutical Compositions

[0254] The present disclosure further provides pharmaceutical compositions comprising an anti-IL-36R antibody or antigen-binding fragment thereof and one or more pharma- ceutically acceptable carriers.

[0218]

[0001] The present disclosure further provides a pharmaceutical composition comprising a polynucleotide encoding an anti-IL-36R antibody or an antigen-binding fragment thereof, and one or more pharma- ceutically acceptable carriers. The antibodies provided herein can also be produced in vivo by delivery of a polynucleotide encoding an antibody or an antigen-binding fragment thereof, such as an in-vitro transcribed mRNA or an expression vector. Methods for delivery of polynucleotides for in vivo antibody expression are known in the art, see, for example, Rybakova, Y. et al, Molecular Therapy, vol. 27 (8), pp. 1415-1423 (2019); Deal, CE et al, Vaccines, 2021, 9, 108.

[0219]

[0002] The present disclosure further provides a pharmaceutical composition comprising an expression vector comprising a polynucleotide encoding an anti-IL-36R antibody or an antigen-binding fragment thereof, and one or more pharma- ceutically acceptable carriers.

[0220]

[0255] In certain embodiments, the expression vector comprises a viral vector or a non-viral vector. Examples of viral vectors include, but are not limited to, adeno-associated viral (AAV) vectors, lentiviral vectors, retroviral vectors, and adenoviral vectors. Examples of non-viral vectors include naked DNA, plasmids, exosomes, mRNA, and the like. In certain embodiments, the expression vector is suitable for gene therapy in humans. Vectors suitable for gene therapy include, for example, adeno-associated viral (AAV) or adenoviral vectors. In certain embodiments, the expression vector comprises a DNA vector or an RNA vector. In certain embodiments, the pharmaceutically acceptable carrier is a polymer excipient, for example, but not limited to, a microsphere, a microcapsule, a polymeric micelle, and a dendrimer. The polynucleotides or polynucleotide vectors of the present disclosure may be encapsulated in, attached to, or coated with polymer-based moieties by methods known in the art (see, e.g., W. Heiser, Nonviral gene transfer techniques, published by Humana Press, 2004; U.S. Pat. No. 6,025,337; Advanced Drug Delivery Reviews, 57(15):2177-2202 (2005)).

[0221]

[0256] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharma- ceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestrants or chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, ingredients known in the art, or various combinations thereof.

[0222]

[0257] Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickening agents, coloring agents, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in compositions comprising the antibodies or antigen-binding fragments thereof and conjugates provided herein reduces oxidation of the antibodies or antigen-binding fragments thereof. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving the stability of the antibodies and maximizing shelf life. Thus, in certain embodiments, pharmaceutical compositions are provided that include one or more antibodies or antigen-binding fragments thereof disclosed herein and one or more antioxidants, such as methionine. Further provided are methods of preventing oxidation, extending shelf life, and / or improving efficacy of the antibodies or antigen-binding fragments provided herein by combining the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0223]

[0258] To further illustrate, pharma- ceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride for injection, Ringer's injection, isotonic dextrose injection, sterile water for injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphates or citrates; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; emulsifying agents such as Polysorbate 80 (TWEEN-80); sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Pharmaceutical compositions in multi-dose containers may contain antimicrobial agents utilized as carriers, including phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0224]

[0259] The pharmaceutical compositions can be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained release formulations, or powders. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0225]

[0260] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form, such as liquid solution, suspension, emulsion, or solid form suitable for producing liquid solution, suspension, emulsion. Preparations for injection may include sterile and / or non-pyrogenic solutions ready for injection, sterile dry soluble products such as lyophilized powders ready for mixing with a solvent immediately before use, including tablets for subcutaneous injection, sterile suspensions ready for injection, sterile dry insoluble products ready for mixing with a vehicle immediately before use, and sterile and / or non-pyrogenic emulsions. The solutions may be aqueous or non-aqueous.

[0226]

[0261] In certain embodiments, unit dose preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration should be sterile and nonpyrogenic, as known and practiced in the art.

[0227]

[0262] In certain embodiments, a sterile lyophilized powder is prepared by dissolving the antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability or other pharmacological factors of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those of skill in the art, at about neutral pH in one embodiment. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of an anti-IL-36R antibody or antigen-binding fragment thereof, or a composition thereof. Overfilling the vial by a small amount (e.g., about 10%) more than needed for a dose or set of doses is permissible to facilitate accurate sample removal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0228]

[0263] The lyophilized powder is reconstituted with water for injection to provide a formulation for use in parenteral administration.In one embodiment, for reconstitution, sterile and / or non-pyrogenic water or other suitable liquid carrier is added to the lyophilized powder.The exact amount depends on the selected therapy given and can be determined empirically.

[0229]

[0264] kit

[0265] In certain embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein. In certain embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and a second therapeutic agent.

[0230]

[0266] In some embodiments, the second therapeutic agent may be one useful for treating psoriasis, such as GPP (generalized pustular psoriasis), PPP (palmoplantar pustulosis), HS (hidradenitis suppurativa), Behcet's disease, etc.

[0231]

[0267] In some embodiments, the second therapeutic agent comprises a hormone-based immunosuppressant. In some embodiments, the second therapeutic agent comprises a glucocorticoid or steroid. Non-limiting exemplary glucocorticoids or steroids include budesonide, flunisolide, triamcinolone acetonide, fluticasone propionate, beclomethasone dipropionate, and ciclesonide.

[0232]

[0268] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating psoriasis. In some embodiments, the second therapeutic agent may be a non-biological agent, such as methotrexate, cyclosporine, fumaric acid esters (FAE), hydroxycarbamide, fumarate (e.g., dimethyl fumarate), retinoids (synthetic forms of vitamin A), glucocorticoids, or steroids. In some embodiments, the second therapeutic agent may be a biologic that blocks immune processes involved in psoriasis, targeting specific aspects of the immune system that contribute to psoriasis. In some embodiments, the biologic is a monoclonal antibody that targets anti-IL17, anti-IL12 / 23, anti-IL23, and / or anti-TNF alpha, etc. Examples of such monoclonal antibodies include, but are not limited to, infliximab, adalimumab, golimumab, certolizumab pegol, ixekizumab, ustekinumab, guselkumab, efalizumab, alefacept, secukinumab, and brodalumab.

[0233]

[0269] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating GPP (generalized pustular psoriasis). In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug, such as a glucocorticoid or steroid, etanercept, PUVA, hydroxyurea, dapsone, cyclosporine A, adalimumab, etretinate, isotretinoin, or acitretin.

[0234]

[0270] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating PPP (palmoplantar pustulosis). In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug, such as a retinoid, cyclosporine, tetracycline, colchicine, Tripterygium wilfordii, Tripterygium hypoglaucum hatch, or an anti-interleukin 23 monoclonal antibody (e.g., guselkumab).

[0235]

[0271] In some embodiments, the second therapeutic agent includes a therapeutic agent or drug for treating HS (hidradenitis suppurativa). In some embodiments, the second therapeutic agent includes a therapeutic agent or drug, such as a corticosteroid, an antibiotic, an antiandrogen, or an anti-inflammatory agent. Examples of antibiotics include, but are not limited to, rifampicin, clindamycin, tetracycline, and minocycline. Examples of antiandrogens include, but are not limited to, spironolactone, flutamide, cyproterone acetate, ethinyl estradiol, finasteride, dutasteride, and metformin. Examples of anti-inflammatory agents include, but are not limited to, TNF inhibitors, such as infliximab, etanercept, and adalimumab.

[0236]

[0272] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating Behcet's disease.In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug, such as topical drugs, corticosteroids, methotrexate, colchicine, thalidomide, azathioprine, cyclophosphamide, cyclosporine, mycophenolate mofetil, and anti-tumor necrosis factor antagonists.Examples of anti-tumor necrosis factor antagonists include, but are not limited to, infliximab, etanercept, and apremilast.

[0237]

[0273] In some embodiments, the second therapeutic agent is a drug that targets IL-17, IL-23, TNF, IL-12, IL-1, or the like.

[0274] Such kits may further include, if desired, for example, a container containing one or more pharma- ceutically acceptable carriers, additional containers, and one or more of a variety of other conventional pharmaceutical kit components, as will be readily apparent to one of skill in the art. Instructions as an insert or label indicating the amounts of the components to be administered, guidelines for administration, and / or guidelines for mixing the components may also be included in the kit.

[0238]

[0275] Chimeric Antigen Receptor (CAR) Compositions

[0276] The present disclosure also provides a chimeric antigen receptor (CAR) comprising the anti-IL-36R antigen binding domain and a T cell activation domain provided herein. A chimeric antigen receptor (CAR) is an engineered chimeric receptor that combines the antigen binding domain of an antibody with one or more signaling domains for T cell activation. Immune cells, for example, T cells and natural killer (NK) cells, can be engineered to express a CAR. T cells expressing a CAR are referred to as CAR-T cells. CARs can mediate antigen-specific cellular immune activity in T cells, allowing the CAR-T cells to eliminate cells (e.g., tumor cells) expressing the targeted antigen. In one embodiment, binding of a CAR-T cell provided herein to an IL-36R expressed on a cell, for example, a cancer cell, results in the proliferation and / or activation of the CAR-T cell, where the activated CAR-T cell can release cytotoxic factors, for example, perforin, granzymes, and granulysin, to initiate cytolysis and / or apoptosis of the cancer cell.

[0239]

[0277] In some embodiments, the T cell activation domain of a CAR comprises a costimulatory signaling domain and a TCR signaling domain, which can be linked to each other in a random or specified order, optionally using a short peptide linker (e.g., a glycine-serine double linker) having, for example, a length of between 2 and 10 amino acids.

[0240]

[0278] In some embodiments, the CAR further comprises a transmembrane domain. When expressed in a cell, the anti-IL-36R antigen binding domain is extracellular and the T cell activation domain is intracellular.

[0241]

[0279] In certain embodiments, the CAR comprises an anti-IL-36R antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a TCR signaling domain, where the antigen-binding domain specifically binds to IL-36R and comprises an antigen-binding fragment of an antibody provided herein. 1. Antigen-binding domain

[0280] In some embodiments, the anti-IL-36R antigen-binding domain of the CAR comprises one or more CDR sequences provided herein, one or more heavy chain variable domains or light chain variable domains provided herein, or one or more antigen-binding fragments derived from any of the anti-IL-36R antibodies provided herein.

[0242]

[0281] In some embodiments, it is beneficial for the antigen-binding domain to be derived from the same species as the CAR will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen-binding domain used in the CAR to be derived from a human antibody or a humanized antibody. In some embodiments, the antigen-binding domain comprises a single chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises, for example, an Fv, Fab, and (Fab') 2 , as well as bifunctional (i.e., bispecific) hybrid antibody fragments (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In certain embodiments, the antigen-binding domain comprises a Fab or scFv. 2. Transmembrane domain

[0282] In certain embodiments, the CAR comprises a transmembrane domain fused to the extracellular antigen-binding domain of the CAR.In one embodiment, the transmembrane domain can be selected to naturally associate with one of the domains in the CAR.In some examples, the transmembrane domain can be selected or modified to avoid binding to the transmembrane domain of other members of the T cell receptor complex.

[0243]

[0283] The transmembrane domain of the CARs provided herein can be derived from the transmembrane domain of any naturally occurring membrane-bound or transmembrane protein, such as the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain of the CAR can also use various human hinges, such as human Ig (immunoglobulin) hinges.

[0244]

[0284] Alternatively, the transmembrane domain of the CAR provided herein may be synthetic, for example, containing mainly hydrophobic residues, such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan, and valine is included at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker between 2 and 10 amino acids in length may form the link between the transmembrane domain of the CAR and the intracellular signaling domain. A glycine-serine doublet provides a particularly preferred linker. 3. TCR signaling domain

[0285] The T cell activation domain of the CAR provided herein comprises a TCR signaling domain.The TCR signaling domain can activate the T cell expressing the CAR to perform at least one of the normal TCR effector functions of T cell, such as cytolytic activity or helper activity, including secretion of cytokines.The TCR signaling domain can be either the full length of the natural intracellular signaling domain or a fragment thereof sufficient to transmit TCR effector function signals.

[0245]

[0286] Exemplary intracellular signaling domains useful in the CARs provided herein include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor binding, as well as any derivative or variant of these sequences and any synthetic sequence having the same functional capability.

[0246]

[0287] The TCR signaling domain that acts in a stimulatory manner can comprise signaling motifs known as immune receptor tyrosine-based activation motifs or ITAMs.The TCR signaling domains that comprise ITAMs that are useful in the CARs provided herein include TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.In certain embodiments, the TCR signaling domain comprises the cytoplasmic signaling sequence derived from CD3-zeta. 4.Costimulatory signaling regions

[0288] In certain embodiments, the T cell activation domain of the CAR provided herein may further comprise a costimulatory signaling region. The costimulatory signaling region may be derived from a costimulatory molecule that acts to mediate TCR activation in an antigen-independent manner and is required for the efficient response of lymphocytes to antigen. Exemplary costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and the ligand that specifically binds to CD83. 5. Polynucleotide sequence encoding CAR

[0289] In one aspect, the present disclosure further provides a nucleic acid sequence encoding a CAR provided herein, comprising a first polynucleotide sequence encoding an antigen-binding domain of the CAR provided herein, and optionally a second polynucleotide sequence encoding a transmembrane domain and a T cell activation domain provided herein. In some embodiments, the sequence encoding the antigen-binding domain is operably linked to the sequence encoding the transmembrane domain and the T cell activation domain. The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, for example, by screening a library from a cell expressing the gene, by inducing the gene from a vector known to contain it, or by directly isolating it from a cell or tissue containing it, using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0247]

[0290] In one aspect, the present disclosure provides a vector comprising a nucleic acid sequence encoding a CAR provided herein.In some embodiments, the vector is a retroviral and lentiviral vector construct expressing the CAR of the present disclosure that can be directly transduced into cells, or an RNA construct that can be directly transfected into cells.

[0248]

[0291] In one aspect, the disclosure provides an isolated cell that comprises a nucleic acid sequence encoding a CAR provided herein and / or expresses a CAR provided herein.

[0249]

[0292] In certain embodiments, the cells that contain the nucleic acid encoding the CAR or express the CAR are selected from the group consisting of T cells, NK cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. In one embodiment, the cells that contain the nucleic acid encoding the CAR or express the CAR exhibit anti-tumor immunity when the antigen-binding domain of the CAR binds to its corresponding antigen. The cytotoxic lymphocytes are preferably autologous cells, although xenogeneic or allogeneic cells may also be used. As used herein, "autologous" refers to any material derived from the same individual that is subsequently reintroduced into that individual.

[0250]

[0293] In one aspect, the present disclosure further provides a method for stimulating a T cell-mediated immune response against an IL-36R-expressing cell or tissue in a subject, the method comprising administering to the subject an effective amount of a cell genetically modified to express a CAR provided herein.

[0251]

[0294] In one aspect, the present disclosure further provides a method for treating a mammal having a disease, disorder, or condition associated with increased expression of IL-36R, comprising administering to the mammal an effective amount of a cell genetically modified to express a CAR provided herein, thereby treating the mammal. In certain embodiments, the cell is an autologous T cell. In certain embodiments, the mammal has been diagnosed with a disease, disorder, or condition associated with increased expression of IL-36R.

[0252]

[0295] How to use

[0296] In another aspect, a method of treating a disease, disorder, or condition in a subject that would benefit from modulating IL-36R activity is provided. In another aspect, a method of treating an IL-36R-related disease or disorder in a subject in need thereof is provided. In another aspect, a method of treating a disease, disorder, or condition that is responsive to IL-36R inhibition in a subject in need thereof is provided.

[0253]

[0297] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or a polynucleotide encoding an antibody or antigen-binding fragment thereof provided herein, and / or a pharmaceutical composition provided herein. In certain embodiments, the subject is a human.

[0254]

[0298] IL-36R is known to play a role in the pathogenesis of some diseases, such as psoriasis. For example, experimental results show that IL-36 (α, β, and γ) is elevated in the serum and lesional skin of psoriasis patients and correlates with disease activity, and IL-36Ra deficiency or overexpression of IL-36 agonist ligands can lead to pustular psoriasis. IL-36R signaling is a key effector in pustular psoriasis. IL-36R signaling plays a pivotal role in hidradenitis suppurativa. In the lesional skin of ichthyosis patients, the level of IL-36 (α, β, and γ) mRNA is increased, and there is widespread expression of IL-36R.

[0255]

[0299] In some embodiments, the IL-36R-related disease or disorder is an IL-36R positive disease or disorder. In some embodiments, the subject to be treated is identified as having an IL-36R positive disease or disorder. In some embodiments, the IL-36R-related disease, disorder or condition is responsive to IL-36R inhibition. In some embodiments, the IL-36R-related disease, disorder or condition is associated with dysregulation of IL-36R-mediated signal transduction, more particularly, associated with upregulation of IL-36R signal transduction.

[0256]

[0300] In some embodiments, the disease or disorder is associated with dysregulation of IL-36R-mediated signaling in cells. In some embodiments, dysregulation of IL-36R-mediated signaling comprises dysregulation of IL-36 (e.g., IL-36α, IL-36β, or IL-36γ), an IL-36R antagonist, and / or IL-38. In some embodiments, dysregulation of IL-36R-mediated signaling comprises overactivation of IL-36 (e.g., IL-36α, IL-36β, or IL-36γ), or oversuppression of an IL-36R antagonist, or oversuppression of IL-38, compared to control levels (e.g., levels in a healthy subject).

[0257]

[0301] In some embodiments, the disease or disorder is an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, or cancer.

[0302] In some embodiments, the inflammatory disease is selected from the group consisting of allergic inflammation of the skin, lungs, and digestive 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. In some embodiments, the inflammatory disease is selected from the group consisting of allergic inflammation of the skin, lungs, and digestive tract, atopic dermatitis (also known as atopic eczema), asthma (allergic and non-allergic), and epithelial-mediated inflammation.

[0258]

[0303] In some embodiments, the autoimmune disease is selected from the group consisting of multiple sclerosis, asthma, type 1 diabetes mellitus, rheumatoid arthritis, scleroderma, Crohn's disease, plaque psoriasis (commonly referred to as psoriasis), hidradenitis suppurativa, generalized pustular psoriasis (GPP), palmoplantar pustulosis (PPP), inflammatory bowel disease, psoriatic arthritis, systemic lupus erythematosus (SLE), ulcerative colitis, ankylosing spondylitis, atopic dermatitis, and acne vulgaris. In some embodiments, the methods provided herein are useful for treating pustular psoriasis, generalized pustular psoriasis, palmoplantar pustulosis (PPP), plaque psoriasis, atopic dermatitis, and acne vulgaris. In some embodiments, the autoimmune disease is selected from the group consisting of psoriasis vulgaris (commonly referred to as psoriasis), hidradenitis suppurativa, generalized pustular psoriasis (GPP), palmoplantar pustulosis (PPP), inflammatory bowel disease, atopic dermatitis, acne vulgaris, and Behcet's disease.

[0259]

[0304] In some embodiments, the respiratory disease is selected from the group consisting of asthma, cystic fibrosis, emphysema, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome.

[0305] In some embodiments, the metabolic disease is selected from the group consisting of obesity, type 2 diabetes, atherosclerosis, and cardiovascular disease.

[0260]

[0306] In some embodiments, the cancer may be 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.

[0261]

[0307] In some embodiments, the disease or disorder is selected from the group consisting of psoriasis, pustular psoriasis, hidradenitis suppurativa, ichthyosis, inflammatory bowel disease (IBD), atopic dermatitis, acne vulgaris, and Behcet's disease.

[0262]

[0308] The presence and / or amount of IL-36R in a biological sample of interest may indicate whether the subject from which the biological sample is derived is likely to be responsive to an anti-IL-36R antibody. Various methods can be used to determine the presence and / or amount of IL-36R in a test biological sample obtained from a subject. For example, the test biological sample may be exposed to an anti-IL-36R antibody or an antigen-binding fragment thereof that binds to and detects the expressed IL-36R protein. Alternatively, IL-36R can also be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarray, serial analysis of gene expression (SAGE), and fluorescent in situ hybridization (FISH). In some embodiments, the test sample is derived from an epithelial tissue. In certain embodiments, the presence or upregulated level of IL-36R in a test biological sample indicates the likelihood of responsiveness. The term "upregulated" as used herein refers to an overall increase in the expression level of IL-36R in a test sample of 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or more, compared to the expression level of IL-36R in a reference sample detected using the same method. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from which the test sample was obtained.

[0263]

[0309] In certain embodiments, IL-36R-related diseases, disorders, or conditions include, but are not limited to, inflammatory diseases, autoimmune diseases, respiratory diseases, metabolic disorders, and cancer.

[0264]

[0310] In certain embodiments, the inflammatory disease is selected from the group consisting of allergic inflammation of the skin, lungs, and gastrointestinal tract, atopic dermatitis (also known as atopic eczema), asthma (allergic and non-allergic), and epithelial-mediated inflammation. In some embodiments, the autoimmune disease is selected from the group consisting of psoriasis vulgaris (commonly referred to as psoriasis), hidradenitis suppurativa, generalized pustular psoriasis (GPP), palmoplantar pustulosis (PPP), inflammatory bowel disease, atopic dermatitis, and acne vulgaris. In some embodiments, the respiratory disease is selected from the group consisting of asthma, cystic fibrosis, emphysema, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome. In some embodiments, the metabolic disease is selected from the group consisting of obesity, type 2 diabetes, atherosclerosis, and cardiovascular disease. In some embodiments, the cancer may be 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.

[0265]

[0311] In some embodiments, the disease or disorder is selected from the group consisting of psoriasis, pustular psoriasis, hidradenitis suppurativa, ichthyosis, inflammatory bowel disease (IBD), atopic dermatitis, acne vulgaris, and Behcet's disease.

[0266]

[0312] The therapeutically effective amount of an antibody or antigen-binding fragment provided herein will depend on a variety of factors known in the art, such as, for example, weight, age, past medical history, current medications, the subject's health status and potential for cross-reactivity, allergies, sensitivities, and adverse side reactions, as well as the route of administration and the extent of disease occurrence. Dosages may be proportionately reduced or increased by the practitioner (e.g., physician or veterinarian) as dictated by these and other circumstances or requirements.

[0267]

[0313] In some embodiments, the antibodies or antigen-binding fragments provided herein may be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the administered dose may vary over the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than subsequent administered doses. In certain embodiments, the administered dose may vary over the course of treatment depending on the subject's response.

[0268]

[0314] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response), and may for example be administered as a single dose or as several divided doses administered over time.

[0269]

[0315] The antibodies or antigen-binding fragments thereof provided herein may be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0270]

[0316] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibodies or antigen-binding fragments thereof disclosed herein may be administered in combination with a second therapeutic agent.

[0271]

[0317] In some embodiments, the second therapeutic agent may be one useful for treating psoriasis, such as GPP (generalized pustular psoriasis), PPP (palmoplantar pustulosis), HS (hidradenitis suppurativa), Behcet's disease, etc.

[0272]

[0318] In some embodiments, the second therapeutic agent comprises a hormone-based immunosuppressant. In some embodiments, the second therapeutic agent comprises a glucocorticoid or steroid. Non-limiting exemplary glucocorticoids or steroids include budesonide, flunisolide, triamcinolone acetonide, fluticasone propionate, beclomethasone dipropionate, and ciclesonide.

[0273]

[0319] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating psoriasis. In some embodiments, the second therapeutic agent may be a non-biological agent, such as methotrexate, cyclosporine, fumaric acid esters (FAE), hydroxycarbamide, fumarate (e.g., dimethyl fumarate), retinoids (synthetic forms of vitamin A), glucocorticoids, or steroids. In some embodiments, the second therapeutic agent may be a biologic that blocks immune processes involved in psoriasis, targeting specific aspects of the immune system that contribute to psoriasis. In some embodiments, the biologic is a monoclonal antibody that targets anti-IL17, anti-IL12 / 23, anti-IL23, and / or anti-TNF alpha, etc. Examples of such monoclonal antibodies include, but are not limited to, infliximab, adalimumab, golimumab, certolizumab pegol, ixekizumab, ustekinumab, guselkumab, efalizumab, alefacept, secukinumab, and brodalumab.

[0274]

[0320] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating GPP (generalized pustular psoriasis). In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug, such as a glucocorticoid or steroid, etanercept, PUVA, hydroxyurea, dapsone, cyclosporine A, adalimumab, etretinate, isotretinoin, or acitretin.

[0275]

[0321] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating PPP (palmoplantar pustulosis). In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug, such as a retinoid, cyclosporine, tetracycline, colchicine, Tripterygium wilfordii, Tripterygium hypoglaucum hatch, or an anti-interleukin 23 monoclonal antibody (e.g., guselkumab).

[0276]

[0322] In some embodiments, the second therapeutic agent includes a therapeutic agent or drug for treating HS (hidradenitis suppurativa). In some embodiments, the second therapeutic agent includes a therapeutic agent or drug, such as a corticosteroid, an antibiotic, an antiandrogen, or an anti-inflammatory agent. Examples of antibiotics include, but are not limited to, rifampicin, clindamycin, tetracycline, and minocycline. Examples of antiandrogens include, but are not limited to, spironolactone, flutamide, cyproterone acetate, ethinyl estradiol, finasteride, dutasteride, and metformin. Examples of anti-inflammatory agents include, but are not limited to, TNF inhibitors, such as infliximab, etanercept, and adalimumab.

[0277]

[0323] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating Behcet's disease.In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug, such as topical drugs, corticosteroids, methotrexate, colchicine, thalidomide, azathioprine, cyclophosphamide, cyclosporine, mycophenolate mofetil, and anti-tumor necrosis factor antagonists.Examples of anti-tumor necrosis factor antagonists include, but are not limited to, infliximab, etanercept, and apremilast.

[0278]

[0324] In some embodiments, the second therapeutic agent is a drug that targets IL-17, IL-23, TNF, IL-12, IL-1, or the like.

[0325] In certain of these embodiments, an antibody or antigen-binding fragment thereof provided herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the antibody or antigen-binding fragment thereof and the additional therapeutic agent may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment thereof that is administered "in combination" with another therapeutic agent may not be administered simultaneously with or in the same composition as the agent. An antibody or antigen-binding fragment thereof that is administered before or after another agent is considered to be administered "in combination" with the agent, as this phrase is used herein, even if the antibody or antigen-binding fragment and the second agent are administered via different routes. When possible, additional therapeutic agents administered in combination with an antibody or antigen-binding fragment thereof disclosed herein are administered according to the schedule listed in the product information sheet for that additional therapeutic agent, or according to protocols known in the art, such as the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)).

[0279]

[0326] In another aspect, the present disclosure further provides a method of modulating the activity of IL-36R in an IL-36R-positive cell, comprising exposing the IL-36R-positive cell to an antibody or antigen-binding fragment thereof provided herein.

[0280]

[0327] In another aspect, the present disclosure provides a method of detecting the presence or amount of IL-36R in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof provided herein and determining the presence or amount of IL-36R in the sample.

[0281]

[0328] In another aspect, the disclosure provides a method of diagnosing an IL-36R-related disease, disorder, or condition in a subject comprising the steps of: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein; b) determining the presence or amount of IL-36R in the sample; and c) correlating the presence or amount of IL-36R with the presence or status of the IL-36R-related disease, disorder, or status in the subject.

[0282]

[0329] In another aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein, optionally conjugated to a detectable moiety, useful for detecting an IL-36R-related disease, disorder, or condition. The kit may further comprise instructions for use.

[0283]

[0330] In another aspect, the present disclosure also provides the use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating, preventing, or ameliorating an IL-36R-related disease, disorder, or condition in a subject, and in the manufacture of a diagnostic reagent for diagnosing an IL-36R-related disease, disorder, or condition.

[0284]

[0331] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below are within the scope of the invention, either in whole or in part. These specific compositions, materials, and methods are not intended to limit the invention, but merely illustrate specific embodiments within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials, and methods without exercising inventive ability and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures described herein while remaining within the boundaries of the invention. It is the intention of the inventors that such variations are included within the scope of the invention. EXAMPLES

[0285] Example 1 Generation of antibodies

[0332] 1.1 Immunization and antibody screening

[0333] Mice from various strains (C57BL / 6, BALB / c, SJL, and CD-1) were immunized with recombinant human IL-36R-his protein (derived from the extracellular domain, amino acid residues 1-337 of SEQ ID NO:211). Those that generated strong titer responses were selected for single B cell isolation. B cells from spleen and lymph nodes were isolated and enriched with microbeads. B cells that recognize hIL-36R were stained and isolated by FACS. Immunoglobulin heavy and light chain sequences of these B cells were cloned and recombinantly expressed. These monoclonal antibodies were then rescreened for blocking hIL-36R binding and signaling. The variable region sequences of selected anti-IL-36R antibodies are listed in Tables 1 and 2 of the present disclosure.

[0286]

[0334] 1.2 Benchmark antibody expression

[0335] IL36R antibodies BI655130, ANB019, and REGN14 were used as benchmarks. Benchmark antibody sequences were obtained from US Patent No. US10550189, US20200017592A1, US10526410B2, and IMGT Information System (code 10845). Coding DNA sequences were synthesized and cloned into pcDNA3.1 vector to construct antibody expression plasmids. Expression plasmids were transfected into CHO-s cells using ExpiCHO™ Expression System (Gibco, A29133). After 14 days, supernatants were harvested and antibodies were purified by affinity chromatography using a Protein A column.

[0287]

[0336] The variable sequences of the benchmark antibodies are listed below:

[0337] VH of BI655130 (SEQ ID NO: 205):

[0338] QVQLVQSGAEVKKPGASVKVSCKASGYSFTSSWIHWVKQAPGQGLEWMGEINPGNVRTNYNENFRNKVTMTVDTSISTAYMELSRLRSDDTAVYYCTVVFYGEPYFPYWGQGTLVTVSS

[0339] VL of BI655130 (SEQ ID NO: 206):

[0340] QIVLTQSPGTLSLSPGERATMTCTASSSVSSSYFHWYQQKPGQAPRLWIYRTSRLASGVPDRFSGSGSGTDFTLTISRLEPEDAATYYCHQFHRSPLTFGAGTKLEIK

[0341] VH of ANB019 (SEQ ID NO:207):

[0342] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMNWVRQAPRQGLEWMGMFHPTGDVTRLNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARTTSMIIGGFAYWGQGTLVTVSS

[0343] VL of ANB019 (SEQ ID NO: 208):

[0344] DIVMTQTPLSLSVTPGQPASISCRSSKSLLHRNAITYFYWYLHKPGQPPQLLIYQMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGGGTKVEIK

[0345] VH of REGN14 (SEQ ID NO:209):

[0346] EVQLVESGGDLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSVISWNSDVIAYSDSVKGRFTISRDNAKNSLYLQMNSLRTEDTALYYCTKGHKWSFFDYWGQGTLVTVSS

[0347] VL of REGN14 (SEQ ID NO:210):

[0348] EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIFNVANRATDIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK Example 2 IL-36R binding assay by Biolayer Interferometry (BLI)

[0349] The antibody identified in Example 1 was diluted in kinetic buffer (PBS pH 7.4, 0.1% BSA + 0.01% Tween-20) to a concentration of 100 nM. Recombinant hIL-36R protein (Acrobio, Cat. No. IL2-H52H6) was diluted in kinetic buffer to obtain a concentration gradient: 500 nM, 250 nM, 125 nM, and a series of 0 nM as a reference control well. The antibody was immobilized on a Protein A biosensor after equilibration. The baseline was detected for 60 seconds. The antibody-antigen binding was then detected for 180 seconds to obtain Kon coefficient data. This was followed by dissociation for 180 seconds in kinetic buffer to obtain Koff coefficient data. Regeneration of the biosensor was performed in 10 mM glycine, pH 2.0 buffer. All kinetic data was collected at 30°C. Data was acquired on a Gator Bioanalysis System.

[0288]

[0350] As shown in FIG. 1 and Table 6, chimeric antibodies 5F7 and 9A6 bind to hIL-36R with KD values ​​of 1.46E-08M and 2.05E-11M, respectively.

[0289] [Table 7]

[0290]

[0351] The blocking activity of the anti-IL-36R antibodies in Table 1 was evaluated using a reporter cell system, Jurkat-IL36R-IL1Racp-NFkB-luc cells.

[0352] As shown in FIG. 18, 5F7 and 9A6 show the highest blocking activity among all the anti-IL-36R antibodies in Table 1.

[0291] Example 3 Evaluation of blocking activity by IL36R reporter assay

[0353] The blocking activity of anti-IL-36R antibodies was evaluated using Jurkat-IL36R-IL1Racp-NFkB-luc cells, a reporter cell line. Jurkat-IL36R-IL1Racp-NFkB-luc cells were harvested and seeded in a 96-well white plate at a concentration of 50000 cells / 25ul per well. 50ul of serially diluted antibodies were added, mixed with the cells, and incubated at 37°C for 30 minutes. 2ng / ml of IL-36α ligand was then added at 25ul / well, mixed with the cells, and incubated at 37°C for 4.5 hours. Afterwards, 50ul / well of One-Glu reagent was added to the plate, and the luminescence signal was read on a Tecan Spark.

[0292]

[0354] As shown in Figure 2, chimeric antibodies 5F7 and 9A6 had IC of 0.4477 nM and 0.3123 nM, respectively. 50 blocked ligand IL-36α-induced IL-36R signaling.

[0293] Example 4 Antibody Humanization

[0355] The antibody clone 5F7 was selected for humanization. First, the most suitable human framework to graft the rodent CDRs was selected using IgBLAST from NCBI. The variable region with high amino acid sequence identity (homologous match or best fit) to the rodent variable region was used. Second, the 5F7 mAb was humanized by grafting three CDRs from the light chain variable region into a human VL that was as homologous as possible to the mouse antibody VL. Similarly, those three CDRs from the heavy chain variable region were grafted into a human VH that was as homologous as possible to the mouse antibody. Furthermore, in the framework regions of the selected human variable region, several amino acid residues were changed to amino acid residues that were present in the mouse variable region (so-called back mutations). The humanized sequence of 5F7 is listed in Tables 3-5 of the present disclosure. The activity of humanized 5F7 was evaluated by BLI and IL-36R reporter assay. The experimental procedures are described in Examples 2 and 3.

[0294]

[0356] As shown in Table 7 and FIG. 3, the binding and blocking activity of the 5F7 original molecule was recapitulated after humanization.

[0295] [Table 8]

[0296] Example 5 Antibody affinity maturation

[0357] To enhance the binding affinity of humanized 5F7 antibody, a combination of CDR1 walking randomization and rational design based approach was used for affinity maturation. First, mutations were introduced into CDR-H1 and CDR-L1 by overlap PCR method using oligonucleotides containing NNK codons at the mutation sites, and DNA sequencing was performed before selection to ensure that the clones derived from the library were not biased. Second, selection experiments were performed three times independently with different soluble IL-36R-his concentrations of 2 μg / mL, 1 μg / mL, and 0.5 μg / mL. After three rounds of selection, the amino acid sequences of randomly selected clones from the final round were analyzed, and then enriched clones were obtained from the soluble IL-36R-his concentration of 0.5 μg / mL. Then, one light chain and seven heavy chains of the above enriched clones were cloned into pCDNA3.1+ with hIgG4 constant region sequence and expressed in CHO cells. Here, the heavy chain constant region had the mutation S228P. The variable region sequences of these clones are listed in Table 4 of the present disclosure. The binding and blocking activities of these clones were evaluated by the Gator Bioanalysis System and IL-36R reporter assay.

[0297]

[0358] Amino acid sequence of the heavy chain constant region (SEQ ID NO:203):

[0359] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0360] Amino acid sequence of the light chain constant region (SEQ ID NO:202):

[0361] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0362] High affinity 5F7 mutants were successfully isolated, as shown in Table 8 and Figure 4. The blocking activity of 5F7 was also evaluated after maturation.

[0298] [Table 9]

[0299] Example 6 Binding affinity testing by SPR

[0363] To evaluate the binding affinity of the antibody to hIL-36R, the binding affinity of the antibody was tested by surface plasmon resonance (SPR) technology using Biacore T200. The assay was performed at 25°C, and the running buffer was HBS-EP+. The diluted antibody was captured on the sensor chip through Fc capture method. Reference antibodies BI655130, ANB019, and REGN14 were produced by recombinant method. The heavy chain variable sequence and the light chain variable sequence are described in SEQ ID NO: 205 and 206 for BI655130, SEQ ID NO: 207 and 208 for ANB019, and SEQ ID NO: 209 and 210 for REGN14. hIL-36R was used as the analyte, followed by injection of the running buffer as the dissociation phase.

[0300]

[0364] As shown in Figure 5 and Table 9, 5F7-2a8 has a 1.52x10 -9 It binds to hIL-36R with a KD value of M.

[0301] [Table 10]

[0302] Example 7 Antibody-hIL36R binding assay by ELISA

[0365] Recombinant hIL36R protein was coated on ELISA plates at a concentration of 1 μg / ml at 100 ul per well overnight. After washing, the plates were blocked with PBS containing 1% BSA + 1% normal goat serum + 0.05% Tween20. Then, 100 ul of serially diluted antibodies were added to the plates and incubated for 1 hour. Specific binding of antibodies to hIL36R was detected by anti-hIgG antibody conjugated to HRP.

[0303]

[0366] As shown in Figure 6, 5F7-2a8 had an EC 50 and binds to recombinant hIL-36R-his. Example 8 Binding to membrane IL-36R.

[0304]

[0367] To evaluate the binding ability of antibodies to cell membrane IL-36R of various species, HEK293 cells were transfected with pcDNA3.1-hIL36R, pcDNA3.1-cynoIL36R, or pcDNA3.1-musIL36R plasmids using Lipofectamine2000 (Invitrogen, Cat. No. 11668-019). After 48 hours, the transfected cells were subjected to FACS analysis for anti-IL-36R antibody binding. Cells were harvested and seeded in 96-well plates at a concentration of 100000 cells / 50ul / well. 50ul of serially diluted antibodies were added to the cells and incubated at 4°C for 1 hour. After that, cells were washed twice with PBS and 100ul of diluted secondary antibody (FITC-labeled anti-hIgG) was added to the cells and incubated at 4°C for 30 minutes. After incubation, cells were washed twice with PBS and analyzed by flow cytometry.

[0305]

[0368] As shown in Figure 7, 5F7-2a8 binds to membrane hIL-36R with an EC50 of 0.1462ug / ml. As shown in Figures 8 and 9, 5F7-2a8 has no cross-reactivity with either cynomolgus or mouse IL-36R.

[0306] Example 9 Cross-reactivity test against cynomolgus monkey and mouse IL-36R

[0369] To test whether 5F7 binds to recombinant cynomolgus / mouse IL-36R protein, the antibody was diluted to a concentration of 100 nM in kinetic buffer (PBS pH 7.4, 0.1% BSA + 0.01% Tween-20). Recombinant cynomolgus IL36R protein (Acrobio, Cat. No. IL2-C52H5) or mouse IL-36R (R&D, Cat. No. 2354-RP) was diluted in kinetic buffer to obtain a concentration gradient: 100 nM, 50 nM, 25 nM, 12.5 nM, and 0 nM as a reference control well. The antibody was immobilized on a protein A biosensor after equilibration. Cynomolgus or mouse IL-36R was used as the analyte, followed by injection of running buffer as the dissociation phase.

[0307]

[0370] As shown in Table 10, 5F7-hu-3 has no cross-reactivity with either cynomolgus or mouse IL-36R.

[0308] [Table 11]

[0309] Example 10 Binding specificity

[0371] To test the binding specificity of 5F7-2a8, IL1R1 is the most homologous related IL-1R family member to IL-36R. Recombinant hIL1R1 protein was used to test whether 5F7-2a8 binds to IL1R1. hIL1R1 protein was coated overnight on ELISA plates at a concentration of 1g / ml with 100ul per well. The plates were then blocked with PBS containing 1% BSA + 1% normal goat serum + 0.05% Tween20. After that, 100ul of serially diluted antibodies were added to the plates and incubated for 1 hour. Specific binding of antibodies to hIL1R1 was detected using anti-hIgG antibody conjugated to HRP.

[0310]

[0372] As shown in FIG. 10, 5F7-2a8 has no binding activity to hIL1R1. Example 11 Antibodies block IL-36α / β / γ-induced IL-8 release in A431 cells

[0373] To evaluate the activity of anti-IL-36R antibodies to inhibit IL-8 release in A431 cells, A431 cells were harvested and seeded in 96-well plates at a concentration of 50000 cells / 50ul / well. 100ul of serially diluted antibodies were added to the cells and incubated at 37°C for 30 minutes. Then, 50ul of 300ng / ml IL-36α or 100ng / mL IL-36β or 100ng / mL IL-36γ was added to the plates and incubated at 37°C for 24 hours. After incubation, the supernatants were transferred to 96-well V-bottom plates and centrifuged at 1500g for 5 minutes. The cell-free supernatants were carefully collected and stored in a -70°C freezer. The concentration of IL-8 in the supernatants was detected using an IL-8 ELSIA kit (DAKEWE, Cat. No. 1110802).

[0311]

[0374] As shown in Figure 11, 5F7-2a8 blocks IL-36α, IL-36β, or IL-36γ-induced IL-8 release in A431 cells with IC50 of 0.1953, 0.1109, and 0.008775ug / ml, respectively. It can be confirmed that 5F7-2a8 strongly inhibits IL-36α-induced IL-8 production in A431 cells. The inhibitory activity of 5F7-2a8 is comparable to that of BI655130, but much more potent than that of ABN019 or REGN14.

[0312] Example 12 Antibodies block IL-36α-induced IL-8 release in HDF cells

[0375] To evaluate the activity of anti-IL-36R antibodies to inhibit IL-8 release in primary human dermal fibroblast (HDF) cells, HDF cells were harvested and seeded in 96-well plates at a concentration of 5000 cells / 50ul / well. 100ul of serially diluted antibodies were added to the plates and incubated at 37°C for 30 minutes. Then, 50ul of 300ng / ml IL-36α was added to the cells and incubated at 37°C for 24 hours. After incubation, the supernatant was transferred to a 96-well V-bottom plate and centrifuged at 1500g for 5 minutes. The cell-free supernatant was carefully collected and stored in a -70°C freezer. The concentration of IL-8 in the supernatant was detected using an IL-8 ELSIA kit (DAKEWE, Cat. No. 1110802).

[0313]

[0376] As shown in Figure 12, 5F7-2a8 blocks IL-36α-induced IL-8 release in HDF cells with IC50 of 0.006958ug / ml. It can be confirmed that 5F7-2a8 strongly inhibits IL-36α-induced IL-8 production in HDF cells. The inhibitory activity of 5F7-2a8 is comparable to that of BI655130, but much more potent than that of ABN019 or REGN14.

[0314] Example 13 Antibodies block IL-36α / β / γ-induced IL-8 / IL-6 / TNF-α release in HEK cells

[0377] To evaluate the activity of anti-IL-36R antibodies to inhibit IL-8 release in primary human epidermal keratinocyte (HEK) cells, the HEK cell stimulation and blocking procedures were as described in Part 11. IL-8, IL-6, and TNF-a concentrations were detected using the Multi-Analyte Flow Assay Kit (Biolegend) as per the manufacturer's instructions.

[0315]

[0378] As shown in Figure 13, 5F7-2a8 blocks IL-36α-induced IL-8 / IL-6 / TNF-a release in HEK cells with IC50 of 0.1293, 0.1098, and 0.05352ug / ml, respectively. As shown in Figure 15, 5F7-2a8 blocks IL-36β-induced IL-8 / IL-6 / TNF-a release in HEK cells with IC50 of 0.04805, 0.01885, and 0.02346ug / ml, respectively. As shown in Figure 16, 5F7-2a8 blocks IL-36γ-induced IL-8 / IL-6 / TNF-a release in HEK cells with IC50 of 0.03264, 0.04458, and 0.005258ug / ml, respectively.

[0316]

[0379] It can be confirmed that 5F7-2a8 strongly inhibits IL-36 (α / β / γ)-induced IL-8, IL-6, and TNFα production in HEK cells. The inhibitory activity of 5F7-2a8 is comparable to that of BI655130, but much more potent than that of ABN019 or REGN14.

[0317] Example 14 Fc mutations to extend antibody half-life

[0380] To extend the half-life of 5F7-2a8, mutations were introduced into the IgG4 Fc domain that enhance antibody-FcRn binding. M252Y / S254T / T256E (YTE) mutations were introduced into the 5F7-2a8-YTE molecule, and T307Q / N434A (QA) mutations were introduced into the 5F7-2a8-QA molecule. The heavy chain constant region sequence of 5F7-2a8-WT with wild-type Fc is shown as SEQ ID NO: 203. The heavy chain constant region of 5F7-2a8-YTE is shown as SEQ ID NO: 201. The heavy chain constant region of 5F7-2a8-QA is shown as SEQ ID NO: 204.

[0318]

[0381] Amino acid sequence of the heavy chain constant region of 5F7-2a8-YTE (SEQ ID NO:201):

[0382] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0383] Amino acid sequence of the heavy chain constant region of 5F7-2a8-QA (SEQ ID NO:204):

[0384] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLQVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHAHYTQKSLSLSLGK

[0385] The light chain constant region sequences of 5F7-2a8-WT, 5F7-2a8-YTE, and 5F7-2a8-QA are identical to SEQ ID NO: 202. The binding affinity of the antibodies to hFcRn was evaluated using the Gator Bio analysis system. Briefly, recombinant hFcRn-his / B2M heterodimer protein (Acrobio, Cat. No. FCM-H82W4) was loaded onto the anti-his sensor, and serially diluted antibodies were used as analytes. Binding and dissociation were performed in pH 6.0 KD buffer.

[0319]

[0386] As shown in FIG. 16 and Table 11, the binding affinity of 5F7-2a8-YTE and 5F7-2a8-QA was increased approximately 10-fold compared to 5F7-2a8-WT.

[0320] [Table 12]

[0321] Example 15 PK analysis of 5F7-2a8-YTE and 5F7-2a8-QA

[0387] To evaluate the pharmacokinetics of 5F7-2a8-YTE and 5F7-2a8-QA in cynomolgus monkeys and hFcRn transgenic mice, 2 animals (1 male and 1 female) per group were administered a single dose of 5mg / kg by injection for PK studies in cynomolgus monkeys. Serum was collected at the following time points after drug administration: 0 hours, 0.5 hours, 2 hours, 8 hours, 24 hours, 48 ​​hours, 3 days, 4 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, and 49 days. Serum antibody concentrations were detected by ELISA. Briefly, recombinant hIL36R protein was coated overnight on ELISA plates at 100ul per well at a concentration of 1μg / ml. After washing, the plates were blocked with PBS containing 1% BSA+1% normal goat serum+0.05% Tween20. Then, 100ul of appropriately diluted serum or antibody at a certain concentration was added to the plate and incubated for 1 hour.The binding of antibody was then detected using anti-hIgG antibody conjugated to HRP.The serum drug concentration was calculated using the standard curve from the sample at a certain concentration.

[0322]

[0388] As shown in FIG. 17, Table 12, and Table 13, the half-lives of 5F7-2a8-YTE and 5F7-2a8-QA were significantly extended in cynomolgus monkeys compared to the benchmark antibody whose Fc does not have FcRn binding enhancement.

[0323] [Table 13]

Claims

1. An anti-IL-36R antibody or antigen-binding fragment thereof, comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, HCDR1 is DYYX １ X ２ (SEQ ID NO: 191), comprising the amino acid sequence of SEQ ID NO: 19, 33, 48, 64, 79, 94, 109, 124 or 139; HCDR2 is LIRNKAAGYTIYYX ３ X ４ X ５ VKG (SEQ ID NO: 192), comprising the amino acid sequence of SEQ ID NO: 20, 34, 49, 65, 80, 95, 110, 125 or 140, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 5, 21, 35, 50, 66, 81, 96, 111, 126 or 141; X １ is M or L, and X ２ is N, H, S or R, and X ３ is S or A, and X ４ is A or D, and X ５ is S or P.

2. light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3; LCDR1 is RASX １８ NINIWLS (SEQ ID NO: 193), comprising the amino acid sequence of SEQ ID NO: 22, 36, 51, 67, 82, 97, 112, 127 or 142; LCDR2 comprises the amino acid sequence of SEQ ID NO: 7, 23, 37, 52, 68, 83, 98, 113, 128 or 113, and LCDR3 is X １９ QSQSYPLT (SEQ ID NO: 194), comprising the amino acid sequence of SEQ ID NO: 24, 38, 53, 69, 84, 99, 114, 129 or 143; X １８ is Q or R, and X １９ is Q or L; The anti-IL-36R antibody or antigen-binding fragment thereof according to claim 1.

3. heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, and / or light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3; (a) HCDR1 is DYYX １ X ２ (SEQ ID NO: 191), HCDR2 is LIRNKAAGYTIYYX ３ X ４ X ５ VKG (SEQ ID NO: 192), HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 is RASX １８ comprising the amino acid sequence of NINIWLS (SEQ ID NO: 193), LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 is X １９ comprising the amino acid sequence of QSQSYPLT (SEQ ID NO: 194), X １ is M or L, and X ２ is N, H, S or R, and X ３ is S or A, and X ４ is A or D, and X ５ is S or P, and X １８ is Q or R, and X １９ is Q or L; (b) HCDR1 comprises the amino acid sequence of SEQ ID NO: 19; HCDR2 comprises the amino acid sequence of SEQ ID NO: 20; HCDR3 comprises the amino acid sequence of SEQ ID NO: 21; LCDR1 comprises the amino acid sequence of SEQ ID NO: 22; LCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 24; (c) HCDR1 comprises the amino acid sequence of SEQ ID NO: 33; HCDR2 comprises the amino acid sequence of SEQ ID NO: 34; HCDR3 comprises the amino acid sequence of SEQ ID NO: 35; LCDR1 comprises the amino acid sequence of SEQ ID NO: 36; LCDR2 comprises the amino acid sequence of SEQ ID NO: 37, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 38; (d) HCDR1 comprises the amino acid sequence of SEQ ID NO: 48; HCDR2 comprises the amino acid sequence of SEQ ID NO: 49; HCDR3 comprises the amino acid sequence of SEQ ID NO: 50; LCDR1 comprises the amino acid sequence of SEQ ID NO: 51; LCDR2 comprises the amino acid sequence of SEQ ID NO: 52, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 53; (e) HCDR1 comprises the amino acid sequence of SEQ ID NO: 64; HCDR2 comprises the amino acid sequence of SEQ ID NO: 65; HCDR3 comprises the amino acid sequence of SEQ ID NO: 66; LCDR1 comprises the amino acid sequence of SEQ ID NO: 67; LCDR2 comprises the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 69; (f) HCDR1 comprises the amino acid sequence of SEQ ID NO: 79; HCDR2 comprises the amino acid sequence of SEQ ID NO: 80; HCDR3 comprises the amino acid sequence of SEQ ID NO: 81; LCDR1 comprises the amino acid sequence of SEQ ID NO: 82; LCDR2 comprises the amino acid sequence of SEQ ID NO: 83, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 84; (g) HCDR1 comprises the amino acid sequence of SEQ ID NO: 94; HCDR2 comprises the amino acid sequence of SEQ ID NO: 95; HCDR3 comprises the amino acid sequence of SEQ ID NO: 96; LCDR1 comprises the amino acid sequence of SEQ ID NO: 97; LCDR2 comprises the amino acid sequence of SEQ ID NO: 98, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 99; (h) HCDR1 comprises the amino acid sequence of SEQ ID NO: 109; HCDR2 comprises the amino acid sequence of SEQ ID NO: 110; HCDR3 comprises the amino acid sequence of SEQ ID NO: 111; LCDR1 comprises the amino acid sequence of SEQ ID NO: 112; LCDR2 comprises the amino acid sequence of SEQ ID NO: 113, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 114; (i) HCDR1 comprises the amino acid sequence of SEQ ID NO: 124; HCDR2 comprises the amino acid sequence of SEQ ID NO: 125; HCDR3 comprises the amino acid sequence of SEQ ID NO: 126; LCDR1 comprises the amino acid sequence of SEQ ID NO: 127; LCDR2 comprises the amino acid sequence of SEQ ID NO: 128, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 129; (j) HCDR1 comprises the amino acid sequence of SEQ ID NO: 139; HCDR2 comprises the amino acid sequence of SEQ ID NO: 140; HCDR3 comprises the amino acid sequence of SEQ ID NO: 141; LCDR1 comprises the amino acid sequence of SEQ ID NO: 142; LCDR2 comprises the amino acid sequence of SEQ ID NO: 113, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 143; (k) HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO:4; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO:6; LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO:8; (l) HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO: 151; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 152; LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153; (m) HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO: 164; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO:6; LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153; (n) HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO: 173; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO:6; LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153; (o) HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO: 151; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO:6; LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153; (p) HCDR1 comprises the amino acid sequence of SEQ ID NO: 180; HCDR2 comprises the amino acid sequence of SEQ ID NO: 151; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 152; LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153; (q) HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO: 151; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 152; LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153; (r) HCDR1 comprises the amino acid sequence of SEQ ID NO: 184; HCDR2 comprises the amino acid sequence of SEQ ID NO: 151; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 152; LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153; (s) HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO: 151; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 152; LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153; (t) HCDR1 comprises the amino acid sequence of SEQ ID NO: 188; HCDR2 comprises the amino acid sequence of SEQ ID NO: 151; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 152; LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153; or (u) HCDR1 comprises the amino acid sequence of SEQ ID NO: 3; HCDR2 comprises the amino acid sequence of SEQ ID NO: 151; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 152; LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 153; The anti-IL-36R antibody or antigen-binding fragment thereof according to claim 2.

4. HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, 180, 184 or 188; HCDR2 comprises the amino acid sequence of SEQ ID NO: 4, 151, 164 or 173; HCDR3 comprises the amino acid sequence of SEQ ID NO:5; LCDR1 comprises the amino acid sequence of SEQ ID NO: 6 or 152; LCDR2 comprises the amino acid sequence of SEQ ID NO:7, and The antibody or antigen-binding fragment thereof according to claim 1, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO: 8 or 153.

5. further comprising one or more of heavy chain framework region 1 (HFR1), HFR2, HFR3 and HFR4, and / or one or more of light chain framework region 1 (LFR1), LFR2, LFR3 and LFR4; (a) HFR1 is X ６ VQLX7ESGGGLVKPGGSLRLSCAASGX ８ X ９ FX １０ (SEQ ID NO: 195) or a homologous sequence having at least 85% sequence identity thereto; HFR2, WX １１ RQAPGKGLEWVX １２ (SEQ ID NO: 196) or a homologous sequence having at least 85% sequence identity thereto; HFR3 is RFTISRDX １３ X １４ KSX １５ LYLQMNSLX １６ X １７ EDTAVYYCVR (SEQ ID NO: 197) or a homologous sequence having at least 85% sequence identity thereto; HFR4 comprises the amino acid sequence of SEQ ID NO: 157 or a homologous sequence having at least 85% sequence identity thereto; LFR1 is X ２０ IVMTQSPX ２１ X ２２ X ２３ SX ２４ SX ２５ GX ２６ RX ２７ TX ２８ X ２９ C (SEQ ID NO: 198) or a homologous sequence having at least 85% sequence identity thereto; LFR2 is WYQQKPGX ３０ APX ３１ LFIY (SEQ ID NO: 199) or a homologous sequence having at least 85% sequence identity thereto; LFR3, GVPX ３２ RFSGSGSGTX ３３ FTLTISSLQX ３４ EDFAX ３５ YYC (SEQ ID NO: 200) or a homologous sequence having at least 85% sequence identity thereto; and LFR4 comprises the amino acid sequence of SEQ ID NO: 161 or a homologous sequence having at least 85% sequence identity thereto; X ６ is Q or E, and X ７ is Q or V, and X ８ is F or Y, and X ９ is A, D or N, and X １０ is T or G, and X １１ is I or V, and X １２ is S or A, and X １３ is N or D, and X １４ is A or S, and X １５ is S or T, and X １６ is R or K, and X １７ is A or T, and X ２０ is D or E, and X ２１ is S or A, and X ２２ is S or T, and X ２３ is L or V, and X ２４ is A or V, and X ２５ is V or P, and X ２６ is D or E, and X ２７ is V or A, and X ２８ is I or L, and X ２９ is T or S, and X ３０ is Q or K, and X ３１ is K or R, and X ３２ is S or A, and X ３３ is D or E, and X ３４ is S or P, and X ３５ The antibody or antigen-binding fragment thereof according to claim 1, wherein is T or V.

6. and / or a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 31, 46, 62, 77, 92, 107, 122, 137, 149, 162, 171, 176, 179, 182, 183, 185, 187, and 189, or a homologous sequence thereof with at least 80% sequence identity; and / or A light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 32, 47, 63, 78, 93, 108, 123, 138, 150, 163, 172, and 177, or a homologous sequence thereof having at least 80% sequence identity; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. further comprising substitutions or modifications of one or more amino acid residues, while retaining binding specificity for human IL-36R; at least one of the substitutions or modifications is in one or more of the non-complementarity determining region (CDR) sequences of the heavy or light chain variable region; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, further comprising an Fc region, optionally a human immunoglobulin (Ig) Fc region, or optionally a human IgG4 Fc region, wherein the Fc region is derived from human IgG4 containing the mutations M252Y / S254T / T256 (YTE) or T307Q / N434A (QA).

9. and / or comprising a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 201, 203 and 204. a light chain constant region comprising the amino acid sequence of SEQ ID NO: 202; The antibody or antigen-binding fragment thereof according to claim 8.

10. 6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, which is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody, a fusion protein, a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, or a bivalent domain antibody.

11. a) a K of 2E-08 M or less (preferably 1E-08 M or less, e.g., 9E-09 M or less, 8E-09 M or less, 7E-09 M or less, 6E-09 M or less, 5E-09 M or less, 4E-09 M or less, 3E-09 M or less, 2E-09 M or less, or 1E-10 M or less) as measured by biolayer interferometry (BLI). ｄ and having binding affinity for recombinant human IL-36R protein; b) a K of 1E-08 M or less (e.g., 9E-09 M or less, 8E-09 M or less, 7E-09 M or less, 6E-09 M or less, 5E-09 M or less, 4E-09 M or less, 3E-09 M or less, 2E-09 M or less, or 1E-10 M or less) as measured by surface plasmon resonance (SPR). ｄ and having binding affinity for recombinant human IL-36R protein; c) an EC200 of 0.1 μg / mL or less (e.g., 0.09 μg / mL, 0.08 μg / mL, 0.07 μg / mL, 0.06 μg / mL, 0.05 μg / mL, 0.04 μg / mL, 0.03 μg / mL, 0.02 μg / mL, 0.01 μg / mL, or 0.005 μg / mL) as measured by enzyme-linked immunosorbent assay (ELISA). ５０ and having binding affinity for recombinant human IL-36R protein; or d) an EC50 of 0.5 μg / mL or less (e.g., 0.4 μg / mL, 0.3 μg / mL, 0.2 μg / mL, 0.19 μg / mL, 0.18 μg / mL, 0.17 μg / mL, 0.16 μg / mL, 0.15 μg / mL, or 0.1 μg / mL) as measured by fluorescence-activated cell sorting (FACS). ５０ and having binding affinity to membrane human IL-36R. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, having one or more binding properties to human IL-36R selected from the group consisting of:

12. a) capable of blocking IL-36R signaling induced by an IL-36R agonist (preferably IL-36α) as measured by an IL-36R reporter assay; b) capable of inhibiting IL-36R agonist-induced IL-8 release in cells, wherein the IL-36R agonist comprises IL-36α, IL-36β, and / or IL-36γ; c) capable of inhibiting IL-36R agonist-induced IL-6 release in cells, wherein the IL-36R agonist comprises IL-36α, IL-36β, and / or IL-36γ; or d) having the ability to inhibit IL-36R agonist-induced TNF-α release in cells, wherein the IL-36R agonist comprises IL-36α, IL-36β, and / or IL-36γ. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, having one or more properties selected from the group consisting of:

13. (i) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, and (ii) one or more pharmaceutically acceptable carriers A pharmaceutical composition comprising:

14. 14. The pharmaceutical composition of claim 13, further comprising an additional therapeutic agent, wherein the additional therapeutic agent is an agent for treating a disease or disorder responsive to IL-36R inhibition.

15. A method for expressing an antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, the method comprising the step of culturing a host cell containing a vector comprising an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof described in any one of claims 1 to 5 under conditions in which the vector is expressed.

16. 10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or a polynucleotide encoding said antibody or antigen-binding fragment thereof, for use in a method for treating, preventing, or alleviating a disease or disorder responsive to IL-36R inhibition in a subject, the method comprising administering to a subject a therapeutically effective amount of said antibody or antigen-binding fragment thereof, or a polynucleotide encoding said antibody or antigen-binding fragment thereof; In particular, said disease or disorder is selected from the group consisting of an inflammatory disease, an autoimmune disease, a respiratory disease, a metabolic disorder, and cancer, wherein: a) the inflammatory disease is selected from the group consisting of 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; b) the autoimmune disease is selected from the group consisting of multiple sclerosis, asthma, type 1 diabetes mellitus, rheumatoid arthritis, scleroderma, Crohn's disease, plaque psoriasis (commonly referred to as psoriasis), hidradenitis suppurativa, generalized pustular psoriasis (GPP), palmoplantar pustulosis (PPP), inflammatory bowel disease, psoriatic arthritis, systemic lupus erythematosus (SLE), ulcerative colitis, ankylosing spondylitis, atopic dermatitis, acne vulgaris, and Behcet's disease; c) the respiratory disease is selected from the group consisting of asthma, cystic fibrosis, emphysema, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome; d) the metabolic disease is selected from the group consisting of obesity, type 2 diabetes, atherosclerosis, and cardiovascular disease; e) the cancer is selected from the group consisting of 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; An antibody or an antigen-binding fragment thereof, or a polynucleotide encoding said antibody or antigen-binding fragment thereof.

17. A method for detecting the presence or amount of IL-36R in a sample, comprising the steps of contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, and determining the presence or amount of IL-36R in the sample.

18. (a) an antigen-binding domain, a transmembrane domain, and a TCR signaling domain; or (b) antigen-binding domain, transmembrane domain, TCR signaling domain, and costimulatory domain A chimeric antigen receptor (CAR) comprising: A chimeric antigen receptor (CAR), wherein the antigen-binding domain specifically binds to IL-36R and comprises the antigen-binding fragment of any one of claims 1 to 5.

19. A method for stimulating a T cell-mediated immune response against IL-36R-expressing cells or tissues in a mammal, the method comprising the step of administering to the mammal an effective amount of cells genetically modified to express the CAR of claim 18.

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