IL-36R binding protein and medical application thereof

Abstract

The invention relates to an IL-36R binding protein and a medical application thereof. In particular to an IL-36R / IL-23 binding protein, an IL-36R binding protein, a method for treating inflammatory diseases and autoimmune diseases by using the IL-36R binding protein, and related pharmaceutical application of the IL-36R binding protein.

Description

IL-36R binding protein and its medical use

[0001] This disclosure claims priority to Chinese patent application 202310148631.7 filed on February 21, 2023, and the entire contents of the aforementioned patent application are incorporated into this disclosure by reference. Technical Field

[0002] The present disclosure relates to the field of biomedicine, and in particular to IL-36R binding proteins and methods for treating cancer and related pharmaceutical uses thereof. Background Art

[0003] Interleukin-36 (IL-36) belongs to the IL-1 superfamily and includes three cytokines with proinflammatory functions: IL-36α, IL-36β, and IL-36γ. IL-36 is primarily secreted by epithelial cells and inflammatory immune cells in an inactive full-length form. At sites of inflammatory disease, IL-36 is cleaved by neutrophil-derived proteases (such as cathepsin G and elastases), releasing the active fragment, which further binds to the receptor to exert downstream functions. The IL-36 receptor is a heterodimer composed of IL-36R (also known as interleukin-1 receptor-like 2, IL1RL2, primarily expressed on epithelial cells and monocytes) and IL-1RAcP. IL-36 binds to IL-36R, recruiting IL-1RAcP and exerting downstream functions by activating signaling pathways such as MAPK and NF-κB. IL-36RA and IL-38 can bind to IL-36R and inhibit the recruitment of IL-1RAcP, thereby blocking the activation of downstream pathways by IL-36 (Elias M et al. JCI. 2021).

[0004] Activation of the IL-36 pathway mediates multiple biological processes: 1) promoting keratinocyte proliferation and inhibiting its differentiation; 2) promoting the release of multiple cytokines (including IL-6, TNFα, IL-8, CCL20, CXCL10, etc.); 3) promoting the maturation and activation of dendritic cells (DC) (Yuan ZC et al. Frontiers Immunol. 2019). Disorders of the IL-36 pathway are associated with a variety of autoimmune diseases. Upregulated IL-36 expression has been observed in the skin lesions of many patients with inflammatory skin diseases (including psoriasis, hidradenitis suppurativa, lichen simplex chronicus, prurigo nodularis, pemphigus, etc.) and in the intestines of patients with inflammatory bowel diseases (ulcerative colitis, Crohn's disease) (Aquino TM et al. J Cutan Pathol. 2021). Literature reports suggest that inactivating mutations in the inhibitory ligand IL-36RA are closely associated with the development of generalized pustular psoriasis (GPP). Approximately 46.8% to 60.5% of Chinese GPP patients harbor inactivating mutations (Li X et al., J Dermatol Sci, 2014). A Japanese study showed that 9 / 11 patients with GPP alone and 2 / 20 patients with GPP and psoriasis vulgaris harbored inactivating mutations (Sugiura K et al., J Invest Dermatol, 2013). In a preclinical IMQ-induced psoriasis model, blocking the IL-36 pathway significantly reduced disease scores and suppressed the expression of downstream inflammatory genes (Mahil SK et al., Sci Trans Med, 2017).

[0005] Interleukin-23 (IL-23) belongs to the IL-12 family and is a heterodimer composed of p19 and p40 subunits. IL-23 is primarily secreted by activated macrophages and dendritic cells. After binding to the IL-23R / IL-12Rβ1 heterodimeric receptor complex, it promotes the differentiation and phenotypic maintenance of type 17 T cells by activating the JAK-STAT pathway and promoting the secretion of multiple type 17 cytokines, such as IL-17A, IL-17F, and IL-22 (Elias M et al. JCI. 2021; Teng MWL et al. Nat Med. 2016). The IL-23 / IL-17 pathway plays a crucial role in various autoimmune diseases.

[0006] Although anti-IL-23 antibodies have good efficacy in psoriasis, some patients still do not respond well to anti-IL-23 antibodies (Ghoreschi, K et al, The Lancet, 2021). The gene expression of the IL-36 pathway is significantly increased in the skin lesions of these patients who do not respond well to IL-23 antibodies. IL-36 is directly involved in skin inflammatory diseases and immune cell recruitment. Targeting the IL-36 and IL-23 pathways simultaneously can potentially increase the onset rate of anti-IL-23 antibodies and expand the beneficiary population. In view of the unmet patient needs during clinical treatment, the present disclosure provides highly active therapeutic anti-IL-36R antibodies that can specifically bind to IL-36R and have excellent antigen binding specificity, affinity, and pharmacokinetic and pharmacodynamic properties that can be used to intervene in or treat diseases related to the IL-36R signaling pathway, especially autoimmune diseases. In addition, the present disclosure provides a therapeutic humanized anti-IL-36R / IL-23 bispecific antibody with novel mechanism, which can specifically bind to IL-36R and IL-23 simultaneously, and has excellent antigen binding specificity, affinity, and pharmacokinetic and pharmacodynamic properties that can be used to intervene in or treat diseases related to the IL-36R / IL-23 signaling pathway, especially autoimmune diseases.

[0007] Summary of the Invention

[0008] The present disclosure provides IL-36R binding proteins, IL-36R / IL-23 binding proteins, their encoding nucleic acids, vectors, host cells, pharmaceutical compositions, methods for treating or preventing diseases, and related pharmaceutical uses.

[0009] IL-36R binding protein

[0010] The present disclosure provides an IL-36R binding protein comprising an immunoglobulin single variable domain comprising:

[0011] CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 3, 12-26, or

[0012] CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 4 and 27-35,

[0013] Wherein, the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, for example, according to the Kabat numbering system.

[0014] In some embodiments, an IL-36R binding protein is provided, comprising any one or any combination of the above-mentioned CDR1, CDR2, and CDR3.

[0015] In some embodiments, an IL-36R binding protein is provided, which comprises an immunoglobulin single variable domain, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 5, 6, and 7, or in SEQ ID NOs: 8, 9, and 10, respectively.

[0016] In some embodiments, the immunoglobulin single variable domain in the aforementioned IL-36R binding protein is humanized, backmutated, affinity matured, T cell epitope (TCE) removed / reduced, antibody deamidation reduced, and / or antibody isomerization reduced.

[0017] In some embodiments, the immunoglobulin single variable domain is obtained by removing / reducing TCEs and has one or more changes in one or more CDRs, which result in reduced immunogenicity of the IL-36R binding protein.

[0018] In some embodiments, the immunoglobulin single variable domain has one, more or any combination of mutations at amino acid residues 1, 27, 28, 29, 30, 37, 44, 45, 47, 49, 71, 74, 76, 78, 94, 103 (numbered according to the Kabat numbering system), for example, one, more or any combination of mutations selected from 1D, 27Y, 28S, 29Y, 30R, 37Y, 44Q, 45R, 47A, 49A, 71Q, 74A, 76T, 78V, 94A, 103S, for example, a combination of mutations in Table 3-1 of the present disclosure.

[0019] In some embodiments, the immunoglobulin single variable domain has one, more or any combination of mutations at amino acid residues 27, 29, 37, 33, 45, 47, 49, 71, and 94 (numbered according to the Kabat numbering system), for example, one, more or any combination of mutations selected from 27N, 29Y, 37F, 44E, 45R, 47G, 49A, 71K, and 94A, for example, a combination of mutations in Table 3-2 of the present disclosure.

[0020] In some embodiments, the immunoglobulin single variable domain has one, more or any combination of mutations selected from F27N, F29Y, V37F, G44E, L45R, W47G, S49A, R71K, and R94A, for example, a combination of mutations listed in Table 3-2 of the present disclosure.

[0021] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the aforementioned IL-36R binding protein is as shown in any one of SEQ ID NOs: 3, 12-26, or has at least 80% or at least 90% sequence identity to any one of SEQ ID NOs: 3, 12-26, respectively. In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the aforementioned IL-36R binding protein is as shown in any one of SEQ ID NOs: 4, 27-35, or has at least 80% or at least 90% sequence identity to any one of SEQ ID NOs: 4, 27-35, respectively.

[0022] In the present disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity; "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.

[0023] In some embodiments, the aforementioned IL-36R binding protein comprises or is an antibody or antigen-binding fragment thereof that specifically binds to a human IL-36R protein or a fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a recombinant antibody or fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a linear antibody, a single-chain antibody, a nanobody, a peptibody, a domain antibody, and a multispecific antibody (bispecific antibody, diabody, triabody, tetrabody, tandem di-scFv, tandem tri-scFv).

[0024] In some embodiments, the immunoglobulin single variable domain in the aforementioned IL-36R binding protein is a single domain antibody or VHH.

[0025] In some embodiments, the present disclosure provides an IL-36R binding protein comprising one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, wherein the immunoglobulin single variable domains can be the same or different and can form dimers or multimers.

[0026] In some embodiments, the aforementioned IL-36R binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is a human IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region having an S228P mutation, such as that shown in SEQ ID NO:11, or having at least 80% or at least 90% sequence identity thereto. In some embodiments, the Fc region has enhanced stability or comprises a mutation that increases the stability of the Fc region compared to a wild-type Fc region. In some embodiments, the Fc region can enable the binding protein to form a dimeric molecule. In some embodiments, the Fc region can extend the in vivo half-life of the binding protein.

[0027] In some embodiments, the immunoglobulin single variable domain in the aforementioned IL-36R binding protein is linked to the Fc region directly or via a linker. The linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length, free of secondary or higher structure. For example, the linker is a flexible linker, such as G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, and more particularly (G4S)2.

[0028] In some embodiments, the IL-36R binding protein of the present disclosure is an anti-IL-36R antibody or an antigen-binding fragment thereof, or a conjugate or fusion protein comprising the antibody or antigen-binding fragment.

[0029] In some embodiments, the aforementioned IL-36R binding protein has at least one activity selected from the group consisting of:

[0030] (a)≤10 -7 K D The value binds to human IL-36R or its epitope;

[0031] (b) having an activity of inhibiting IL-17 secretion; preferably, with an IC of less than 0.03 nM (e.g., less than 0.03 nM, less than 0.02 nM, less than 0.01 nM or less) 50 In some embodiments, the IC 50 The value is detected by the method of Example 5 of the present disclosure;

[0032] (c) having the activity of inhibiting the proliferation of BaF3-IL-23R cells; preferably, with an IC of less than 0.5 nM (e.g., less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, less than 0.1 nM or less) 50 In some embodiments, the IC 50 The value was detected by the method of Example 9;

[0033] (d) having an inhibitory effect on STAT pathway activity; preferably, with an IC of less than 10 nM (e.g., less than 9 nM, less than 8 nM or less) 50 In some embodiments, the IC 50 The value was detected by the method of Example 9;

[0034] (e) having an inhibitory effect on human IL-36 stimulatory factor activity; preferably, with an IC of less than 0.5 nM (e.g., less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, less than 0.1 nM or less) 50 In some embodiments, the IC 50 The values ​​were determined by the method of Example 8.

[0035] In some embodiments, the aforementioned IL-36R binding proteins of the present disclosure bind to the K D The value can be ≤1×10 -7 M, for example, ≤1×10 -8 M, or ≤1×10 -9 M, or ≤1×10 -10 M.

[0036] In some embodiments, the aforementioned IL-36R binding proteins disclosed herein encompass variants having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any one of SEQ ID NOs: 3, 12-26, or 4, 27-35; the amino acid mutations may be conservative substitutions, replacements, or modifications, and / or deletions or additions that do not affect function; the amino acid mutations may occur in CDR regions and / or FR regions.

[0037] In some embodiments, an anti-IL-36R antibody or antigen-binding fragment thereof is provided that binds to or competes for binding to the same epitope as the immunoglobulin single variable domain in the aforementioned IL-36R binding protein of the present disclosure.

[0038] In some embodiments, anti-IL-36R antibodies or antigen-binding fragments thereof are provided that block the binding of the immunoglobulin single variable domain in the aforementioned IL-36R binding protein of the present disclosure to IL-36R (e.g., human IL-36R).

[0039] In some embodiments, an anti-IL-36R antibody or antigen-binding fragment thereof is provided, wherein the binding of the antibody to IL-36R (e.g., human IL-36R) is blocked by the immunoglobulin single variable domain in the aforementioned IL-36R binding protein of the present disclosure.

[0040] In some embodiments, a protein or molecule is provided that comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) immunoglobulin single variable domains of any of the aforementioned IL-36R binding proteins disclosed herein, wherein the immunoglobulin single variable domains are the same or different. For example, the protein or molecule is a conjugate, and the conjugate can, for example, comprise any detectable label.

[0041] IL-36R / IL-23 binding protein

[0042] The present disclosure provides IL-36R / IL-23 binding proteins that specifically bind to human IL-36R and / or human IL-23p19 subunit.

[0043] In some embodiments, an IL-36R / IL-23 binding protein is provided, comprising a first antigen binding domain that specifically binds to IL-36R, and a second antigen binding domain that specifically binds to IL-23 (e.g., IL-23p19 subunit).

[0044] Regarding the first antigen-binding domain that specifically binds to IL-36R:

[0045] In some embodiments, the first antigen-binding domain in the IL-36R / IL-23 binding protein comprises or is an immunoglobulin single variable domain as described above in the IL-36R binding protein of the present disclosure.

[0046] In some embodiments, the immunoglobulin single variable domain comprises:

[0047] CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 3, 12-26, or

[0048] CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 4 and 27-35,

[0049] Wherein, the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system, for example, according to the Kabat numbering system.

[0050] In some specific embodiments, the immunoglobulin single variable domain comprises any one or any combination of the above-mentioned CDR1, CDR2 and CDR3.

[0051] In some specific embodiments, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown as SEQ ID NO: 5, 6, 7, or as SEQ ID NO: 8, 9, 10, respectively.

[0052] In some embodiments, the immunoglobulin single variable domain in the aforementioned IL-36R binding protein is humanized, backmutated, affinity matured, T cell epitope (TCE) removed / reduced, antibody deamidation reduced, and / or antibody isomerization reduced.

[0053] In some specific embodiments, the immunoglobulin single variable domain is humanized. The heavy chain framework region (FR) of the human germline template used for humanization is derived from IGHV3-23*04, IGHV3-74*01, and / or IGHJ1*01. In some embodiments, FR1, FR2, and FR3 are derived from IGHV3-23*04, and FR4 is derived from IGHJ1*01. In some embodiments, FR1, FR2, and FR3 are derived from IGHV3-74*01, and FR4 is derived from IGHJ1*01.

[0054] In some specific embodiments, the immunoglobulin single variable domain has one, more or any combination of mutations at amino acid residues 1, 27, 28, 29, 30, 37, 44, 45, 47, 49, 71, 74, 76, 78, 94, 103 (numbered according to the Kabat numbering system), for example, one, more or any combination of mutations selected from 1D, 27Y, 28S, 29Y, 30R, 37Y, 44Q, 45R, 47A, 49A, 71Q, 74A, 76T, 78V, 94A, 103S, for example, a combination of mutations in Table 3-1 of the present disclosure.

[0055] In some specific embodiments, the immunoglobulin single variable domain has one, more or any combination of mutations at amino acid residues 27, 29, 37, 33, 45, 47, 49, 71, and 94 (numbered according to the Kabat numbering system), for example, one, more or any combination of mutations selected from 27N, 29Y, 37F, 44E, 45R, 47G, 49A, 71K, and 94A, for example, a combination of mutations in Table 3-2 of the present disclosure.

[0056] In some specific embodiments, the amino acid sequence of the immunoglobulin single variable domain is as shown in any one of SEQ ID NOs: 3, 12-26, or has at least 80% or at least 90% sequence identity to any one of SEQ ID NOs: 3, 12-26, respectively. In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the aforementioned IL-36R binding protein is as shown in any one of SEQ ID NOs: 4, 27-35, or has at least 80% or at least 90% sequence identity to any one of SEQ ID NOs: 4, 27-35, respectively.

[0057] In some embodiments, the immunoglobulin single variable domain is a single domain antibody or VHH.

[0058] In some embodiments, the first antigen-binding domain that specifically binds to IL-36R comprises a fab or VH and VL of BI655130 or H4H14706P2, which are incorporated herein by reference in their entirety by reference to WO2013074569A1 and WO2020018503A. In some embodiments, the first antigen-binding domain that specifically binds to IL-36R comprises a VH and a VL, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 36, and the VL comprises the LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 37; for example, the VH is the VH of SEQ ID NO: 36 or is at least 80% or at least 90% identical thereto, and the VL is the VL of SEQ ID NO: 37 or is at least 80% or at least 90% identical thereto. In some specific embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 38, and the VL comprises LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 39; for example, the VH is the VH in SEQ ID NO: 38 or has at least 80% or at least 90% identity thereto, and the VL is the VL in SEQ ID NO: 39 or has at least 80% or at least 90% identity thereto.

[0059] Regarding the second antigen binding domain that specifically binds to IL-23 (e.g., IL-23 p19 subunit):

[0060] In some embodiments, the second antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0061] In some embodiments, in the second antigen binding domain,

[0062] VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 40, and VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 41; or

[0063] The VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequence of any one of SEQ ID NOs: 52-57, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequence of any one of SEQ ID NOs: 58-61;

[0064] Wherein, the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, for example, according to the Kabat numbering system.

[0065] In some embodiments, in the second antigen binding domain,

[0066] VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 42-44, and VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 45-47;

[0067] VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 50, 43, and 44, and VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 45-47;

[0068] VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 51, 43, and 44, and VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 45-47.

[0069] In some embodiments, the second antigen binding domain is humanized, backmutated, affinity matured, T cell epitope (TCE) removed / reduced, antibody deamidation reduced, and / or antibody isomerization reduced.

[0070] In some embodiments, the second antigen binding domain has a mutation at amino acid residues 1, 30, 37, 44, 49, 73, 89, and / or 93 (numbered according to the Kabat numbering system) in VH, and / or a mutation at amino acid residues 4, 17, 36, 58, 60, and / or 68 (numbered according to the Kabat numbering system) in VL. For example, VH has one, more, or any combination of mutations among 1E, 30T, 37V, 44G, 49G, 73N, 89R, and 93V, and / or VL has one, more, or any combination of mutations among 4L, 17Q, 36F, 58I, 60A, and 68R.

[0071] In some embodiments, in the second antigen binding domain,

[0072] VH comprises an amino acid sequence as set forth in SEQ ID NO:40, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO:41, or at least 80% or at least 90% identical thereto;

[0073] VH comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 52-57, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 58-61, or at least 80% or at least 90% identical thereto;

[0074] For example, VH comprises the amino acid sequence shown in SEQ ID NO: 56, and VL comprises the amino acid sequence shown in SEQ ID NO: 60.

[0075] In some specific embodiments, the second antigen-binding domain comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 62 or 64, or is at least 80% or at least 90% identical thereto; the amino acid sequence of the light chain is as shown in SEQ ID NO: 63 or 65, or is at least 80% or at least 90% identical thereto.

[0076] In some embodiments, the second antigen-binding domain that specifically binds to IL-23 comprises a fab or VH and VL of Risankizumab or Guselkumab. In some embodiments, the second antigen-binding domain that specifically binds to IL-23 comprises a VH and a VL, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 66, and the VL comprises the LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 67; for example, the VH is the VH of SEQ ID NO: 66 or is at least 80% or at least 90% identical thereto, and the VL is the VL of SEQ ID NO: 67 or is at least 80% or at least 90% identical thereto. In some specific embodiments, the second antigen-binding domain that specifically binds IL-23 comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 77, and the VL comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 78; for example, the VH is the VH of SEQ ID NO: 77 or is at least 80% or at least 90% identical thereto, and the VL is the VL of SEQ ID NO: 78 or is at least 80% or at least 90% identical thereto.

[0077] About IL-36R / IL-23 Binding Protein:

[0078] The following exemplifies the IL-36R / IL-23 binding proteins of the present disclosure:

[0079] In some embodiments, the IL-36R / IL-23 binding protein has one or more (e.g., 2, 3, 4, 5, 6) first antigen-binding domains that specifically bind to IL-36R, and / or one or more (e.g., 2, 3, 4) second antigen-binding domains that specifically bind to IL-23. In some specific embodiments, the IL-36R / IL-23 binding protein has two first antigen-binding domains that specifically bind to IL-36R and two second antigen-binding domains that specifically bind to IL-23.

[0080] In some embodiments, in the IL-36R / IL-23 binding protein, the valency ratio of the first antigen-binding domain that specifically binds to IL-36R to the second antigen-binding domain that specifically binds to IL-23 is between 6:1 and 1:3 (e.g., 4:1 to 1:2), for example, 1:1, 1:2, 2:1, 1:3, or 3:1.

[0081] In some embodiments, in the IL-36R / IL-23 binding protein, the first antigen-binding domain that specifically binds to IL-36R is located at the N-terminus and / or C-terminus of the second antigen-binding domain that specifically binds to IL-23.

[0082] In some embodiments, the IL-36R / IL-23 binding protein further comprises a human immunoglobulin Fc region, for example, the Fc region is a human IgG1, IgG2, or IgG4 Fc region.

[0083] In some embodiments, the Fc region can enable the binding protein to form a dimeric molecule.

[0084] In some embodiments, the Fc region comprises a mutation that extends the in vivo half-life, which is determined by the FcRn binding affinity. The extension of half-life can allow for a reduction in the amount of drug administered to the patient and / or a reduction in the frequency of administration. For example, the Fc region comprises an M252Y, S254T, and / or T256E mutation.

[0085] In some specific embodiments, the Fc region can be an Fc region with reduced effector function, for example, the Fc region can have a mutation, and exemplary IgG Fc regions with reduced effector function include those with the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1).

[0086] In some embodiments, the Fc region is an Fc region with increased stability, for example, an Fc region of human IgG4 having an S228P mutation.

[0087] In some embodiments, the amino acid sequence of the Fc region is as shown in SEQ ID NO: 11, 48, 76, or is at least 80% or at least 90% identical thereto.

[0088] In some embodiments, the Fc region of the IL-36R / IL-23 binding protein comprises a first subunit Fc1 and a second subunit Fc2 that are capable of associating with each other.

[0089] In some embodiments, Fc1 and Fc2 contain amino acid mutations that result in Fc1 preferentially pairing with Fc2 or forming heterodimers with Fc2 compared to Fc1. In some embodiments, the mutations are located in the CH3 of Fc1 and Fc2. In some embodiments, the amino acid mutations in Fc1 and Fc2 result in greater electrostatic complementarity than a wild-type mutant lacking the mutations. In some embodiments, the amino acid mutations in Fc1 and Fc2 result in greater steric complementarity than a wild-type mutant lacking the mutations.

[0090] In some embodiments, in Fc1 and Fc2, for example, within the CH3 / CH3 interface, one or more amino acid residues in the CH3 domain of Fc1 are mutated with one or more amino acid residues having a larger side chain volume, thereby generating a protrusion (or knob) on the surface of the CH3 domain of Fc1, and one or more, preferably two or three, amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated with amino acid residues having a smaller side chain volume, thereby generating a depression (or hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1. In some embodiments, the import residue with a larger side chain volume is phenylalanine (F), tyrosine (Y), arginine (R), or tryptophan (W). In some embodiments, the import residue with a smaller side chain volume is serine (S), alanine (A), valine (V), or threonine (T).

[0091] In some specific embodiments, the Fc1 comprises at least one or at least two amino acid mutations selected from T366S, L368A and Y407V (hole mutation modification), and the Fc2 comprises T366W (knob mutation modification); or the Fc1 comprises T366W (knob mutation modification), and the Fc2 comprises at least one or at least two amino acid mutations selected from T366S, L368A and Y407V (hole mutation modification).

[0092] In some embodiments, Fc1 and Fc2, for example, CH3, may contain a natural non-cysteine ​​to cysteine ​​mutation, such as S354C in Fc1 and Y349C in Fc2; or Y349C in Fc1 and S354C in Fc2.

[0093] In some embodiments, Fc1 and Fc2, for example, in the Fc1CH3 / Fc2CH3 interface, comprise the following amino acid mutations or combinations thereof: T366Y / Y407T; T366W / Y407A; T366Y / Y407T; T394W / F405A; T366Y / F405AT394W / Y407T; T366W / F407T; 5WT394S / Y407A; F405W / T394S; D399C / K392C; T366W / T366S / L368A / Y407V; T366W / D399C / T366S / L368A / K392C / Y407V; T366W / K392C / T366S / D399C / L368A / Y407V; S354C / T366W / Y349C / T366S / L368A / Y407V; Y349C / T366W / S354C / T366S / L368A / Y407V; E356C / T366W / Y349C / T366S / L368A / Y407V; Y349C / T366W / E356C / T366S / L368A / Y407V; E357C / T366W / Y349C / T366S / L368A / Y407V; and Y349C / T366W / E357C / T366S / L368A / Y407V.

[0094] In some embodiments, Fc1 and Fc2 further comprise amino acid mutations that form an electrostatic interaction interface between Fc1 and Fc2 (e.g., CH3 and CH3). Amino acid mutations that form an electrostatic interaction interface are, for example, selected from the following: K370E / D399K / K439D / D356K / E357K / K409D; K409D / D399K; K409E / D399K; K409E / D399R; K409D / D399R; D339K / E356K; D399K / E356K / K409D / K392D; D 399K / E356K / K409D / K439D; D399K / E357K / K409D / K370D; D399K / E356K / E357K / K409D / K 392D / K370D; D399K / E357K / K409D / K392D; K392D / K409D / D399K; and K409D / K360D / D399K.

[0095] In some embodiments, Fc1 and / or Fc2 comprise domains from different antibody subtypes, such as CH3 from different antibody subtypes.

[0096] In addition, the present disclosure cites WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012058768, WO2013157954, and WO2013096291 for modifying the CH3 region of the Fc region to enhance heterodimerization.

[0097] In some embodiments, in the IL-36R / IL-23 binding protein, the first antigen-binding domain that specifically binds IL-36R and the second antigen-binding domain that specifically binds IL-23 are linked directly or via a linker. In some embodiments, the second antigen-binding domain that specifically binds IL-23 is linked directly or via a linker to an Fc region.

[0098] In some specific embodiments, the linker includes but is not limited to (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h The amino acid sequence shown, wherein m, n are each independently selected from integers of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), and h is independently selected from integers of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20). In some embodiments, the linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structure. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, for example (G4S)2, (G4S)3.

[0099] In some specific embodiments, the IL-36R / IL-23 binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains are as follows from N-terminus to C-terminus:

[0100] (1) First polypeptide chain: [first antigen-binding domain that specifically binds to IL-36R]-[Linker 1]a-[VH of the second antigen-binding domain that specifically binds to IL-23]-CH1-Fc region; Second polypeptide chain: [VL of the second antigen-binding domain that specifically binds to IL-23]-CL;

[0101] (2) First polypeptide chain: [VH that specifically binds to the second antigen-binding domain of IL-23]-CH1-Fc region; Second polypeptide chain: [First antigen-binding domain that specifically binds to IL-36R]-[Linker 1]a-[VL that specifically binds to the second antigen-binding domain of IL-23]-CL;

[0102] (3) First polypeptide chain: [VH that specifically binds to the second antigen-binding domain of IL-23]-CH1-Fc region-[Linker 1]a-[first antigen-binding domain that specifically binds to IL-36R]; Second polypeptide chain: [VL that specifically binds to the second antigen-binding domain of IL-23]-CL;

[0103] (4) First polypeptide chain: [VH that specifically binds to the second antigen-binding domain of IL-23]-CH1-Fc region; Second polypeptide chain: [VL that specifically binds to the second antigen-binding domain of IL-23]-[Linker 1]a-CL [first antigen-binding domain that specifically binds to IL-36R];

[0104] (5) First polypeptide chain: [VH that specifically binds to the second antigen-binding domain of IL-23]-CH1-Fc region-[Linker 1]a-[First antigen-binding domain that specifically binds to IL-36R]; Second polypeptide chain: [VL that specifically binds to the second antigen-binding domain of IL-23]-CL-[Linker 2]b-[First antigen-binding domain that specifically binds to IL-36R]

[0105] (6) First polypeptide chain: [first antigen-binding domain that specifically binds to IL-36R]-[Linker 1]a-[VH of the second antigen-binding domain that specifically binds to IL-23]-CH1-Fc region; Second polypeptide chain: [first antigen-binding domain that specifically binds to IL-36R]-[Linker 2]b-[VL of the second antigen-binding domain that specifically binds to IL-23]-CL

[0106] Wherein, - represents a peptide bond, and a linker is a polypeptide capable of achieving a linking function. Linker 1 and Linker 2 may be the same or different; a and b may be independently selected from 1 or 0, for example, both a and b are 1. The linker is selected from, for example, G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, and ASGS, for example, (G4S)2 and (G4S)3.

[0107] In some embodiments, an IL-36R / IL-23 binding protein is provided, comprising a first and a second polypeptide chain. In some specific embodiments, the amino acid sequence of the first polypeptide chain is as set forth in any one of SEQ ID NOs: 68, 70, 71, 74, and 75, or has at least 80% or at least 90% sequence identity thereto, and the amino acid sequence of the second polypeptide chain is as set forth in any one of SEQ ID NOs: 69, 72, and 73, or has at least 80% or at least 90% sequence identity thereto.

[0108] In some embodiments, an IL-36R / IL-23 binding protein is provided, comprising a first and a second polypeptide chain:

[0109] (1) The amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 68 or 70, or has at least 80% or at least 90% sequence identity thereto, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 69, or has at least 80% or at least 90% sequence identity thereto;

[0110] (2) the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 71, or has at least 80% or at least 90% sequence identity thereto, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 72 or 73, or has at least 80% or at least 90% sequence identity thereto;

[0111] (3) The amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 74 or 75, or has at least 80% or at least 90% sequence identity thereto, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 69, or has at least 80% or at least 90% sequence identity thereto.

[0112] In some embodiments, the IL-36R / IL-23 binding protein of the present disclosure has at least one activity selected from the group consisting of:

[0113] (a)≤10 -7 K D The value binds to human IL-36R or its epitope;

[0114] (b) ≤10 -7 K D The value binds to the human IL-23p19 subunit or its epitope;

[0115] (c) having an activity of blocking IL-23 / IL-23R binding; preferably, the IC50 value of blocking human IL-23 / IL-23R binding is less than 0.6 nM (e.g., less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM or less), the IC 50The value is detected by ELISA method; in some embodiments, the IC 50 The value is detected by the method of Example 5 of the present disclosure;

[0116] (d) having an activity of inhibiting IL-17 secretion; preferably, with an IC of less than 0.03 nM (e.g., less than 0.03 nM, less than 0.02 nM, less than 0.01 nM or less) 50 In some embodiments, the IC 50 The value is detected by the method of Example 5 of the present disclosure;

[0117] (e) having the activity of inhibiting the proliferation of BaF3-IL-23R cells; preferably, with an IC of less than 0.5 nM (e.g., less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, less than 0.1 nM or less) 50 In some embodiments, the IC 50 The value was detected by the method of Example 9;

[0118] (f) having an inhibitory effect on STAT pathway activity; preferably, with an IC of less than 10 nM (e.g., less than 9 nM, less than 8 nM or less) 50 In some embodiments, the IC 50 The value was detected by the method of Example 9;

[0119] (g) having an inhibitory effect on human IL-36 stimulatory factor activity; preferably, with an IC of less than 0.5 nM (e.g., less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, less than 0.1 nM or less) 50 In some embodiments, the IC 50 The value was detected by the method of Example 8;

[0120] (h) capable of alleviating or treating inflammatory diseases caused by excessive expression of IL-36 and / or IL-23, such as psoriasis; preferably, in a mouse animal model, it can inhibit mouse ear swelling induced by human IL-36 and human IL-23, with an inhibition ratio of, for example, at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more; in some embodiments, the inhibition ratio is achieved by detecting IL-36α, IL-23, IL-17F, S100A8, S100A9, or KRT16, such as the detection method in Example 10.

[0121] In some embodiments, the IL-36R / IL-23 binding protein of the present disclosure encompasses variants, wherein the variant has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to the first and second polypeptide chains in any combination of (1) to (3) above; the amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function.

[0122] In some embodiments, IL-36R / IL-23 binding proteins are provided that bind to or compete for binding to IL-23 and / or IL-36R, or bind to or compete for binding to the same epitope of IL-23 and / or IL-36R as the aforementioned IL-36R / IL-23 binding proteins of the present disclosure.

[0123] In some embodiments, an IL-36R / IL-23 binding protein is provided that blocks the aforementioned IL-36R / IL-23 binding protein of the present disclosure from binding to IL-23 and / or IL-36R.

[0124] In some embodiments, a protein or molecule is provided, comprising any of the aforementioned IL-36R / IL-23 binding proteins disclosed herein. For example, the protein or molecule is a conjugate, and the conjugate may comprise any detectable label.

[0125] Composition or complex of IL-36R binding protein and IL-23 binding protein

[0126] The present disclosure provides compositions or complexes comprising an IL-36R binding protein and an IL-23 binding protein, for example, comprising an anti-IL-36R antibody or an antigen-binding fragment thereof and an anti-IL-23 antibody or an antigen-binding fragment thereof.

[0127] In some embodiments, the IL-36R binding protein is the aforementioned IL-36R binding protein of the present disclosure, and the IL-23 binding protein comprises a VH and a VL, wherein the VH comprises the HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO:40, and the VL comprises the LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in SEQ ID NO:41; or, the VH comprises the HCDR1, HCDR2, and HCDR3 of the amino acid sequence set forth in any one of SEQ ID NOs:52-57, and the VL comprises the LCDR1, LCDR2, and LCDR3 of the amino acid sequence set forth in any one of SEQ ID NOs:58-61; wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems, for example, according to the Kabat numbering system.

[0128] In some specific embodiments, in the IL-23 binding protein,

[0129] VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 42-44, and VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 45-47;

[0130] VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 50, 43, and 44, and VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 45-47;

[0131] VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 51, 43, and 44, and VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 45-47.

[0132] In some specific embodiments, in the IL-23 binding protein,

[0133] VH comprises an amino acid sequence as set forth in SEQ ID NO:40, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as set forth in SEQ ID NO:41, or at least 80% or at least 90% identical thereto;

[0134] VH comprises an amino acid sequence as shown in any one of SEQ ID NOs: 52-57, or at least 80% or at least 90% identical thereto, and VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 58-61, or at least 80% or at least 90% identical thereto.

[0135] Exemplarily, a composition or complex is provided, comprising an IL-36R binding protein and an IL-23 binding protein, wherein the IL-36R binding protein comprises or is VHH 213 or a humanized and backmutated sequence thereof (e.g., VHH213_Hu07) in Examples 1-3 of the present disclosure, or is VHH162 or a humanized and backmutated sequence thereof (e.g., VHH162_Hu11); and the IL-23 binding protein comprises or is the anti-IL-23 antibody (e.g., Hu29-19) in Example 4 of the present disclosure.

[0136] Polynucleotides and vectors

[0137] The present disclosure provides polynucleotides encoding the IL-36R binding protein and IL-36R / IL-23 binding protein of the present disclosure. The nucleic acids of the present disclosure may be RNA, DNA, or cDNA. According to some embodiments of the present disclosure, the nucleic acids of the present disclosure are substantially isolated nucleic acids.

[0138] The nucleic acids disclosed herein may also be in the form of, present in, and / or part of, a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may particularly be an expression vector, i.e., a vector that provides for expression of the IL-36R binding protein or IL-36R / IL-23 binding protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Such expression vectors typically comprise at least one nucleic acid disclosed herein, operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the skill of the art. Examples of regulatory elements and other elements useful or necessary for expression of the IL-36R binding protein or IL-36R / IL-23 binding protein disclosed herein include promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

[0139] The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0140] host cells

[0141] The present disclosure provides recombinant host cells that express or are capable of expressing one or more IL-36R binding proteins, IL-36R / IL-23 binding proteins, and / or polynucleotides or vectors of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0142] Examples of bacterial cells include cells of gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0143] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0144] Examples of mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0145] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0146] Preparation method

[0147] The present disclosure provides a method for preparing an IL-36R binding protein and an IL-36R / IL-23 binding protein, comprising: expressing the target protein in a host cell as described above, and isolating the target protein from the host cell. Optionally, a purification step may be included, for example, purification using an A or G Sepharose FF column containing an adjusted buffer, washing away non-specifically bound components, eluting the bound antibodies using a pH gradient method, detecting using SDS-PAGE, and collecting. Optionally, filtration and concentration are performed using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product must be immediately frozen, such as at -70°C, or freeze-dried.

[0148] Methods for producing and purifying antibodies are well known in the art and can be found in, for example, the Cold Spring Harbor Manual of Antibody Laboratory Techniques (Chapters 5-8 and 15).

[0149] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded and cultured in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.

[0150] Composition

[0151] The present disclosure provides compositions comprising the aforementioned IL-36R binding proteins and IL-36R / IL-23 binding proteins of the present disclosure. For example, pharmaceutical compositions are provided, comprising an amount of the aforementioned IL-36R binding proteins and IL-36R / IL-23 binding proteins effective for treating, alleviating, or preventing a disease, and at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0152] In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the IL-36R binding protein or IL-36R / IL-23 binding protein in a unit dose, or the amount of the IL-36R binding protein or IL-36R / IL-23 binding protein in a unit dose of the pharmaceutical composition may be 0.1 to 2000 mg, and in some embodiments, 1 to 1000 mg.

[0153] In some embodiments, an article of manufacture or product is provided, comprising the aforementioned IL-36R binding protein or IL-36R / IL-23 binding protein. Optionally, the article of manufacture comprises a container and a label. Containers such as bottles, syringes, and test tubes contain a composition effective for treating a condition. The label on or associated with the container indicates that the composition is used to treat the selected condition.

[0154] In some embodiments, the aforementioned disease is an inflammatory disease or an autoimmune disease.

[0155] Methods of treatment and pharmaceutical uses

[0156] The present disclosure provides methods for using the aforementioned IL-36R binding proteins, L-36R / IL-23 binding proteins, polynucleotides, and compositions (including pharmaceutical compositions) for treating, alleviating, preventing, or diagnosing diseases or disorders.

[0157] In some embodiments, a method for ameliorating, alleviating, treating, or preventing a disease is provided, comprising administering to a subject an ameliorative, alleviating, therapeutic, or preventive effective amount of the aforementioned IL-36R binding protein, IL-36R / IL-23 binding protein, polynucleotide, or composition (including a pharmaceutical composition).

[0158] In some embodiments, the IL-36R binding protein, IL-36R / IL-23 binding protein polynucleotide, and composition (including pharmaceutical composition) disclosed herein are used for the preparation of a medicament for improving, alleviating, treating, or preventing a disease.

[0159] In some embodiments, the aforementioned disease is a disease or disorder associated with overexpression of IL-36 and / or IL-23.

[0160] In some embodiments, the aforementioned disease is a disease or condition mediated by IL-36R.

[0161] In some embodiments, the aforementioned disease is an inflammatory disease or an autoimmune disease, such as psoriasis.

[0162] Detection

[0163] The present disclosure provides uses for detecting IL-36R binding proteins, IL-36R / IL-23 binding proteins, polynucleotides, and compositions. The present disclosure also provides methods, systems, or devices for detecting IL-36R or IL-23 in vivo or in vitro, comprising treating a sample with the aforementioned binding proteins, polynucleotides, and compositions of the present disclosure.

[0164] In some embodiments, an in vitro detection method, system, or device may include, for example:

[0165] (1) contacting a sample with an IL-36R binding protein, IL-36R / IL-23 binding protein, polynucleotide, or composition of the present disclosure;

[0166] (2) detecting a complex formed between the aforementioned binding protein, polynucleotide, and sample; and / or

[0167] (3) contacting a reference sample (e.g., a control sample) with the binding protein and nucleic acid; and

[0168] (4) Determining the extent of complex formation by comparison with a reference sample. A change (e.g., a statistically significant change) in complex formation in the sample compared to the control sample indicates the presence of IL-36R and IL-23 in the sample.

[0169] In some embodiments, kits are provided, comprising the aforementioned IL-36R binding protein, IL-36R / IL-23 binding protein, and polynucleotides, and instructions for diagnostic use. The kits may also contain at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the IL-36R binding protein and IL-36R / IL-23 binding protein can be formulated as a pharmaceutical composition.

[0170] Definition of terms

[0171] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in the present disclosure, all other technical and scientific terms used in the present disclosure have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0172] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0173] "IL-23" (also known as IL23) is mainly produced by activated dendritic cells, macrophages and monocytes. It is a member of the IL-12 heterodimeric cytokine family and consists of two subunits: p19 (also known as IL-23p19 subunit) and p40 (also known as IL-23 p40 subunit). The p40 subunit is a subunit that is shared with IL-12 (J Immunol. 2018 Sep 15; 201(6): 1605-1613.).

[0174] "IL-36R binding protein" encompasses any protein that specifically binds to IL-36R or any molecule comprising such a protein, including but not limited to antibodies, antigen-binding fragments thereof, or conjugates or fusion proteins thereof, as defined herein, directed against IL-36R. In some embodiments, an "IL-36R binding protein" may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, or more) single-domain antibodies that specifically bind to IL-36R as described in the embodiments of the present disclosure. In some embodiments, the "IL-36R binding protein" of the present disclosure may also comprise, in addition to an immunoglobulin single variable domain that binds to IL-36R, a linker and / or a moiety with effector function, such as a half-life extending moiety (e.g., an immunoglobulin single variable domain that binds to serum albumin) and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region. In some embodiments, an "IL-36R / IL-23 binding protein" encompasses a bispecific antibody against IL-36R and IL-23 as described in the embodiments of the present disclosure.

[0175] "IL-36R / IL-23 binding protein" encompasses any protein that specifically binds to IL-23 and IL-36R, or any molecule comprising such a protein, including but not limited to antibodies, polypeptides, fusion proteins of antibodies and polypeptides, or conjugates or fusion proteins thereof. In some embodiments, "IL-36R / IL-23 binding protein" encompasses the anti-IL-36R and IL-23 bispecific antibodies of the disclosed embodiments.

[0176] "Antibodies" encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies may refer to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant regions of immunoglobulins' heavy chains differ, resulting in different antigenicity. Based on this, immunoglobulins can be divided into five classes, or so-called immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Igs of the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of heavy chain disulfide bonds, such as IgG, which can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of antibody heavy and light chains vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0177] The antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic, or modified forms thereof, with monoclonal antibodies being particularly suitable for use in a number of embodiments. In general, the antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" refers generally to products such as cells or nucleic acids, proteins, or vectors, indicating that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cells are derived from cells so modified. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form or express native genes that are abnormally expressed, underexpressed, or not expressed at all.

[0178] "Antigen-binding fragment" encompasses single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0179] For the determination or definition of CDRs, the deterministic depiction of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions. The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the position of certain structural loop regions. (See, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd.). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from primary sequence (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In the conformational definition, the positions of the CDRs can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166).In addition, other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or residue group does not significantly affect antigen binding. As used in this disclosure, CDRs can refer to CDRs defined by any method known in the art (including combinations of methods). The correspondence between the various numbering systems is well known to those skilled in the art.

[0180] A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0181] "Immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional tetrapeptide chain structure antibody or a heavy chain antibody chain), or a polypeptide consisting essentially of such a globular region. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule, consisting of a two-layer sandwich of about seven antiparallel beta-sheet strands arranged in two beta-sheets, optionally stabilized by conserved disulfide bonds.

[0182] An "immunoglobulin variable domain" is an immunoglobulin domain that essentially consists of four "framework regions," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3," respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity for an antigen by having an antigen-binding site.

[0183] "Antibody framework (FR)" refers to the portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain.

[0184] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without the VH / VL interactions required between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, e.g., camelized human VH), IgNAR, domains, (single domain) antibodies that are VH domains or derived from VH domains (such as dAbs), and antibodies that are VH domains or derived from VH domains. TM ) and (single domain) antibodies (such as dAbs) as the VL domain or derived from the VL domain TM ). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0185] "VHH" is also called heavy chain single domain antibody, VHH, V H H domains, VHH antibody fragments, VHH antibodies, nanobodies, are variable domains of antigen-binding immunoglobulins known as "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). "VHH" is used to distinguish the variable domains from the heavy chain variable domains (referred to herein as "VH domains" or VH) and light chain variable domains (referred to herein as "VL domains" or VL) present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to an epitope without the need for additional antigen-binding domains (this is in contrast to the VH or VL domains in conventional tetrapeptide chain structure antibodies, in which case the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. H H domain", "VHH antibody fragment", "VHH antibody", as well as" "Nanobody" is a trademark of Ablynx NV, Ghent, Belgium. VHHs include, but are not limited to, natural antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained by phage display technology. The total number of amino acid residues in a VHH will generally be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in the present disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17 June, 2009 and WO94 / 04678.

[0186] As is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions numbered according to Kabat may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or coding conventions include Chothia, IMGT, and AbM.

[0187] "Humanized antibodies," also known as CDR-grafted antibodies, are antibodies produced by transplanting non-human CDR sequences into the human variable region framework. This can overcome the strong immune response induced by chimeric antibodies due to the presence of a large number of non-human protein components. To avoid a simultaneous decrease in immunogenicity and activity, minimal reverse mutations can be performed on the fully human variable region to maintain activity. Examples of "humanization" include "humanizing" a Camelidae-derived VHH domain by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in a VH domain of a conventional human tetrapeptide antibody (also referred to as "sequence optimization" in this disclosure; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by one or more mutations that provide improved VHH properties, such as removal of potential post-translational modification sites). A humanized VHH domain may contain one or more fully human framework region sequences, and in some embodiments, may contain human framework region sequences from IGHV3. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework), i.e., CDR "grafting" onto other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for the CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies that have been further affinity-matured by phage display to CDRs. In addition, to avoid a decrease in immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequences can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0188] An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for the antigen, compared to a parent antibody that does not possess such alterations. For example, an "affinity matured" IL-36R binding protein or anti-IL-36R antibody has one or more alterations in one or more CDRs that result in increased affinity for the antigen, compared to its parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example, as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3):889-896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0189] Typically, the IL-36R binding proteins, IL-36R / IL-23 binding proteins of the present disclosure will be expressed as preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (K D ), and / or with at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen or target protein to be bound (i.e. IL-36R, IL-23, CD16A). Any -4 M's K DValues ​​are generally considered to indicate nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays) as described herein.

[0190] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules can be determined by determining the dissociation constant (K D K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K values ​​were compared with those for non-target interactions (e.g., control antibodies known not to bind IL-36R, IL-23, or CD16A). D The value is evaluated.

[0191] A "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0192] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0193] "Nucleic acid" or "polynucleotide" are used interchangeably in this disclosure to refer to any DNA or RNA molecule, whether single-stranded or double-stranded, and, in the case of single-stranded, its complementary sequence, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0194] "Host cell" includes individual cells or cell cultures that can be or have been recipients of vectors for incorporating polynucleotide inserts. Host cells include the progeny of a single host cell, and due to natural, accidental or intentional mutations, the progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such designations include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included.

[0195] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. "Inhibit growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0196] "Administer," "apply," and "treat" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, for example, therapeutic, pharmacokinetics, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. When applied to humans, veterinary medicine, or research subjects, it refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0197] "Treatment" means administering an internal or external therapeutic agent, such as a binding protein or a pharmaceutical composition thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0198] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0199] "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and the description includes instances where the event or circumstance occurs or does not occur. "And / or" should be taken as specifically disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" as used in phrases such as "A and / or B" in the present disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive meaning, rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to." In the context of mutations contained in the Fc region in the present disclosure, " / " means "and," for example, "L234A / L235A" means "L234A and L235A," i.e., the Fc contains L234A and L235A mutations; the amino acid positions of the mutations in the Fc region of the present disclosure are all defined according to the EU numbering system.

[0200] The "subject" and "patient" of the present disclosure refer to mammals, especially primates, and especially humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0201] Figures 1A and 1B show the binding activity of anti-IL-36R single-domain antibodies VHH162 and VHH213 and their humanized antibodies to human IL-36R on the surface of CHO-K1 cells.

[0202] Figure 2 shows the competition binding results between VHH162_Hu09 and VHH213_Hu01 and with control antibodies BI655130 and H4H14706P2.

[0203] FIG3A shows the experimental results of the severity score of erythema in imiquimod-induced psoriasis animals, FIG3B shows the experimental results of the severity score of skin desquamation in imiquimod-induced psoriasis animals, and FIG3C shows the comprehensive score results of erythema and skin desquamation in imiquimod-induced psoriasis animals.

[0204] Figure 4A shows the experimental results of right ear thickness in animals with human IL-23-induced psoriasis, Figure 4B shows the experimental results of area under the curve in animals with human IL-23-induced psoriasis (in the accompanying figure, ***P<0.001 compared with the negative control), and Figure 4C shows the experimental results of right ear weight in animals with human IL-23-induced psoriasis (in the accompanying figure, **P<0.01, ***P<0.001 compared with the negative control).

[0205] FIG5 is a schematic diagram of the structure of the anti-IL-36R and anti-IL-23 bispecific antibody disclosed herein.

[0206] Figure 6A shows the results of ELISA detection of the binding activity of bispecific antibodies IL-36R-IL-23-01 and IL-36R-IL-23-02 to human IL-23, and Figure 6B shows the results of FACS detection of the binding activity of bispecific antibodies IL-36R-IL-23-01 and IL-36R-IL-23-02 and anti-IL-36R single domain antibodies VHH162_Hu11 and VHH213_Hu07 to cell surface human IL-36R.

[0207] Figures 7A to 7D show the results of the inhibitory effects of IL-36R-IL-23-01 and IL-36R-IL-23-02 on IL-36-induced IL-8 secretion by A431.

[0208] Figures 8A and 8B show the results of the inhibitory effects of IL-36R-IL-23-01 and IL-36R-IL-23-02 on IL-23-induced proliferation of BaF3-mIL-23R cells.

[0209] Figure 9 shows the inhibitory effects of IL-36R-IL-23-01 and IL-36R-IL-23-02 on the IL-23-induced DB-STAT3-Luc2 reporter gene system.

[0210] Figure 10A is a flow chart of the study on the human IL-36 and human IL-23-induced ear swelling model of human IL-36R transgenic mice, Figure 10B shows the effects of IL-36R-IL-23-01 and VHH213_Hu07 on ear weight in the human IL-36 and human IL-23-induced ear swelling model of mice, and Figure 10C shows the effects of IL-36R-IL-23-01 and VHH213_Hu07 on downstream gene expression in the human IL-36 and human IL-23-induced ear swelling model of mice.

[0211] Figure 11 shows the effects of IL-36R-IL-23-01 and VHH213_Hu07 on downstream gene expression in the mouse ear swelling model induced by human IL-36 and human IL-23. DETAILED DESCRIPTION

[0212] The following examples further illustrate the present disclosure, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[0213] Example 1. Screening of anti-IL-36R antibodies

[0214] 1. Immune antigens, screening antigens, and cell line construction

[0215] The human IL-36R extracellular domain with a human Fc tag at the C-terminus (CJ62) was used as the immunizing antigen, and the human IL-36R extracellular domain with both a His tag and an Avi tag at the C-terminus (Karabinin, IL1-HM4L2), the biotinylated human IL-36R extracellular domain (Karabinin, IL1-HM4L2B), or the cynomolgus macaque IL-36R extracellular domain with a His tag at the C-terminus (Karabinin, IL1-CM1L2) was used as the screening antigen. Specific immunizing and screening antigen information is shown in Table 1.

[0216] Table 1. IL-36R antigen information for immunization and screening

[0217] The FlpIn system was used to construct a CHO-K1 cell line stably expressing the full-length human IL-36R and monkey IL-36R proteins. TM CHO-K1 cells (Thermo, R75807) were plated at 2E5 cells / well in a six-well plate (culture medium: F12 + 10% FBS + 100 μg / mL Zeocin). After overnight culture, cells were transfected with a mixture of plasmids overexpressing human IL-36R or monkey IL-36R full-length protein and pOG44 (Thermo, V6005) using Lipo3000 (GIBCO, L3000001). Fresh culture medium (F12 + 10% FBS) was replaced 24 hours after transfection. 48 hours after transfection, cells were digested and 800 μg / mL Hygromycin B (Gibco, 10687010) was added for resistance screening. After negative cells completely disappeared, the remaining cells were digested and a portion was analyzed by flow cytometry. Cells with a positive rate exceeding 95% were expanded and frozen for subsequent antibody binding screening.

[0218] The human and monkey IL-36R sequences are as follows, where the underlined portion is the extracellular region sequence, with amino acids starting and ending at Asp20-Arg335.

[0219] >Human IL-36R sequence (Q9HB29-1) (SEQ ID NO: 1)

[0220] Monkey IL-36R (EHH61702.1) (SEQ ID NO: 2)

[0221] 2. Camel Immunization, Nanobody Phage Display Library Construction, and Anti-human IL-36R Nanobody Screening

[0222] 1) Camel Immunization

[0223] Two healthy camels (Camelus bactrianus) were immunized using the human Fc-tagged extracellular domain of human IL-36R (CJ62) as the immunizing antigen. For the initial immunization on day 0, 200 μg of human IL-36R antigen was mixed with complete Freund's adjuvant (CFA) and injected subcutaneously. On days 14, 28, and 42, 200 μg of human IL-36R antigen was mixed with incomplete Freund's adjuvant (IFA) and injected subcutaneously. Serum titers were measured on days 42 and 56. Nanobody phage display libraries were constructed from peripheral lymphocytes of each camel.

[0224] 2) Phage library construction

[0225] After the fourth immunization, 50 mL of peripheral blood was collected from two camels, and PBMCs were isolated. RNA was extracted from the PBMCs and reverse transcribed to obtain total cDNA. Antibody genes were amplified using primers with camel antibody signature sequences. VHH gene fragments were amplified using VHH signature sequences and cloned into a phage library vector (pSCD-2). The ligation products were electroporated into competent Escherichia coli TG1 cells. A library capacity of 1.1 × 10 9 The transfected cells were infected with M13KO7 and cultured overnight to release VHH-expressing phage particles. The supernatant was collected, sterilized by filtration at 4°C, and stored at -80°C until further use.

[0226] 3) Nanobody (VHH) screening

[0227] The VHH phage library was screened using liquid-phase affinity panning, using biotinylated human IL-36R (kain, IL1-HM4L2B) as the antigen. The phage library was mixed with the antigen, and the complexes of human IL-36R antigen and phage were captured using streptavidin-coupled agarose microspheres. After washing with PBS / 0.05% Tween, bound phage were eluted from the microspheres using Glycine Elution Buffer (0.2M Glycine-HCl, pH 2.2, 1mg / mL BSA). The eluted phage were amplified and then subjected to a second round of screening. The two rounds of screening used 10μg / mL and 5μg / mL of human IL-36R antigen, respectively. After two rounds of screening, clones were selected from the titer determination plates, identified by ELISA, sequenced, and analyzed to obtain the sequences of the human IL-36R antigen-binding Nanobodies VHH162 and VHH213, as shown below. The CDRs are underlined, see Table 2.

[0228] >VHH162 (SEQ ID NO: 3)

[0229] >VHH213 (SEQ ID NO:4)

[0230] Table 2. Anti-IL-36R Nanobody CDR Sequences (defined by the Kabat numbering system)

[0231] The variable region sequences of the Nanobodies listed in Table 2 were linked to human IgG4 Fc fragments (including the hinge region and with the S228P mutation, according to the EU numbering system) to construct VHH-Fc antibodies. Plasmids were constructed and transiently transfected into HEK293 cells for secretory expression. The antibodies in the supernatant were purified using a protein A column, washed with PBS, and eluted with 0.1 M glycine buffer (pH 2.5). The eluted protein was dialyzed into PBS buffer (pH 7.4). The human IgG4 Fc sequence is as follows:

[0232] >hIgG4-Fc (containing S228P mutation) (SEQ ID NO: 11)

[0233] Example 2. Humanized modification of anti-IL-36R antibody

[0234] Antibody humanization was performed using the CDR-grafting method. The variable regions of the parental IL-36R antibody were aligned with fully human germline genes in the IMGT or NCBI / igblast databases. A human germline gene with high homology to the anti-IL-36R antibody was selected as the humanization template. The camelid antibody CDRs were recombined with the corresponding humanized template framework regions to form the humanized sequence. Residues in the FR regions that affect antibody stability and affinity were backmutated, and mutations were designed to address immunogenicity risk sites and sites with deamidation and isomerization risk.

[0235] 1. Humanized framework selection and backmutation design of VHH162

[0236] The humanized heavy chain templates for camel-derived nanobody VHH162 are IGHV3-23*04 and IGHJ1*01. The camel-derived nanobody heavy chain CDRs were recombined with the corresponding humanized template framework regions to form a human FR1-camel-derived CDR1-human FR2-camel-derived CDR2-human FR3-camel-derived CDR3-human FR4 antibody heavy chain variable region sequence, wherein FR1, FR2, and FR3 are derived from IGHV3-23*04, and FR4 is derived from IGHJ1*01. The resulting humanized variable region sequences (underlined CDR sequences, italicized FR sequences, defined according to the Kabat numbering system) are as follows:

[0237] >VHH162-graft (humanized VHH) (SEQ ID NO: 12)

[0238] Based on the VHH162-graft, back mutations were designed. See Table 3-1 for back mutations.

[0239] Table 3-1. VHH162 backmutation design (sites defined according to the Kabat numbering system)

[0240] The humanized sequence of VHH162 after backmutation (the underlined CDR sequence is defined according to the Kabat numbering system) is as follows:

[0241] >VHH162_Hu00 (SEQ ID NO: 13)

[0242] >VHH162_Hu01 (SEQ ID NO: 14)

[0243] >VHH162_Hu02 (SEQ ID NO: 15)

[0244] >VHH162_Hu03 (SEQ ID NO: 16)

[0245] >VHH162_Hu04 (SEQ ID NO: 17)

[0246] >VHH162_Hu05 (SEQ ID NO: 18)

[0247] >VHH162_Hu06 (SEQ ID NO: 19)

[0248] >VHH162_Hu07 (SEQ ID NO: 20)

[0249] >VHH162_Hu08 (SEQ ID NO: 21)

[0250] >VHH162_Hu09 (SEQ ID NO: 22)

[0251] >VHH162_Hu10 (SEQ ID NO: 23)

[0252] >VHH162_Hu11 (SEQ ID NO: 24)

[0253] >VHH162_Hu12 (SEQ ID NO: 25)

[0254] >VHH162_Hu13 (SEQ ID NO: 26)

[0255] The CDR sequences of the above humanized and backmutated VHH162 are identical to the CDR sequences of camel-derived VHH162.

[0256] 2. Humanized framework selection and backmutation design of VHH213

[0257] The humanized heavy chain template of camel-derived nanobody VHH213 is IGHV3-74*01 and IGHJ1*01, with FR1, FR2, and FR3 derived from IGHV3-74*01 and FR4 derived from IGHJ1*01. The following sequence was obtained:

[0258] >VHH213-graft (humanized VHH) (SEQ ID NO: 27)

[0259] Based on the VHH213-graft, back mutations were designed. See Table 3-2 for back mutations.

[0260] Table 3-2. VHH213 back mutation design (sites defined according to the Kabat numbering system)

[0261] The humanized sequence of VHH213 after back mutation (the underlined CDR sequence is defined according to the Kabat numbering system) is as follows:

[0262] >VHH213_Hu00 (SEQ ID NO: 28)

[0263] >VHH213_Hu01 (SEQ ID NO: 29)

[0264] >VHH213_Hu02 (SEQ ID NO: 30)

[0265] >VHH213_Hu03 (SEQ ID NO: 31)

[0266] >VHH213_Hu04 (SEQ ID NO: 32)

[0267] >VHH213_Hu05 (SEQ ID NO: 33)

[0268] >VHH213_Hu06 (SEQ ID NO: 34)

[0269] >VHH213_Hu07 (SEQ ID NO: 35)

[0270] The CDR sequences of the humanized and backmutated VHH213 sequences are identical to those of the camel-derived VHH213.

[0271] The humanized and backmutated sequences of VHH162 and VHH213 were linked to a human IgG4 Fc fragment (including the hinge region and carrying the S228P mutation according to the EU numbering system) to construct a VHH-Fc antibody. After constructing the humanized antibody recombinant plasmid, HEK293 cells were transiently transfected and secreted for antibody expression. The antibody in the supernatant was purified using a protein A column, washed with PBS, and eluted with 0.1 M glycine buffer (pH 2.5). The eluted protein was dialyzed into PBS buffer (pH 7.4). After testing, the target antibody was obtained.

[0272] Example 3. Identification of the affinity of humanized anti-IL-36R antibodies to IL-36R antigen

[0273] 1. FACS detection of binding of humanized anti-IL-36R antibody to cell surface IL-36R

[0274] To test the ability of humanized anti-IL-36R antibodies to bind to human IL-36R expressed on the cell membrane, different concentrations of candidate anti-IL-36R antibodies were added to 1E5 / well of CHO-K1 cells overexpressing human IL-36R and incubated on ice for 1 hour. After washing twice with FACS buffer (1% FBS, DPBS), 100 μL of AF488-goat anti-human IgG (H+L) fluorescent secondary antibody (1:1000, Invitrogen, A11013) was added and incubated on ice for 30 minutes. After washing twice with FACS buffer, the cells were resuspended in 150 μL of FACS buffer and the fluorescence signal was detected using a flow cytometer (BD, FACSCelesta). EC values ​​were calculated.50 .

[0275] Boehringer Ingelheim's anti-human IL-36R antibody BI655130 (WO2013074569A1, obtained by autonomous expression based on the sequence in the patent) and Regeneron's anti-human IL-36R antibody H4H14706P2 (WO2020018503A2, obtained by autonomous expression based on the sequence in the patent) were used as controls. The sequences are as follows:

[0276] >BI655130 heavy chain sequence (SEQ ID NO: 36)

[0277] >BI655130 light chain sequence (SEQ ID NO: 37)

[0278] >H4H14706P2 heavy chain sequence (SEQ ID NO: 38)

[0279] >H4H14706P2 light chain sequence (SEQ ID NO: 39)

[0280] The binding results of the VHH162 humanized antibodies VHH162_Hu08, VHH162_Hu09, and VHH162_Hu11, as well as the VHH213 humanized antibodies VHH213_Hu01 and VHH213_Hu07, to human IL-36R are shown in Figures 1A and 1B and Tables 4-1 and 4-2. VHH162_Hu08, VHH162_Hu09, VHH162_Hu11, VHH213_Hu01, and VHH213_Hu07 exhibited strong binding activity to human IL-36R on the surface of CHO-K1 cells, comparable to the binding activity of VHH213 on human IL-36R on the surface of CHO-K1 cells.

[0281] Table 4-1. EC values ​​of humanized antibodies VHH162 and VHH213 binding to human IL-36R detected by FACS 50 value

[0282] Note: NA not applicable

[0283] Table 4-2. EC values ​​of humanized antibodies VHH162 and VHH213 binding to human IL-36R detected by FACS 50 value

[0284] Note: NA not applicable

[0285] 2. SPR Detection of Binding of Humanized Anti-IL-36R Antibody to IL-36R Antigen

[0286] Surface plasmon resonance (SPR) was used to detect the affinity of anti-IL-36R antibodies to IL-36R antigen.

[0287] Instrument used Biacore TM 8k (GE Healthcare). A CM5 sensor chip (Cat. #29149603, GE) was used for the experiment, and the mobile phase was HBS-EP+ buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20). Anti-human IgG (Fc) antibody was prepared at a 30μg / mL solution in 10mM sodium acetate buffer (pH 5.0). The Immobilization program was selected for automated amino-coupled immobilization of the anti-human IgG (Fc) antibody channel. Each test antibody was prepared as a ligand in HBS-EP+ buffer and captured using the anti-human IgG (Fc) antibody on the chip channel. Human IL-36R antigen protein (Kaika protein, IL1-HM4L2) was used as the analyte and prepared with HBS-EP+ buffer solution. The analyte was diluted 2-fold and flowed through the experimental channel and reference channel at a flow rate of 30 μL / min. The binding time was 1 minute and the dissociation time was 15 minutes. The regeneration buffer 10mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) was run at a flow rate of 10 μl / min for 30 seconds. The association rate Ka and dissociation rate Kd, as well as the dissociation constant (i.e., affinity K) were calculated. D ). See Table 5 for the results.

[0288] Table 5. SPR affinity data of anti-IL-36R antibodies binding to human IL-36R antigen

[0289] The results of binding to human IL-36R antigen showed that VHH162_Hu11 and VHH213_Hu07 had strong affinity for human IL-36R, and their affinity was similar to that of BI655130 and H4H14706P2 for human IL-36R.

[0290] 3. Competitive Binding Results of Humanized Anti-IL-36R Antibody and Control Antibody

[0291] Competitive binding analysis of anti-IL-36R antibody and human IL-36R protein was performed by Biacore TM8k (GE Healthcare) instrument was used for the experiment. A CM5 sensor chip was used for the experiment, and the mobile phase was HBS-EP+ buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20). Following the instructions for the His Capture Kit (Cat.#29234602, Cytiva) and the Amino Coupling Kit (Cat.#BR100633, GE), anti-His antibodies were covalently coupled to a CM5 biosensor chip (Cat.#29149603, GE) to affinity capture human IL-36R protein (Kaika protein, IL1-HM4L2). Antibody 1 was then injected until saturation, i.e., the signal no longer increased, and then antibody 2 was injected. Biacore was used to analyze the IL-36R protein. TM Binding curves were generated using an 8k instrument to monitor the reaction signal in real time. After each dissociation cycle, the biochip was washed and regenerated with 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54). Data were fitted using a tandem dual model using Biacore Insight Evaluation 3.0 software.

[0292] The results are shown in Figure 2 . The antigenic epitopes of VHH162_Hu09 overlap with those of VHH213_Hu01, and are different from those of H4H14706P2 and BI655130.

[0293] Example 4. Preparation of anti-human IL-23p19 monoclonal antibody

[0294] 1. Preparation of Mouse Antibodies

[0295] SJL mice were immunized with human IL-23 protein (Sino Biological, CT048-H08H). After immunization three times, blood was collected to determine the titer of the antibody in the serum. Mice with high antibody titers in the serum and titers approaching a plateau were selected for spleen cell fusion. The fused hybridoma cells were plated in a 96-well cell culture plate and cultured in a 37°C, 5% CO2 incubator. The cell culture supernatant was tested by ELISA. The screened positive clones were amplified and frozen for seed preservation, and subcloned until a single cell clone was obtained. The selected hybridoma clones were further prepared and purified using a serum-free cell culture method. The obtained hybridoma antibodies were tested for binding to human IL-23 protein and blocking of the receptor by ELISA (see Example 5 of the present disclosure), and hybridoma cell lines with good binding activity and blocking activity were selected.

[0296] A monoclonal hybridoma cell line, mAb29, was selected. The process was as follows: hybridoma cells in the logarithmic growth phase were harvested, RNA was extracted using Trizol (Invitrogen, Cat# 15596-018), and reverse transcribed into cDNA. PCR amplification of the cDNA template was performed and the resulting DNA sequence was sequenced by a sequencing company. The resulting DNA sequence corresponds to the following amino acid sequence for the antibody variable region, and the CDR sequences are shown in Table 6.

[0297] mAb29 heavy chain variable region (SEQ ID NO: 40)

[0298] mAb29 light chain variable region (SEQ ID NO:41)

[0299] Table 6. Heavy and light chain CDR sequences of mAb29 (defined under Kabat numbering convention)

[0300] The VH / VK sequences of the mAb29 variable region sequence were amplified by PCR, respectively, and then homologously recombined with the expression vector pHr (with a signal peptide and a hIgG1 / hkappa constant region gene (CH1-Fc / CL) fragment). For example, the human heavy chain IgG1 constant region sequence is shown in SEQ ID NO: 48, and the human light chain kappa constant region sequence is shown in SEQ ID NO: 49. A recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc-pHr / VL-CL-pHr was constructed to obtain the chimeric antibody Ch29.

[0301] >Human IgG1 heavy chain constant region (containing L234A / L235A mutations) (SEQ ID NO: 48)

[0302] >Human light chain kappa constant region (SEQ ID NO: 49)

[0303] 2. Humanization

[0304] By comparing the IMGT human antibody heavy and light chain variable region germline gene database, highly homologous heavy and light chain variable region germline genes were selected as templates. The CDRs of the murine antibody were grafted onto the corresponding human templates, forming a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable region sequence was then fused with the human constant region sequence to generate a humanized antibody. The following example describes the humanization of mAb29. The amino acid residues of the antibody's CDRs were determined and annotated using the Kabat numbering system.

[0305] Highly homologous germline heavy and light chain variable region genes were selected as templates. The humanized light chain templates for the murine antibody mAb29 were IGKV4-1*01 and IGKJ4*01. The FR1, FR2, FR3 of the human germline light chain IGKV4-1*01 and the JK4 region of IGKJ4*01 (as FR4) were selected as the humanized antibody light chain framework regions. The humanized heavy chain templates were IGHV2-26*01 and IGHJ6*01. The FR1, FR2, FR3 of the human germline heavy chain IGHV2-26*01 and the JH6 region of IGHJ6*01 (as FR4) were selected as the humanized antibody light chain framework regions. First, the CDRs of the murine antibody mAb29 were transplanted into the corresponding selected human templates, replacing the CDR regions of the human templates. Then, amino acid residues 4, 17, 36, 58, 60, and / or 68 (numbered according to the Kabat numbering system) of the light chain variable region of the humanized antibody were mutated, and amino acid residues 1, 30, 37, 44, 49, 73, 89, and / or 93 (numbered according to the Kabat numbering system) of the heavy chain variable region were mutated. In addition, amino acid residue 1 of HCDR1: SYAIS (SEQ ID NO: 42) of the heavy chain variable region was mutated to N or Q to obtain new HCDR1: NYAIS (SEQ ID NO: 50) or QYAIS (SEQ ID NO: 51). The sequences of the humanized antibody variable regions of mAb29 are as follows:

[0306] >hAb29VH1(Graft+Q1E,A49G,A93V)(SEQ ID NO:52)

[0307] >hAb29VH2(Graft+Q1E, S30T, A49G, T73N, A93V) (SEQ ID NO:53)

[0308] >hAb29VH3(Graft+Q1E, S30T, I37V, A49G, T73N, T89R, A93V) (SEQ ID NO:54)

[0309] >hAb29VH4(Graft+S30T, I37V, A44G, A49G, T73N, T89R, A93V) (SEQ ID NO:55)

[0310] >hAb29VH5(Graft+S30T, I37V, A44G, A49G, T73N, T89R, A93V+S31N) (SEQ ID NO:56)

[0311] >hAb29VH6(Graft+S30T, I37V, A44G, A49G, T73N, T89R, A93V+S31Q) (SEQ ID NO:57)

[0312] >hAb29VL1(Graft+Y36F, G68R)(SEQ ID NO:58)

[0313] >hAb29VL2(Graft+M4L, Y36F, V58I, G68R) (SEQ ID NO:59)

[0314] >hAb29VL3(Graft+M4L, Y36F, V58I, D60A, G68R) (SEQ ID NO:60)

[0315] >hAb29VL4(Graft+M4L, E17Q, Y36F, V58I, D60A, G68R) (SEQ ID NO: 61)

[0316] Note: The sequence order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The underlined part of the sequence is the CDR region (CDR amino acid residues are determined by the Kabat numbering system), and the rest is the FR region.

[0317] The expression vectors for the antibody light chain and heavy chain are constructed separately, and the humanized antibody light / heavy chains are cross-paired and combined. After transfection into 293E cells, the culture supernatant is collected and purified to obtain a humanized full-length antibody. The humanized antibody heavy chain constant region can be selected from IgG1, IgG2, IgG3, and IgG4 constant regions. Exemplarily, the humanized antibody heavy chain variable region of the aforementioned mAb29 is fused with the human heavy chain IgG1 constant region (sequence as shown in SEQ ID NO: 48) to form the antibody full-length heavy chain, and the humanized antibody light chain variable region is fused with the human light chain κ constant region (sequence as shown in SEQ ID NO: 49) to form the antibody full-length light chain, to obtain the mAb29 humanized antibody shown in Table 7 below:

[0318] Table 7. mAb29 humanized antibody

[0319] Exemplary, full-length sequences of the humanized antibody heavy and light chains are as follows:

[0320] >Hu29-19 heavy chain (SEQ ID NO: 62)

[0321] >Hu29-19 light chain (SEQ ID NO: 63)

[0322] >Hu29-24 heavy chain (SEQ ID NO: 64)

[0323] >Hu29-24 light chain (SEQ ID NO: 65)

[0324] Note: Italics indicate constant regions, and underlines indicate mutations.

[0325] Example 5. Functional verification of anti-human IL-23p19 monoclonal antibody

[0326] 1. Affinity test

[0327] 1) ELISA binding assay

[0328] The binding activity of the test molecule to IL-23p19 and BAFF was detected by ELISA (coating the test molecule). The specific method is as follows:

[0329] The sample to be tested was diluted to 2 μg / mL with PBS buffer at pH 7.4, added to a 96-well ELISA plate at a volume of 100 μL / well, and incubated at 4°C overnight. After discarding the liquid, 300 μL of 5% skim milk (BD, 232100) diluted with PBS was added to each well for blocking and incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded, and the plate was washed 3 times with PBST buffer (pH 7.4 PBS containing 0.1% tween-20), and 100 μL of gradient diluted IL-23 (Sino Biological, CT048-H08H) or BAFF (ACROBiosystems, BAF-H52D4) solution was added to each well and incubated at 37°C for 1 hour. After incubation, the plate was washed 3 times with PBST. To detect binding to IL-23 and BAFF, 100 μL of Anti-His-HRP (Sino biological, 105327-MM02T-H, 1:2000 dilution) was added to each well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, 100 μL of TMB colorimetric substrate (KPL, 5120-0077) was added to each well and incubated at room temperature for 10-15 minutes. 50 μL of 1M H2SO4 was added to each well to terminate the reaction. The absorbance at 450 nm was read using a microplate reader. The binding curve of the antibody and antigen was fitted using software to calculate the EC 50Value. Among them, RCT-18 is telitacicept (RC18, Rongchang Biopharmaceuticals). The results of the binding experiment of anti-IL-23 antibodies and their fusion proteins to human IL-23 are shown in Table 8-1.

[0330] Table 8-1. Results of IL-23 protein binding experiments

[0331] The experimental results show that the anti-IL-23 antibodies disclosed herein can specifically bind to human IL-23 protein.

[0332] 2) Biacore test

[0333] A certain amount of the sample to be tested was affinity-captured using a biosensor chip Protein A (GE, 29127556). A series of antigen concentration gradients were then passed over the chip surface. The reaction signals were monitored in real time using Biacore (GE, 8K) to generate binding and dissociation curves. After each dissociation cycle, the biochip was washed and regenerated with 10 mM glycine-HCl solution, pH 1.5 (GE, BR-1003-54). The experimental data were fitted using BIAevaluation version 4.1 software using a 1:1 model to determine affinity values. The relevant antigen protein used in this test is as follows: human IL-23 (Sino biological, CT048-H08H). Affinity test results are shown in Tables 8-2 and 8-3.

[0334] Table 8-2. Affinity test results of anti-IL-23 antibodies binding to human IL-23p19

[0335] Table 8-3. Affinity test results of other anti-IL-23 antibodies binding to human IL-23p19

[0336] The experimental results show that the anti-IL-23 antibodies constructed in the present disclosure can bind to human IL-23p19 with high affinity.

[0337] The sequence of the anti-IL-23 antibody control molecule Risankizumab (see WHO Drug Information Vol. 30, No. 1, 2016) in the present disclosure is as follows:

[0338] >Risankizumab heavy chain (SEQ ID NO: 66)

[0339] Risankizumab light chain (SEQ ID NO: 67)

[0340] 2. Ligand and receptor blocking experiments

[0341] The blocking activity of anti-IL-23 antibodies against IL-23 / IL-23R was detected by ELISA. The specific method is as follows:

[0342] The receptor protein was diluted to 2 μg / mL in PBS (Source Bio, B320) at pH 7.4 and added to a 96-well ELISA plate (Corning, 3590) at a volume of 100 μL / well. The plate was incubated overnight at 4°C. After discarding the liquid, 200 μL of 1% Casein blocking solution (Thermo, 37528) was added to each well for blocking and incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded and the plate was washed three times with PBST buffer (PBS, pH 7.4 containing 0.1% tween-20) before use. A fixed concentration of biotin-labeled ligand protein was mixed with a gradient dilution of the antibody or fusion protein and pre-incubated at 37°C for 30 minutes before being added to the blocked ELISA plate and incubated at 37°C for 1.5 hours. After incubation, wash the plate three times with PBST, add 100 μL of streptavidin-HRP (Invitrogen, 434323, 1:4000 dilution) to each well, and incubate at 37°C for 1 hour. Remove the supernatant, wash the plate three times with PBST, and add 100 μL of TMB colorimetric substrate (KPL, 5120-0077) to each well. Incubate at room temperature for 10-15 minutes. Add 50 μL of 1 M H2SO4 to each well to terminate the reaction. Read the absorbance at 450 nm using a microplate reader. Use software to fit the curve of inhibition of ligand and receptor binding, and calculate the IC 50 The sources of the ligand and receptor proteins used in this test example are as follows: IL-23 (Sino Biological, CT048-H08H), IL-23R (Sino Biological, 13840-H02H), and BAFF-R (Sino Biological, 16079-H02H).

[0343] The results of the anti-IL-23 antibody blocking experiment on IL-23 / IL-23R are shown in Table 9-1 and Table 9-2.

[0344] Table 9-1. Results of the IL-23 / IL-23R blocking experiment with anti-IL-23 antibodies and their fusion proteins

[0345] Table 9-2. Results of IL-23 / IL-23R blocking experiments with other anti-IL-23 antibodies

[0346] The experimental results show that the anti-IL-23 antibody constructed in the present disclosure can effectively block IL-23 / IL-23R binding.

[0347] 3. BaF3-IL-23R Cell Proliferation Assay

[0348] The in vitro activity of the antibody was tested using a BaF3-IL-23R cell proliferation assay. The assay method is as follows:

[0349] BaF3 cells (Cobioer, CBP60474) stably expressing IL-23R and IL-12Rβ1 were plated in a 96-well cell plate (Corning, 3903) with a volume of 50 μL per well and 4000 cells. The gradient dilution of the test sample was mixed with a fixed concentration of IL-23 protein (Sino Biological, CT048-H08H), added to the cell culture plate, and cultured in a 37°C incubator for 72 hours. The cell culture plate was then removed, 50 μL of Celltiter Glo detection solution (Promega, G755B) was added to each well, placed in a shaker for 10 minutes, taken out and allowed to stand at room temperature for 10 minutes, and the bioluminescent signal was detected using a microplate reader (PerkinElmer, Victor3). The data were fitted with an inhibition curve using software to calculate the IC 50 value.

[0350] The experimental results of anti-IL-23 antibody inhibiting the proliferation of BaF3-IL-23R cells are shown in Table 10-1 and Table 10-2.

[0351] Table 10-1. Results of experiments on the inhibition of BaF3-IL-23R cell proliferation by anti-IL-23 antibodies and their fusion proteins

[0352] Table 10-2. Results of experiments on inhibition of BaF3-IL-23R cell proliferation by other anti-IL-23 antibodies

[0353] The experimental results show that the anti-IL-23 antibodies disclosed herein have strong activity in inhibiting the proliferation of BaF3-IL-23R cells.

[0354] 4. IL-17 secretion assay

[0355] The IL-17 secretion assay was used to test the effect of anti-IL-23 antibodies on the inhibition of IL-23-induced T cell differentiation. The experimental method is as follows:

[0356] In a 96-well plate (Corning, 3599), 100 μL of 2 μg / mL anti-mouse CD3 antibody (BioLegend, 100238) and 2 μg / mL anti-mouse CD28 antibody (BioLegend, 102116) were added to each well and incubated at 37°C for 1 hour. The cells were washed twice with PBS and then used for further analysis. Mouse spleens were triturated and centrifuged at 4°C for 5 minutes to collect the lower layer of cells. The cells were washed once with wash solution (PBS + 2% FBS + 2mM EDTA) and centrifuged. The supernatant was removed and RBC Lysis Buffer (Invitrogen, 00-4333-57) was added. The cells were allowed to stand at room temperature for 5 minutes until the red blood cells were completely lysed. The cells were centrifuged again and resuspended for counting. The cell suspension was sorted using the Mouse CD4 Cells Kit (Invitrogen, 11415D). The isolated CD4+ T cells were resuspended in RPMI 1640 Medium (Gibco, 11875119) + 10% FBS (Gibco, 10099-141) and counted for later use. The cell suspension was plated in a coated 96-well plate. A fixed concentration of IL-23 (R&D Systems, 1290-IL-010) was mixed with serially diluted antibodies or fusion proteins and preincubated for 1 hour before addition to the 96-well plate. The cells were cultured in a 37°C cell culture incubator for 48 hours. The 96-well plate was removed and centrifuged at 1000 rpm for 3 minutes. The supernatant was collected and the IL-17 content in the supernatant was measured using the Mouse IL-17 DuoSet ELISA Kit (R&D Systems, DY421). The experimental results are shown in Table 11.

[0357] Table 11. Results of the IL-17 secretion inhibition experiment

[0358] The experimental results show that the anti-IL-23 antibodies disclosed herein all have strong activity in inhibiting IL-17 secretion.

[0359] 5. Imiquimod-induced psoriasis animal model experiment

[0360] The in vivo efficacy of anti-IL-23 antibodies was evaluated using an imiquimod cream (IMQ)-induced psoriasis animal model.

[0361] SPF female C57BL / 6 hIL-23A / hIL12B transgenic mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd.), 8 to 9 weeks old, were randomly divided into groups of 5 mice each. The backs of the mice were depilated before the experiment. From day 0 to day 5, the sham-operated group received an even application of 80 mg of petrolatum (Unilever); the model group received an even application of 80 mg of imiquimod cream (Bemerson) for 6 consecutive days. On day 0 and day 3, the test samples (20 mpk for risankizumab, Hu29-19, Hu29-24, and Hu38-4) were injected intraperitoneally 1 hour before application of imiquimod cream. Risankizumab served as a positive control, and PBS served as a negative control. The doses were administered twice. The severity of the rash and desquamation on the backs of the mice was scored daily, and the experiment was terminated on day 6.

[0362] The severity of the rash was scored as follows: no rash, 0 points; slightly red, 1 point; red but not dark, 2 points; dark red, 3 points; and very red, 4 points. Desquamation was scored as follows: no desquamation, 0 points; small areas of fine dandruff, 1 point; moderately thick dandruff, 2 points; large areas of rough, thickened dandruff, 3 points; and large areas of large, patchy dandruff, 4 points.

[0363] The experimental results are shown in Figures 3A to 3C. The experimental results show that compared with the positive control Risankizumab, the anti-IL-23 antibody constructed in the present disclosure has stronger in vivo efficacy, and the scores of rash severity and skin desquamation severity are lower than those of Risankizumab.

[0364] 6. Experiment on human IL-23-induced psoriasis animal model

[0365] The in vivo efficacy of the anti-IL-23 antibody constructed in the present disclosure was evaluated by using a human IL-23-induced psoriasis animal model. SPF female C57BL / 6J mice (Weitong Lihua Experimental Animal Technology Co., Ltd.) were 6 to 8 weeks old. After anesthesia, the mice were anesthetized and the thickness of the right ear and body weight were measured. The mice were grouped according to the right ear thickness and body weight data, with 6 mice in each group. The day of grouping was recorded as day 0. Starting from day 1, 1 μg of human IL-23 protein (R&D Systems, 1290-IL-500 / CF) was injected intradermally into the right ear of the mice every day for a total of 7 days. The normal group of mice was injected with PBS. The test drug was injected intraperitoneally on day 0 and day 3, and the weight and right ear thickness of the mice were measured and recorded on days 0, 2, 4, 6, and 8, respectively. The alleviating effect of the test drug on the model was evaluated by the changes in the right ear thickness. The experiment ended on day 8, and the ear pieces with a diameter of 8 mm were collected and weighed.

[0366] The experimental results are shown in Figures 4A to 4C, which show that the anti-IL-23 antibody Hu29-19 constructed in the present invention has a stronger in vivo efficacy than the control molecule Risankizumab in the human IL-23-induced psoriasis animal model, specifically: at a dose of 3mpk, the ear thickness of the Hu29-19 group was lower than that of the control Risankizumab; the statistical results of the area under the curve showed that at a dose of 3mpk, the effect of Hu29-19 was stronger than that of Risankizumab; the statistical results of the mouse ear weight showed that the ear weight of the Hu29-19 1mpk group was the same as that of the Risankizumab 3mpk group, and the ear weight of the Hu29-19 3mpk group was lower than that of Risankizumab.

[0367] Example 6. Construction of anti-IL-36R and anti-IL-23 bispecific antibodies

[0368] The anti-IL-23 antibody Hu29-19 (variable region sequences are hAb29VH5 (SEQ ID NO: 56) and hAb29VL3 (SEQ ID NO: 60)) used to construct the bispecific molecule, the humanized anti-human IL-36R Nanobody is VHH213_Hu07 (SEQ ID NO: 35) or VHH162_Hu11 (SEQ ID NO: 24) sequence, and the human IgG1 heavy chain constant region and human kappa light chain constant region sequences are used to construct the anti-IL-36R and anti-IL-23 bispecific antibody molecules according to the molecular format schematic shown in Figure 5. In the molecular formats shown in Figures 5, Structures A and B, the anti-IL-36R VHH sequences are fused to the N-termini of the anti-IL-23 antibody heavy chain and light chain, respectively, and connected by a (G4S)2 linker. The heavy chain constant region of the constructed anti-IL-36R and anti-IL-23 bispecific antibodies contains L234A and L235A mutations (according to the EU numbering system). The names and construction methods of the bispecific antibodies are shown in Table 12.

[0369] Table 12. Construction rules and nomenclature of anti-IL-36R and anti-IL-23 bispecific antibodies

[0370] In the following sequences, the underlined part is the linker and the italicized part is the constant region sequence.

[0371] >IL-36R-IL-23-01 heavy chain (SEQ ID NO: 68)

[0372] >IL-36R-IL-23-01 light chain (SEQ ID NO: 69)

[0373] >IL-36R-IL-23-02 heavy chain (SEQ ID NO: 70)

[0374] >IL-36R-IL-23-02 light chain

[0375] It is SEQ ID NO: 69.

[0376] >IL-36R-IL-23-03 heavy chain (SEQ ID NO: 71)

[0377] >IL-36R-IL-23-03 light chain (SEQ ID NO: 72)

[0378] >IL-36R-IL-23-04 heavy chain

[0379] It is SEQ ID NO: 71.

[0380] >IL-36R-IL-23-04 light chain (SEQ ID NO: 73)

[0381] In addition, YTE mutations (M252Y, S254T and T256E mutations, according to the EU numbering system) were introduced into the Fc of the anti-IL-36R and anti-IL-23 bispecific antibodies IL-36R-IL-23-01 and IL-36R-IL-23-02 to construct IL-36R-IL-23-01-YTE and IL-36R-IL-23-02-YTE molecules.

[0382] >IL-36R-IL-23-01-YTE heavy chain (SEQ ID NO: 74)

[0383] >IL-36R-IL-23-02-YTE heavy chain (SEQ ID NO: 75)

[0384] >IL-36R-IL-23-01-YTE, IL-36R-IL-23-02-YTE light chain

[0385] Both are SEQ ID NO: 69.

[0386] >IgG1 Fc (containing L234A / L235A / M252Y / S254T / T256E mutations) (SEQ ID NO: 76)

[0387] Example 7. Affinity of anti-IL-36R and anti-IL-23 bispecific antibodies

[0388] 1. Binding to IL-23 (ELISA)

[0389] To evaluate the binding of anti-IL-36R and anti-IL-23 bispecific antibodies to the IL-23 antigen, the binding of anti-IL-36R and anti-IL-23 bispecific antibodies to the IL-23 antigen was detected by ELISA. Human IL-23 protein (Biopsy, ILB-H52W5) was dissolved in PBS to 2 μg / mL, and 100 μL / well was added to a 96-well plate and incubated at 4°C overnight to coat the antigen. Wash three times with PBST (PBS + 0.05% tween 20) solution. Add 200 μL / well blocking solution (PBST / 1% BSA solution) and incubate at 37°C for 1 hour for blocking. After washing three times with PBST solution, different concentrations of anti-IL-23 monoclonal antibody or anti-IL-36R and anti-IL-23 bispecific antibody (diluted in blocking solution) were added and incubated at 37°C for 2 hours. Wash three times with PBST solution. Add 100 μL of HRP-conjugated anti-human IgG Fc secondary antibody (1:10000, GenScript, A01854) solution and incubate at 37°C for 1 hour. After washing three times with PBST solution, add 100 μL / well TMB solution and react at room temperature for 5-10 minutes. Add 100 μL of stop solution and measure the OD450 value with a multifunctional microplate reader to calculate the EC 50 .

[0390] As controls, AbbVie's anti-human IL-23 antibody Risankizumab and Johnson & Johnson's anti-human IL-23 antibody Guselkumab, as well as the Hu29-19 full-length antibody (SEQ ID NOs: 62 and 63) were used.

[0391] >Guselkumab heavy chain (SEQ ID NO: 77)

[0392] >Guselkumab light chain (SEQ ID NO: 78)

[0393] The results are shown in Figure 6A and Table 13-1. The binding affinities of IL-36R-IL-23-01 and IL-36R-IL-23-02 to human IL-23 antigen are comparable to that of Hu29-19 and higher than those of the control antibodies Risankizumab and Guselkumab.

[0394] Table 13-1. ELISA detection of EC values ​​for the binding of bispecific antibodies to human IL-2350 value

[0395] 2. Binding to IL-23 (SPR)

[0396] The affinity of the anti-IL-36R and anti-IL-23 bispecific antibodies for the IL-23 antigen was determined using SPR. The binding of the monoclonal or bispecific antibody to be tested was performed using the same experimental method as in Example 3, with the human IL-23 antigen protein (Biopsy, ILB-H52W5) as the analyte. The results are shown in Table 13-2.

[0397] Table 13-2. SPR affinity of bispecific antibodies to human IL-23 antigen

[0398] The results showed that each bispecific antibody had a strong affinity for human IL-23 antigen, among which IL-36R-IL-23-01, IL-36R-IL-23-02 and Hu29-19 had similar affinities for human IL-23 antigen.

[0399] 3. Binding to cell surface IL-36R (FACS)

[0400] The binding ability of the anti-IL-36R and anti-IL-23 bispecific antibodies to human IL-36R expressed on the cell membrane was detected by FACS. The experimental method was as follows: different concentrations of candidate test antibodies were added to 1E5 / well of CHO-K1 cells overexpressing human IL-36R and incubated on ice for 1 hour. After washing twice with FACS buffer (1% FBS, DPBS), 100 μL of AF488-goat anti-human IgG (H+L) fluorescent secondary antibody (1:1000, Invitrogen, A11013) was added and incubated on ice for 30 minutes. After washing twice with FACS buffer, the cells were resuspended in 150 μL of FACS buffer and the fluorescence signal was detected using a flow cytometer (BD, FACSCelesta). The EC 50 .

[0401] The results are shown in Figure 6B and Table 13-3. IL-36R-IL-23-01 and IL-36R-IL-23-02 have strong binding activity to human IL-36R expressed on the surface of CHO-K1 cells, and their binding affinities are similar to those of VHH213_Hu07, VHH162_Hu11, and control antibodies BI655130 and H4H14706P2.

[0402] Table 13-3. EC values ​​of bispecific antibodies binding to human IL-36R detected by FACS 50 value

[0403] 4. Binding to IL-36R (SPR)

[0404] The affinity of anti-IL-36R and anti-IL-23 bispecific antibodies to IL-36R antigen was detected by SPR. Each bispecific antibody to be tested was used as a ligand, and the human IL-36R antigen protein (Kacha protein, IL1-HM4L2) was used as the analyte. The experimental method was as follows: the instrument used Biacore TM 8k (GE Healthcare). A CM5 sensor chip (Cat. #29149603, GE) was used for the experiment, and the mobile phase consisted of HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). Anti-human IgG (Fc) antibody was prepared at a 30 μg / mL solution in 10 mM sodium acetate buffer (pH 5.0). The Immobilization program was selected for automated amino-coupled immobilization of the anti-human IgG (Fc) antibody channel. Each test antibody was prepared as a ligand in HBS-EP+ buffer and captured using the anti-human IgG (Fc) antibody on the chip channel. Human IL-36R antigen protein (Kaika protein, IL1-HM4L2) was used as the analyte and prepared with HBS-EP+ buffer solution. The analyte was diluted 2-fold and flowed through the experimental channel and reference channel at a flow rate of 30 μL / min. The binding time was 1 minute and the dissociation time was 15 minutes. The regeneration buffer 10mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) was run at a flow rate of 10 μL / min for 30 seconds. The association rate Ka and dissociation rate Kd, as well as the dissociation constant (i.e., affinity K) were calculated. D ). See Table 13-4 for the results.

[0405] Table 13-4. SPR affinity of bispecific antibodies to IL-23 antigen

[0406] The results showed that all bispecific antibodies had strong affinity for human IL-36R antigen, and their affinity was similar to that of VHH162_Hu11 and VHH213_Hu07 for human IL-36R antigen.

[0407] Example 8. Inhibitory effect of anti-IL-36R and anti-IL-23 bispecific antibodies on IL-36 stimulatory factors activating IL-36R function

[0408] The inhibitory activity of an anti-IL-36R and anti-IL-23 bispecific antibody on IL-36-induced IL-8 secretion was evaluated using the human skin squamous cell line A431. A431 cells (ATCC, HRC00439) were plated at 3E4 / well in a 96-well plate and cultured overnight. Different concentrations of the test antibody and IL-36 (IL36α (CR61), IL36β (CR59), or IL36γ (CM77, NOVOProtein) were added to each well. After 24 hours of culture, the supernatant was collected and IL-8 levels were measured using an IL-8 kit (Cisbio, 62HIL08PEG) and a multi-function microplate reader (Perkin Elmer, Envision 2105).

[0409] The results are shown in Figures 7A, 7B and Tables 14-1 and 14-2. IL-36R-IL-23-01, IL-36R-IL-23-02, IL-36R-IL-23-03 and IL-36R-IL-23-04 were able to significantly block human IL-36-induced IL-8 secretion from A431.

[0410] Table 14-1. Inhibitory effects of IL-36R-IL-23-01 and IL-36R-IL-23-02 on the function of human IL-36 stimulatory factor

[0411] Table 14-2. Inhibitory effects of IL-36R-IL-23-03 and IL-36R-IL-23-04 on the function of human IL-36 stimulatory factor

[0412] Example 9. Inhibitory effect of anti-IL-36R and anti-IL-23 bispecific antibodies on IL-23 stimulatory factors activating IL-23R function

[0413] 1. Inhibitory effect on human IL-23-induced proliferation of Ba / F3-IL-23 cells

[0414] The CellTiter Glo assay was used to evaluate the inhibitory effect of anti-IL-36R and anti-IL-23 bispecific antibodies on human IL-23 protein-induced proliferation of BaF3-mIL-23R cells. BaF3-mIL-23R cells (BaF3 cells stably expressing IL-23R and IL-12Rβ1 (Cobioer, CBP60474)) were plated at 1E4 cells / well (50 μL) in a 96-well plate. 25 μL of various concentrations of anti-IL-36R and anti-IL-23 bispecific antibodies were incubated with 25 μL of human IL-23 (Bipsix, ILB-H52W5, final concentration 150 ng / mL) for half an hour before addition to the cell culture plates. After 72 hours of incubation, 50 μL of CellTiter Glo detection solution (Promega, G7573) was added to each well, and bioluminescence signals were detected using a multi-function microplate reader (Perkin Elmer, Envision 2105).

[0415] The results are shown in Figures 8A and 8B and Tables 15-1 and 15-2. IL-36R-IL-23-01 and IL-36R-IL-23-02 significantly inhibited the proliferation of BaF3-mIL-23R cells induced by human IL-23 protein.

[0416] Table 15-1. Inhibitory effect of IL-36R-IL-23-01 on IL-23-induced proliferation of BaF3-mIL-23R cells

[0417] Table 15-2. Inhibitory effect of IL-36R-IL-23-02 on IL-23-induced proliferation of BaF3-mIL-23R cells

[0418] 2. Inhibitory effect on STAT3 activation signal induced by human IL-23 in DB-STAT3-Luc2 reporter gene system

[0419] The luciferase reporter system was used to evaluate the inhibitory effect of anti-IL-36R and anti-IL-23 bispecific antibodies on the STAT3 activation signal induced by human IL-23 protein in DB-STAT3 luc2 cells.

[0420] DB-STAT3-Luc2 cells (DB cell line stably expressing the STAT3-Luc2 reporter gene system (ATCC, CRL-2289)) were plated in 96-well plates at 3E4 cells / well (50 μL). After incubation for half an hour, 25 μL of anti-IL-36R and anti-IL-23 bispecific antibodies at varying concentrations and 25 μL of human IL-23 (Biopsy, ILB-H52W5, final concentration 1 μg / mL) were added to the cell culture plates. After overnight incubation, 100 μL of Bright-Glo (Promega, E2620) detection solution was added to each well, and bioluminescent signals were detected using a multi-function microplate reader (Perkin Elmer, Envision 2105).

[0421] The results are shown in Figure 9 and Table 16. The results showed that in the DB-STAT3-Luc2 reporter gene system, the anti-IL-36R and anti-IL-23 bispecific antibodies IL-36R-IL-23-01 and IL-36R-IL-23-02 significantly inhibited the activation of STAT3 signaling induced by human IL-23 protein, and the inhibitory effect was comparable to that of the anti-IL-23 monoclonal antibody HU29-19.

[0422] Table 16. Detection of the inhibitory effect of bispecific antibodies on IL-23-induced DB-STAT3-Luc2 reporter gene system

[0423] Example 10. Inhibitory effect of bispecific antibodies on mouse ear swelling induced by human IL-36 and human IL-23

[0424] This model is used to simulate inflammatory diseases caused by excessive IL-36 or IL-23 in humans.

[0425] Female human IL-36R transgenic mice, 6–8 weeks old, were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. Housing environment: SPF; Production license: SCXK(Su)-2021-0003; Human IL-36R Transgenic Mouse Certificate Number: 320726210100341485.

[0426] 1. Inhibitory effect of bispecific antibodies in the mouse acute ear swelling model

[0427] An acute ear swelling model was established in mice by subcutaneously injecting human IL-36 and human IL-23 into the ears of IL-36R humanized transgenic mice to evaluate the in vivo activity of the anti-IL-36R and anti-IL-23 bispecific antibody.

[0428] The experimental process is shown in Figure 10A. After arrival, the animals were adaptively fed for 7 days and randomly divided into groups. On day 0 and day 3 of the experiment, the mice were intraperitoneally injected with equimolar amounts of anti-IL-36R antibodies, anti-IL-23 antibodies, or anti-IL-36R and anti-IL-23 bispecific antibodies. From day 1 to day 6 of the experiment, PBS or 1 μg recombinant human IL-36α protein and 3 μg recombinant human IL-23 protein were subcutaneously injected into the right ear of the mice every day, and an equal volume of PBS was injected into the left ear. The mouse grouping and dosing regimen are shown in Table 17. At the end of the experiment, the weight of the right ear of the mouse was measured, and part of the ear tissue was preserved with RNAlater (Invitrogen, AM7020) for downstream gene expression detection.

[0429] Table 17. Dosing regimen of human IL-36 and human IL-23 to induce ear swelling in human IL-36R transgenic mice

[0430] The results are shown in Figures 10B and 10C. The bispecific antibody IL-36R-IL-23-01 significantly inhibited the increase in right ear weight induced by human IL-36 and human IL-23 in mice (Figure 10B) and reduced the expression levels of inflammatory disease factors and antimicrobial peptides (Figure 10C). Specifically, IL-36R-IL-23-01 had a stronger regulatory effect on downstream gene expression in ear tissue than the anti-IL-36R antibody VHH213_Hu07 and the anti-IL-23 antibody HU29-19 (Figure 10C) (Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 in the accompanying figure).

[0431] 2. Inhibitory effect of bispecific antibodies compared to monoclonal antibodies in the mouse acute ear swelling model

[0432] The experimental process is shown in Figure 10A. After arrival, the animals were adaptively fed for 7 days and randomly divided into groups. On day 0 of the experiment, the mice were intraperitoneally injected with equimolar amounts of anti-IL-36R antibodies, anti-IL-23 antibodies, anti-IL-36R and anti-IL-23 bispecific antibodies, or anti-IL-36R antibodies combined with anti-IL-23 antibodies. From day 1 to day 6 of the experiment, PBS or 1 μg recombinant human IL-36 protein and 3 μg recombinant human IL-23 protein were subcutaneously injected into the right ear of the mice every day, and an equal volume of PBS was injected into the left ear. The mouse grouping and dosing regimen are shown in Table 18. At the end of the experiment, the weight of the right ear of the mouse was measured, and part of the ear tissue was preserved with RNAlater (Invitrogen, AM7020) for downstream gene expression detection.

[0433] Table 18. Dosing regimen of human IL-36 and human IL-23 to induce ear swelling in human IL-36R transgenic mice

[0434] The results are shown in Figure 11. The regulatory effect of the bispecific antibody IL-36R-IL-23-01 (12 mpk) on the expression of downstream genes in mouse ear tissue was significantly stronger than that of the equimolar dose of anti-IL-23 antibody Hu29-19 (10 mpk) and anti-IL-36R antibody VHH213_Hu07 (5.3 mpk), and was equivalent to the combination group of Hu29-19 (10 mpk) and VHH213_Hu07 (5.3 mpk). At the same time, IL-36R-IL-23-01 showed a significant dose effect. High-dose IL-36R-IL-23-01 (24 mpk) had a stronger regulatory effect on multiple genes (such as S100A8 and S100A9, both of which are antimicrobial peptides, upregulated in inflammatory responses, and involved in cytokine recruitment and cytokine expression) than low-dose IL-36R-IL-23-01 (12 mpk) and the combination of Hu29-19 (10 mpk) and VHH213_Hu07 (5.3 mpk) (in the accompanying figures, compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001).

[0435] Although specific embodiments of the present disclosure have been described above, those skilled in the art will appreciate that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the claims.

Claims

1. An IL-36R / IL-23 binding protein comprising a first antigen binding domain that specifically binds to IL-36R and a second antigen binding domain that specifically binds to IL-23; in, The first antigen binding domain comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 3, 12-26, or CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 4 and 27-35, Wherein, the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

2. The IL-36R / IL-23 binding protein according to claim 1, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 5, 6, 7, or 8, 9, 10, respectively.

3. The IL-36R / IL-23 binding protein according to claim 1 or 2, wherein the immunoglobulin single variable domain is humanized, backmutated, affinity matured, T cell epitope removed, antibody deamidation reduced and / or antibody isomerization reduced; Preferably, the heavy chain framework region of the human germline template used in the humanization process is derived from IGHV3-23*04, IGHV3-74*01 and / or IGHJ1*01.

4. The IL-36R / IL-23 binding protein according to any one of claims 1 to 3, wherein the amino acid sequence of the immunoglobulin single variable domain is as shown in any one of SEQ ID NOs: 3, 12-26, or has at least 80%, at least 90% sequence identity thereto, or as shown in any one of SEQ ID NOs: 4, 27-35, or has at least 80%, at least 90% sequence identity thereto; Preferably, the immunoglobulin single variable domain is an anti-IL-36R Nanobody or VHH.

5. The IL-36R / IL-23 binding protein according to any one of claims 1 to 4, wherein The second antigen-binding domain that specifically binds to IL-23 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 52-57, and the VL comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 58-61, or The VH comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO:40, and the VL comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in SEQ ID NO:41, The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, The VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 50, 43, and 44, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 45-47; The VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 51, 43, and 44, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 45-47; The VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs:42-44, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs:45-47.

6. The IL-36R / IL-23 binding protein according to claim 5, wherein The VH comprises an amino acid sequence as shown in any one of SEQ ID NOs: 52-57 or having at least 80%, at least 90% identity thereto, and the VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 58-61 or having at least 80%, at least 90% identity thereto, or The VH comprises an amino acid sequence as shown in SEQ ID NO:40, or having at least 80%, at least 90% identity thereto, and the VL comprises an amino acid sequence as shown in SEQ ID NO:41, or having at least 80%, at least 90% identity thereto; Preferably, the VH comprises the amino acid sequence shown in SEQ ID NO:56, and the VL comprises the amino acid sequence shown in SEQ ID NO:

60.

7. An IL-36R / IL-23 binding protein, comprising a first antigen binding domain that specifically binds to IL-36R and a second antigen binding domain that specifically binds to IL-23; preferably, the first antigen binding domain and the second antigen binding domain are as defined in any one of claims 1 to 6.

8. The IL-36R / IL-23 binding protein of any one of claims 1 to 7, further comprising an Fc region of an immunoglobulin; Preferably, the Fc region is the Fc region of human IgG1, human IgG2 or human IgG4; More preferably, the Fc region is an Fc region of human IgG4 having an S228P mutation, or an Fc region of human IgG1 having an L234A and / or L235A mutation, or an Fc region of human IgG1 having an M252Y, S254T and / or T256E mutation.

9. The IL-36R / IL-23 binding protein according to any one of claims 1 to 8, further comprising a linker; Preferably, the amino acid sequence of the linker is as follows (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h As shown, m and n are each independently selected from integers of 1-8, and h is independently selected from integers of 1-20; More preferably, the linker is a linker represented by (G4S)2 or (G4S)3.

10. The IL-36R / IL-23 binding protein according to any one of claims 1 to 9, comprising a first polypeptide chain and a second polypeptide chain selected from the following: (1) the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO:68 or 70, or has at least 80%, at least 90% sequence identity thereto, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO:69, or has at least 80%, at least 90% sequence identity thereto; (2) The amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 71 or has at least 80% to The amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO:72, 73 or has at least 80%, at least 90% sequence identity thereto; (3) The amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO:74 or 75, or has at least 80%, at least 90% sequence identity thereto, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO:69, or has at least 80%, at least 90% sequence identity thereto.

11. An IL-36R binding protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 3, 12-26, or CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 4 and 27-35, in, The CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

12. The IL-36R binding protein according to claim 11, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 5, 6, 7, or 8, 9, 10, respectively.

13. The IL-36R binding protein according to claim 11 or 12, wherein the immunoglobulin single variable domain is humanized, backmutated, affinity matured, T cell epitope removed, antibody deamidation reduced and / or antibody isomerization reduced; Preferably, the heavy chain framework region of the human germline template used in the humanization process is derived from IGHV3-23*04, IGHV3-74*01 and / or IGHJ1*01.

14. The IL-36R binding protein according to any one of claims 11 to 13, wherein the amino acid sequence of the immunoglobulin single variable domain is as shown in any one of SEQ ID NOs: 3, 12-26, or has at least 80%, at least 90% sequence identity thereto, or as shown in any one of SEQ ID NOs: 4, 27-35, or has at least 80%, at least 90% sequence identity thereto; Preferably, the IL-36R binding protein is an anti-IL-36R nanobody or VHH.

15. The IL-36R binding protein of any one of claims 11 to 14, further comprising an immunoglobulin Fc region; Preferably, the Fc region is the Fc region of human IgG1, human IgG2 or human IgG4.

16. A polynucleotide encoding the IL-36R / IL-23 binding protein of any one of claims 1 to 10, or the IL-36R binding protein of any one of claims 11 to 15; Preferably, the polynucleotide is DNA or RNA.

17. A vector comprising the polynucleotide of claim 16.

18. A host cell containing or expressing the polynucleotide according to claim 16 or the vector according to claim 17.

19. A method for preparing an IL-36R / IL-23 binding protein or an IL-36R binding protein, comprising: Expressing the polynucleotide of claim 16 or the vector of claim 17 in the host cell of claim 18, and isolating the expressed IL-36R / IL-23 binding protein or IL-36R binding protein from the host cell; Optionally, the method further comprises the step of purifying the IL-36R / IL-23 binding protein and the IL-36R binding protein.

20. A pharmaceutical composition comprising the IL-36R / IL-23 binding protein of any one of claims 1 to 10, the IL-36R binding protein of any one of claims 11 to 15, and at least one pharmaceutically acceptable excipient, diluent or carrier.

21. A method for treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the IL-36R / IL-23 binding protein of any one of claims 1 to 10, the IL-36R binding protein of any one of claims 11 to 15, the polynucleotide of claim 16, the vector of claim 17, or the pharmaceutical composition of claim 20; Preferably, the disease is an inflammatory disease or an autoimmune disease; More preferably, the disease is psoriasis.

22. Use of the IL-36R / IL-23 binding protein of any one of claims 1 to 10, the IL-36R binding protein of any one of claims 11 to 15, the polynucleotide of claim 16, the vector of claim 17 or the pharmaceutical composition of claim 20 for preparing a drug for treating or preventing a disease; Preferably, the disease is an inflammatory disease or an autoimmune disease; More preferably, the disease is psoriasis.

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