Therapeutic derivatives of interleukin-22

Abstract

The present invention relates to novel derivatives of interleukin-22 (IL-22), particularly those comprising fatty acids covalently attached to the IL-22 protein, and their use in therapy.

Description

[Technical Field]

[0001] The present invention relates to novel derivatives of interleukin-22 (IL-22), and in particular derivatives comprising fatty acids covalently bound to the IL-22 protein. The present invention also encompasses methods for their production and use in therapies including metabolism, treatment, prevention, and improvement of diseases, disorders, and conditions of the liver, lungs, intestines, kidneys, and skin. [Background technology]

[0002] IL-22 is a 146-amino acid protein with a molecular weight of 17 kDa. It belongs to the IL-10 family of cytokines and selectively activates a heterodimer receptor consisting of the ubiquitously expressed IL-10 receptor B subunit (IL-10RA2) and the IL-22 receptor A subunit (IL-22RA1), which is expressed exclusively in epithelium. It is released from immune cells but is a unique cytokine in that it selectively targets epithelial cells. Therefore, the signaling pathway induced by IL-22 can be relevant in various tissues (targets include skin, intestine, lung, liver, kidney, pancreas, and thymus), but IL-22 specifically activates them in an epithelial-specific manner. The soluble binding protein IL-22BP neutralizes IL-22, thereby regulating its action.

[0003] IL-22 is released in response to signals that reflect chemical or mechanical injury, such as aryl hydrocarbon receptor activation in response to environmental toxins or tryptophan intermediates, and pattern recognition receptor activation, such as Toll-like receptor 4, in response to proteins, fragments, and debris from dying cells or invading pathogens. IL-22 release is further stimulated by certain cytokines, particularly IL-23 and, to a lesser extent, IL-1β. Thus, IL-22 is secreted in response to signals that also reflect pathogen infection and immune activation.

[0004] The action of IL-22 is the result of the organized involvement of several activity / pathway. IL-22 acts in epithelial barrier tissues and organs at the time of injury to protect cells and maintain barrier function (e.g., through activation of anti-apoptotic gene programs). It also accelerates repair (e.g., by inducing the proliferation of mature cells and the activation of stem cells), prevents fibrosis (e.g., by reducing epithelial-mesenchymal transition, antagonizing the NLRP3 inflammasome, and inducing hepatic stellate cell senescence), and controls inflammation (e.g., by inducing antimicrobial peptides and chemotactic signals). IL-22 has been reported to be able to treat a variety of medical conditions, including those often observed in diabetic or overweight mammals, such as hyperglycemia, dyslipidemia, and hyperinsulinemia.

[0005] However, IL-22 is normally rapidly eliminated from the body by the kidneys, which limits its use in clinical practice. Therefore, known methods to extend the half-life of circulating IL-22 attempt to artificially enlarge its size to over 70 kDa to avoid renal clearance. Conjugation of IL-22 to Fc antibody fragments is currently the best solution to this problem; Genentech and Generon Shanghai both have long-acting IL-22-Fc fusions in clinical development. Modification of IL-22 with polyethylene glycol (PEGylation) is another known method to avoid renal clearance.

[0006] However, these existing solutions are not without their drawbacks. Available data suggest that PEG itself is immunogenic, and that PEG-containing vacuoles are observed in cells when PEGylated biologics are used. Reduced activity and heterogeneity are also disadvantages of PEGylation. While Fc fusion technology is very well known, the addition of Fc antibody fragments implies a major structural change to IL-22, which affects its properties beyond extending its half-life. Since Fc fusion increases the size of the protein from approximately 17 kDa to approximately 85 kDa, properties such as diffusion rate, distribution, and receptor engagement kinetics may be affected. For example, some Fc fusions are absorbed slowly and / or are too large for administration via a given route. Genentech and Generon have also reported moderate reversible skin reactions as dose-limiting adverse effects of IL-22-Fc fusions. Furthermore, potency may be affected through steric hindrance caused by the large fusion partner.

[0007] Therefore, there is still a need in the field for novel biocompatible modifiers of IL-22 that improve the circulating half-life and demonstrate optimized pharmacokinetic and pharmacodynamic properties compared to the native molecule. Ideally, they should maintain the potency and other properties of the native molecule and avoid the toxicity, immunogenicity, and any other adverse reactions exhibited by known derivatives. [Overview of the project]

[0008] In the first embodiment, a derivative of IL-22 is provided, which includes a fatty acid covalently bonded to the IL-22 protein.

[0009] In one embodiment of the present invention, a fatty acid is covalently bonded to the IL-22 protein by a linker.

[0010] Fatty acids are given by formula I: HOOC-(CH2) x -CO-*, It may be a fatty acid, e.g., a C12, C14, C16, or C20 fatty acid (wherein x is an integer in the range of 10-18, arbitrarily 12-18, 14-16, or 16-18, and * indicates the binding site of the IL-22 protein or linker). It may be a fatty acid, e.g., a C12, C14, C16, C18, or C20 fatty acid. Advantageously, the fatty acid is a C16 or C18 fatty acid, most advantageously it is a C18 fatty acid.

[0011] The IL-22 protein may be native mature human IL-22 (hereinafter, "hIL-22") or a variant thereof. The variant may be a substitutional form of hIL-22, optionally substituted at positions 1, 21, 35, 64, 113 and / or 114. It may include substitutions of hIL-22 selected from the group consisting of A1C, A1G, A1H, N21C, N21D, N21Q, N35C, N35D, N35H, N35Q, N64C, N64D, N64Q, N64W, Q113C, Q113R, K114C and K114R. Advantageously, the variant contains a Cys residue at position 1 of hIL-22.

[0012] The variant may be an extended form of hIL-22. It may include an N-terminal peptide, such as an N-terminal trimer. Advantageously, the variant includes an N-terminal GPG.

[0013] The linker may contain one or more amino acids, optionally including glutamic acid (Glu) and / or lysine (Lys). The linker may contain an oxyethyleneglycine unit or a plurality of linked oxyethyleneglycine units, optionally 2 to 5 such units, preferably 2 units. The linker may contain one or more oligo(ethylene glycol) (OEG) residues. It may contain an ethylenediamine (C2DA) group and / or an acetamide (Ac) group. Advantageously, the linker may contain a combination of all the aforementioned elements. In particular, the linker may be γGlu-OEG-OEG-C2DA-Ac, γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc, or γGlu-OEG-OEG-εLys-αAc.

[0014] The linker can be a Cys-reactive linker that binds to a Cys residue in hIL-22 or a variant thereof. It can be bound at positions -7, -5, 1, 6, 33, 113, 114 or 153 of hIL-22 or a variant thereof (where positions -7, -5, etc. are as defined herein). By way of example, the linker can be bound to a Cys residue substituted at position 1, 6, 33, 113 or 114 of hIL-22. It can be bound to a Cys residue at positions -5, -7 or 153 relative to hIL-22. Advantageously, the linker is bound to a Cys residue substituted at position 1 of hIL-22.

[0015] In one embodiment, the derivative comprises a C14, C16, C18 or C20 diacid covalently linked by a linker to a variant of hIL-22, the variant comprising an N-terminal G-P-G and a Cys residue at position 1 of hIL-22, and the linker being optionally bound to said Cys residue. Exemplary derivatives of the present invention are those identified herein as derivatives 1-10.

[0016] In a second aspect, there is provided a process for preparing a derivative of the first aspect, the process comprising covalently attaching a fatty acid to an IL-22 protein.

[0017] In a third aspect, there is provided a pharmaceutical composition comprising a derivative of the first aspect and a pharmaceutically acceptable vehicle.

[0018] In a fourth aspect, there is provided a derivative of the first aspect or a pharmaceutical composition of the third aspect for use in therapy.

[0019] In a fifth aspect, there is provided a derivative of the first aspect or a pharmaceutical composition of the third aspect for use in a method of treating a disease, disorder or condition of metabolism, liver, lung, intestine, kidney or skin.

[0020] The metabolic disease, disorder or condition can be obesity, type 1 diabetes, type 2 diabetes, dyslipidemia, hyperglycemia or hyperinsulinemia.

[0021] Liver disease, disorder, or condition may be non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, alcoholic hepatitis, acute liver failure, chronic liver failure, acute exacerbation of chronic liver failure (ACLF), acute liver injury, acetaminophen-induced hepatotoxicity, sclerosing cholangitis, biliary cirrhosis, or a pathological condition caused by surgery or transplantation.

[0022] Lung diseases, disorders, or conditions may include chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, idiopathic pulmonary fibrosis, acute distress syndrome, chemical injury, viral infection, bacterial infection, or fungal infection.

[0023] Intestinal diseases, disorders, or conditions may include inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, graft-versus-host disease (GvHD), chemical injury, viral infection, or bacterial infection.

[0024] Kidney disease, disorder, or condition may be acute kidney disease or chronic kidney disease.

[0025] Skin diseases, disorders, or conditions may include wounds, inflammatory diseases, or GvHD. [Brief explanation of the drawing]

[0026] [Figure 1] The diagram shows (A) C18 diacid, (B) C16 diacid, and (C) C14 diacid, respectively, linked to a linker containing a Cys reactive unit. These combinations of fatty acids and linkers are used in the derivatives of the present invention, which are identified herein as derivatives 1 to 10. [Figure 2] The structure of the derivative of the present invention, identified herein as derivative 1, is shown. [Figure 3] The structure of the derivative of the present invention, identified herein as derivative 6, is shown. [Figure 4] The structure of the derivative of the present invention, identified herein as derivative 10, is shown. [Figure 5] This shows the effect of daily administration of hIL-22 and a comparative IL-22 variant with only skeletal variations (identified as comparative 3 herein) on blood glucose in an 8-day study in a diabetic mouse model (mean ± SEM). [Figure 6] The effects of daily administration of the derivative of the present invention (identified as derivative 1 herein) compared to the IL-22-Fc fusion (specifically, human Fc fused to hIL-22 at the N-terminus; hereafter "hFc-hIL-22") on (A) blood glucose and (B) food intake in a 16-day study in a diabetic mouse model (mean ± SEM; * indicates p < 0.05 using an unpaired t-test). [Figure 7A] This shows the effects of daily administration of derivatives 1 and hFc-hIL-22 on three different target-engaging biomarkers in a 16-day study in a diabetic mouse model (mean ± SEM; *** indicates (A) p < 0.0002 using an unpaired t-test). [Figure 7B] This shows the effects of daily administration of derivatives 1 and hFc-hIL-22 on three different target-engaging biomarkers in a 16-day study in a diabetic mouse model (mean ± SEM; *** indicates (B) p < 0.0003 using an unpaired t-test). [Figure 7C] This shows the effects of daily administration of derivatives 1 and hFc-hIL-22 on three different target-engaging biomarkers in a 16-day study in a diabetic mouse model (mean ± SEM; *** indicates (C)p<0.0026 using an unpaired t-test). [Figure 8] The dose-response curves (mean ± SEM) for daily administration of the derivative of the present invention (identified as derivative 6 herein) (three different doses) compared to derivative 1 and hFc-hIL-22 in a 13-day study in a diabetic mouse model are shown for blood glucose. [Figure 9A]The effects of derivatives 1 and 6 in preventing liver injury in a mouse model of acetaminophen (APAP)-induced liver injury are shown, as revealed by the plasma levels of two different liver enzymes. Using Dunnett's single-factor linear model, compared to vehicle + APAP, * means p<0.05 and ** means p<0.01. [Figure 9B] The effects of derivatives 1 and 6 in preventing liver injury in a mouse model of acetaminophen (APAP)-induced liver injury are shown, as revealed by the plasma levels of two different liver enzymes. Using Dunnett's single-factor linear model, compared to vehicle + APAP, * means p<0.05 and ** means p<0.01. [Figure 10A] This shows the effects of derivatives 1 and 6 on (A) prevention of apoptosis in an APAP-induced liver injury mouse model. NS indicates no statistical significance. [Figure 10B] (B) The effects of derivatives 1 and 6 on cell proliferation in an APAP-induced liver injury mouse model are shown. NS indicates no statistical significance. [Figure 11A] This study demonstrates the efficacy of derivative 6 compared to prednisolone in (A) the prevention and / or reduction of pneumonia in a rat model of bleomycin-induced lung injury. [Figure 11B] (B) The effect of derivative 6 compared to prednisolone in the prevention and / or reduction of pulmonary fibrosis in a rat model of bleomycin-induced lung injury. [Figure 11C] (C) The efficacy of derivative 6 compared to prednisolone in the prevention and / or reduction of pulmonary fibrosis in a rat model of bleomycin-induced lung injury. [Figure 12] This shows the effect of derivative 6 in preventing colitis in a mouse model of dextran sulfate sodium (DSS)-induced colitis. **** indicates p<0.0001 compared to the vehicle (containing DSS). [Figure 13]This shows the effect of derivative 6 compared to hFc-hIL-22 in preventing mucosal epithelial wounds in a mouse model of DSS-induced colitis. Magnification 4x, scale bar = 500 μm. [Figure 14] This shows the level of plasma-regenerated islet-derived protein 3 gamma (Reg3g) in a mouse model of DSS-induced colitis as an indicator of target engagement (Reg3g is a target engagement marker for IL-22). [Figure 15A] The effect of derivative 1 in preventing liver injury in a mouse model of concanavalin A (ConA)-induced liver injury is evident from the serum levels of two different liver enzymes. [Figure 15B] The effect of derivative 1 in preventing liver injury in a mouse model of concanavalin A (ConA)-induced liver injury is evident from the serum levels of two different liver enzymes. [Figure 16] This study demonstrates the effect of derivative 6 on body weight in diet-induced obese mice compared to hFc-hIL-22 and semaglutide, a known fatty acid conjugation GLP-1 derivative. [Modes for carrying out the invention]

[0027] In the following, Greek letters are represented by their symbols rather than their written names. For example, α = alpha, ε = epsilon, γ = gamma, and μ = mu. Amino acid residues can be identified by their full names, three-letter codes, or one-letter codes, all of which are completely equivalent.

[0028] As used herein, the term “IL-22 derivative” refers to an IL-22 protein having a covalently bonded fatty acid. The term encompasses both derivatives in which the fatty acid is directly covalently bonded to the IL-22 protein and those in which the covalent bond is mediated by a linker.

[0029] Covalent bonding of fatty acids is a recognized technique for extending the half-lives of peptides and proteins, and is a method of encapsulating fatty acids from peptides or proteins. It is known from marketed products for type 1 and type 2 diabetes, such as the insulins Levemir® (detemir) and Tresiba® (degludec), as well as the glucagon-like peptide-1 (GLP-1) derivatives Victoza® (liraglutide) and Ozempic® (semaglutide).

[0030] Fatty acid binding enables binding to albumin, thereby preventing renal excretion and providing some degree of steric protection against proteolysis. Advantageously, it offers minimal modification to IL-22 compared to Fc fusion or PEGylation. In this regard, while Fc fusion and PEGylation aim to increase the size of IL-22 beyond the renal clearance threshold, derivatives containing covalently bound fatty acids to the IL-22 protein retain a small size similar to that of the original IL-22 protein. Therefore, because fatty acid binding is minimal modification, the resulting derivatives are thought to maintain native-like properties, including distribution, diffusion rate, and receptor engagement (binding, activation, and trafficking), minimizing the risk of immunogenicity.

[0031] As described above, fatty acid binding has demonstrated therapeutic efficacy in insulin and GLP-1 derivatives for diabetes. However, IL-22 is a very different protein in terms of its size, sequence, and biological properties. Therefore, it was not intuitive to the inventors that fatty acids could be covalently bound to IL-22 while maintaining therapeutic effects. What was particularly surprising was that such minimal modification to IL-22 could result in high potency (close to that of hIL-22) combined with an extremely long circulating half-life.

[0032] Therefore, in a first aspect, the present invention relates to a derivative of IL-22 comprising a fatty acid covalently bonded to the IL-22 protein. The fatty acid may be covalently bonded to the IL-22 protein directly or via a linker, and may itself be devised from various subunits. The term “IL-22 protein” as used herein may mean a native IL-22 protein, e.g., hIL-22, or a variant thereof. A “variant” may be a protein having an amino acid sequence similar to that of the native protein, as further defined herein.

[0033] Human IL-22 protein is naturally synthesized along with a 33-amino acid signal peptide for secretion. Mature human IL-22 protein (i.e., hIL-22) is 146 amino acids long and has 80.8% sequence identity with mouse IL-22 (the latter being 147 amino acids long). The amino acid sequence of hIL-22 is identified herein as Sequence ID No. 1. Like other IL-10 family members, the IL-22 structure contains six α-helices (referred to as helices A-F).

[0034] Therefore, the derivatives of the present invention may have the native amino acid sequence of hIL-22. Alternatively, they may have one or more amino acid sequence variations within the native sequence. They may additionally or alternatively include one or more amino acid sequence variations relative to (i.e., outside of) the native sequence. Therefore, in one embodiment, the derivative includes a fatty acid covalently bonded to hIL-22 or a variant thereof.

[0035] Expressions such as “internal,” “relative to,” “corresponding to,” and “equivalent to” are used herein to characterize the sites of fatty acid alterations and / or covalent bonds in the IL-22 protein by reference to the sequence of a native protein, e.g., hIL-22. In Sequence ID No. 1, the first amino acid residue of hIL-22 (alanine (Ala)) is assigned as position 1.

[0036] Therefore, the variations within the hIL-22 sequence are variations to any of the residue numbers 1-146 in SEQ ID NO: 1. For example, a Glu substitution for the native Asp at residue 10 in hIL-22 is represented herein as "D10E". If the derivative also has a covalently bonded fatty acid at position 10, this is referred herein as a bond at residue "10E".

[0037] However, variations in the sequence of hIL-22 are external variations to residue numbers 1-146 in Sequence ID No. 1. For example, derivative 2 as defined herein contains an N-terminal peptide of 15 amino acids in length. The residues in the N-terminal peptide are numbered negatively, starting from the residue bound to residue 1 in hIL-22. That is, the first residue in the N-terminal peptide bound to residue 1 in hIL-22 is represented as "-1". Thus, derivative 2 has a covalently bonded fatty acid at the seventh residue in the N-terminus, starting from position -1, which is Cys, and therefore the covalent site for derivative 2 is referred to herein as "-7C". However, naturally, the numbering used in the sequence listing of derivative 2 starts from 1 according to WIPO standard ST.25; thus, position 1 in the sequence listing of derivative 2 is actually residue -7 as referred to herein.

[0038] Two, three, four, five or more variations may be generated within the native sequence to form derivatives of the present invention. In this regard, for example, variations of 10, 15, 20, 25, 50, 75, more than 100, or even more than 125 may be generated. Any of residues 1 to 146 in the native sequence may be modified. Exemplary residues for variation are residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 63, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 7 7, 78, 79, 82, 83, 84, 86, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 126, 127, 128, 129, 130, 132, 133, 134, 135, 137, 138, 139, 141, 143, 144, 145 and / or 146. Variations at residues 1, 21, 35, 64, 113 and / or 114 are particularly advantageous.

[0039] Variations within a native sequence are typically amino acid substitutions. The term “substitution,” as used herein, may mean replacing one amino acid in a native protein with another. These substitutions can be conserved or non-conserved.Examples of substitutions include A1C, A1G, A1H, P2C, P2H, I3C, I3H, I3V, S4H, S4N, S5H, S5T, H6C, H6R, C7G, R8G, R8K, L9S, D10E, D10S, K11C, K11G, K11V, S12C, N13C, N13G, F14S, Q15C, Q15E, Q16V, P17L, Y18F, I19Q, T20V, N21C, N21D, N21Q, R22S, F24H, M25E, M25L, L26S, A27L, E29P, A30Q, L32C, L32R, A3 3C, A33N, D34F, N35C, N35D, N35H, N35Q, N36Q, T37C, T37I, D38L, V39Q, R40W, L41Q, I42P, E44R, K45A, F47T, H48G, H48R, G49N, V50S, S51C, M52A, M52C, M52L, M52V, S53C, S53K, S53Y, E54D, E54F, R55Q, R55V, C56Q, L58K, M59I, Q61E, V62D, L63C, N64C, N64D, N64Q, N64W, F65G, L67Q , E69D, E69L, V70S, L71C, F72D, F72L, P73C, P73L, Q74T, R77I, F78Q, Q79E, M82Y, Q83G, E84R, V86A, F88N, A90P, A90T, R91C, R91K, R91Y, L9 2R, S93Y, N94C, N94Q, R95K, R95Q, L96E, S97K, T98C, T98N, T98S, C99V, H100S, E102S, G103D, D104Y, D105Y, L106E, L106Q, H107L, H107N, I1 These are 08L, Q109Y, R110C, R110K, N111K, V112E, Q113C, Q113R, K114C, K114R, L115V, K116Y, D117E, T118G, V119A, K120H, K121R, L122A, G123V, G126Y, E127C, I128V, K129V, G132Y, E133Q, L134P, D135M, L137D, F138R, M139L, M139R, L141Q, N143S, A144E, C145E, I146R and / or I146V.Advantageously, the substitution can be selected from the group consisting of A1C, A1G, A1H, N21C, N21D, N21Q, N35C, N35D, N35H, N35Q, N64C, N64D, N64Q, N64W, Q113C, Q113R, K114C, and K114R. Surprisingly, the substitutions used in this invention do not adversely affect IL-22 activity.

[0040] Specific combinations of substitutions include (i) A1G, N21D, N35D and N64D; (ii) A1G, I3V, S4N, S5T, H6R, R8K, D10E, K11V, T20V, H48R, M52A, S53K, E54D, R55Q, E69D, F72L, A90T, R91K, R95Q, T98S, E102S, L106Q, H107N, R110K, Q113R, K114R, D117E and I146V; (iii) A1G, I3V, S4N, S5T, H6R, R8K, D10E, K11V, T20V, H48R, M52A, S53K, E54 D, R55Q, E69D, F72L, A90T, R91K, R95Q, T98S, E102S, L106Q, H107N, R110K, Q113R, K114R, D117E and I146V; (iv) A1G, N35Q and N64Q; (v) A1G and N64C; (vi) A1G and Q113C; (vii) A1G and K114C; (viii) A1G and M25L; (ix) A1G and M52L; (x) A1G and M139L; (xi) A1G and N36Q; (xii) A1G and D117E; (xiii) A1G and N21Q; (xiv) A1G and N35Q; (xv)A1G and N64Q; (xvi)A1G, N21Q and N35Q; (xvii)A1G, N21Q and N64Q; (xviii)A1G and N21Q, N35Q and N64Q; (xix)A1G and K11C; (xx)A1G and N13C; (xxi)N35Q and N64Q; (xxii)A1C, N35Q and N64Q; (xxiii)H6C, N35Q and N64Q; (xxiv)I3C, N35Q and N64Q; (xxv)P2C, N35Q and N64Q; (xxvi)L32C, N35Q and N64Q; (xxvii )N35Q, M52C and N64Q; (xxviii)N13C, N35Q and N64Q; (xxix)N21C, N35Q and N64Q; (xxx)N35Q, N64Q and N94C; (xxxi)N35Q, N64Q and P73C; (xxxii)N35Q, N64Q and Q113C; (xxxiii)N35Q, N64Q and R91C; (xxxiv)N35Q, N64Q and R110C; (xxxv)S12C, N35Q and N64Q; (xxxvi)N35Q, S51C and N64Q; (xxxvii)N35Q, S53C and N64Q;(xxxviii)N35Q, T37C and N64Q; (xxxix)N35Q, N64Q and T98C; (xxxx)Q15C, N35Q and N64Q; (xxxxi)N35C and N64Q; (xxxxii)H6C, N35Q and N64Q; (xxxxiii)A33C, N35Q and N64Q; and (xxxxiv)A1H, P2H, I3H, S4H, S5H, C7G, R8G, L9S, D10S, K1 1G, N13G, F14S, Q15E, Q16V, P17L, 18F, Y19Q, N21Q, R22S, F24H, M25E, L26S, A27L, E29P, A30Q, L32R, A33N, D34F, N 35H, T37I, D38L, V39Q, R40W, L41Q, I42P, E44R, K45A, F47T, H48G, G49N, V50S, M52V, S53Y, E54F, R55V, C56Q, L58K , M59I, Q61E, V62D, L63C, N64W, F65G, L67Q, E69L, V70S, L71C, F72D, P73L, Q74T, R77I, F78Q, Q79E, M82Y, Q83G, E8 4R, V86A, F88N, A90P, R91Y, L92R, S93Y, N94Q, R95K, L96E, S97K, T98N, C99V, H100S, G103D, D104Y, D105Y, L106E, This includes H107L, I108L, Q109Y, R111K, V112E, L115V, K116Y, D117E, T118G, V119A, K120H, K121R, L122A, G123V, G126Y, E127C, I128V, K129V, G132Y, E133Q, L134P, D135M, L137D, F138R, M139R, L141Q, N143S, A144E, C145E, and I146R. Any combination of substitutions is conceivable and forms part of the present invention.

[0041] Derivatives of the first embodiment may typically include amino acid substitutions in which Cys is substituted for a native residue at any of the positions arbitrarily identified above, e.g., positions 1, 2, 3, 6, 11, 12, 13, 15, 21, 32, 33, 35, 37, 51, 52, 53, 63, 64, 71, 73, 91, 94, 98, 110, 113, 114 and / or 127. Advantageously, the IL-22 protein contained in derivatives of the first embodiment contains a Cys residue at position 1 of hIL-22. A1C substitutions combined with substitutions at two glycosylation sites at positions 35 and 64 are particularly advantageous because they lead to faster uptake without adversely affecting potency or half-life (see derivatives 6 and 10 in Examples 1 and 2).

[0042] Alternatively, or additionally, variations within the native sequence may be amino acid insertions. Up to 5, 10, 15, 20, 25, 30, 35, 40, 45, or even up to 50 amino acids may be inserted into the native sequence. Trimers, pentamers, septamers, octamers, nonamers, and 44-mers are particularly advantageous in this regard. Exemplary sequences are shown in Table 1. Insertions may be made at any position in the native sequence, but those in helix A (e.g., residue 30), loop CD (e.g., residue 75), helix D (e.g., residue 85), and / or helix F (e.g., residue 124) are preferred.

[0043] [Table 1]

[0044] Sequence variations of the amino acid sequence of hIL-22, if present, typically involve elongation, such as the addition of a peptide at the N-terminus. The peptide may consist of up to 5, 10, 15, 20, 25, 30, 35, 40, 45, or even up to 50 amino acids. Monomers, trimers, octamers, 13-mers, 15-mers, 16-mers, 21-mers, and 28-mers are particularly advantageous in this regard. Exemplary sequences are shown in Table 2. Preferably, the IL-22 protein contained in the derivatives of the first embodiment contains an N-terminal GPG. In a particularly preferred example, the derivatives of the first embodiment contain both the Cys residue at position 1 of hIL-22 (SEQ ID NO: 1) and the N-terminal GPG. This has been found to produce derivatives with very good half-lives and potency (see derivatives 1, 3, and 5 in Examples 1 and 2).

[0045] [Table 2]

[0046] Sequence variations of the hIL-22 amino acid sequence, if present, may include the addition of a peptide at the C-terminus. The peptide may consist of up to 5, 10, 15, 20, 25, 30, 35, 40, 45, or even up to 50 amino acids. A septamer having the amino acid sequence GSGSGSC (SEQ ID NO: 15) is particularly advantageous in this respect.

[0047] The derivatives of the present invention may comprise both the N-terminal and C-terminal peptides in addition to the native or variant hIL-22 amino acid sequence described herein. Any combination of the N-terminal and C-terminal peptides described herein is envisioned and expressly included in the present invention.

[0048] The present invention is understood to extend to any derivative of IL-22, including fatty acids covalently bonded to hIL-22 or its variants. A “variant” can be a protein having at least 10% sequence identity with hIL-22. In one embodiment, the variant has at least 20%, or even more than 30%, sequence identity with hIL-22. The variant may have a “substantial amino acid sequence” of hIL-22, which may mean a sequence having at least 40% sequence identity with the amino acid sequence of hIL-22. Thus, in one embodiment, a derivative of the first embodiment has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% amino acid sequence identity with hIL-22. Exemplary IL-22 protein variants incorporated into specific derivatives of the present invention disclosed in the experimental section are shown in SEQ ID NOs: 16-21.

[0049] Those skilled in the art know how to calculate the degree of identity between two amino acid sequences. The alignment of the two sequences must first be prepared, and then the sequence identity value must be calculated. The degree of identity between two sequences can take different values ​​depending on (i) the method used to align the sequences, e.g., structural alignment from ClustalW, BLAST, FASTA, Smith-Waterm (performed by different programs), or 3D comparison; and (ii) the parameters used by the alignment method, e.g., local vs. global alignment, the pair score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, e.g., functional form and constants.

[0050] After alignment, there are many different ways to calculate the degree of identicality between two sequences. For example, the degree of identicality can be divided by (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of non-gap positions; or (iv) the number of equivalent positions excluding overhangs. Furthermore, it is understood that the degree of identicality also strongly depends on length. Therefore, the shorter the pair of sequences, the higher the sequence identicality, which can be expected to occur by chance.

[0051] Therefore, it is understood that accurate alignment of amino acid sequences is a complex process. The well-regarded multiple alignment program ClustalW[48,49] is a preferred method for generating protein multiple alignments according to the present invention. Preferred parameters for ClustalW may be: for protein alignment: gap open penalty = 10.0, gap elongation penalty = 0.2, and matrix = Gonnet. For DNA and protein alignment: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will know that these and other parameters may need to be modified for optimal sequence alignment.

[0052] Preferably, the degree of identity between the two amino acid sequences can then be calculated from such alignment as (N / T)*100 (where N is the number of positions in which the sequences share identical residues, and T is the total number of compared positions, including gaps but excluding overhangs). Thus, the most preferred method for calculating the degree of identity between two sequences comprises (i) creating a sequence alignment using the ClustalW program with a suitable set of parameters, e.g., those shown above; and (ii) inserting the values ​​of N and T into the following formula: Sequence Identity = (N / T)*100.

[0053] Alternative methods for identifying similar sequences are known to those skilled in the art.

[0054] Preferably, the derivatives of the first embodiment contain 200 or fewer amino acids. For example, the derivatives contain less than 190, less than 180, less than 170, less than 160, or even less than 150 amino acids. Preferably, the derivatives contain at least 146 amino acids, which is the number of amino acids in hIL-22. It may contain at least 150 amino acids, at least 160 amino acids, at least 170 amino acids, or even at least 180 amino acids. The derivatives of the present invention may contain proteins of any length within the above range, but they are typically 146 to 180 amino acids in length.

[0055] The derivatives of the present invention contain fatty acids covalently bonded to the IL-22 protein, whether or not they have a native or variant amino acid sequence. Typically, the fatty acids are covalently bonded to the IL-22 protein by a linker. Preferably, the fatty acids and linker are linked to each other via an amide bond, and the linker is covalently bonded to the IL-22 protein. Thus, the fatty acids and linker can exist as side chains on the IL-22 protein. It was surprising to the inventors that the covalently bonded fatty acids did not adversely affect IL-22 activity. It was particularly surprising that the fatty acid bond was associated with further advantages, such as an extension of the half-life.

[0056] The fatty acid can be any suitable fatty acid. In particular, the fatty acid is of formula I: HOOC-(CH2) x -CO-*, It may be a fatty acid, e.g., a C12, C14, C16, or C20 fatty acid (wherein x is an integer in the range of 10-18, arbitrarily 12-18, 14-16, or 16-18, and * indicates the binding site of the IL-22 protein or linker). It may be a fatty acid, e.g., a C12, C14, C16, C18, or C20 fatty acid. Advantageously, the fatty acid is a C16 or C18 fatty acid, most advantageously it is a C18 fatty acid.

[0057] For example, -(CH2) in equation I x- may be a linear alkylene where x is 10. This fatty acid can conveniently be called a C12 diacid, i.e., a fatty dicarboxylic acid having 12 carbon atoms. Alternatively, -(CH2) in formula I x - may be a linear alkylene where x is 12. This fatty acid can conveniently be called a C14 diacid, i.e., a fatty dicarboxylic acid having 14 carbon atoms. In a similar manner, -(CH2) in formula I x - can be a linear alkylene where x is 14(C16 diacid), 16(C18 diacid), or 18(C20 diacid). Preferably, the derivative of the first embodiment includes C14, C16, C18, or C20 diacid; more preferably, C16 or C18 diacid, and even more preferably, C18 diacid.

[0058] Diacitors can form non-covalent associations with albumin, thereby promoting the circulation of their derivatives in the bloodstream. Shorter diacitors (e.g., C16 diacitors) have lower albumin affinity and therefore shorter half-lives than longer diacitors (e.g., C18 diacitors). However, they are still long-acting derivatives with predicted half-lives exceeding one day in humans.

[0059] The fatty acid binding also stabilizes the IL-22 protein against proteolysis. The resulting half-life is typically similar to that of the IL-22-Fc fusion (i.e., significantly improved compared to hIL-22).

[0060] Derivatives of the first embodiment may include specific combinations of fatty acids and IL-22 protein. For example, C14, C16, C18, or C20 diacids may bind to an IL-22 protein containing a Cys residue at position 1 of hIL-22 and / or N-terminal GPG. In one example, a derivative of the first embodiment includes a C18 diacid, and the IL-22 protein contains both a Cys residue at position 1 of hIL-22 and N-terminal GPG.

[0061] As described above, the fatty acid is preferably linked to a linker that binds to the IL-22 protein. The linker may comprise several linker elements, each containing one or more amino acids, e.g., one or more Glu and / or Lys residues. The linker may comprise an oxyethyleneglycine unit or a plurality of linked oxyethyleneglycine units, optionally 2 to 5 such units, preferably 2 units. One or more OEG residues, C2DA and / or Ac groups may be substituted or additionally included. The linker may comprise a Cys-reactive unit. When used herein, "Cys-reactive unit" may mean a functional unit capable of reacting with the sulfur atom of Cys to produce a carbon-sulfur covalent bond. The Cys-reactive unit may have any of several forms, but preferably comprises a carbon atom bonded to a leaving group, the leaving group replacing the sulfur atom of Cys during the formation of the carbon-sulfur bond. The leaving group may be a halogen, optionally a bromine atom. This bromide leaving group can be alpha relative to the actamide functional group; advantageously, it is a bromoacetamide functional group. Alternatively, the leaving group can be a functionalized hydroxyl group in mesylate or tosylate form, or a non-functionalized hydroxyl group. Furthermore, the leaving group can be maleimide or other functional groups. Exemplary linkers include γGlu-OEG-OEG-C2DA-Ac, γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc, and γGlu-OEG-OEG-εLys-αAc, but any suitable linker may be used.

[0062] Fatty acids, or linkers, can bind to any amino acid residue in the IL-22 protein. Examples in this regard are residues -7, -5, 1, 6, 33, 113, 114, and 153 in or relative to the hIL-22 amino acid sequence. Native residues are typically substituted with Cys or Lys to allow for the binding of fatty acids or linkers. Alternatively, fatty acids or linkers may be bound to native Cys or Lys residues. Preferably, fatty acids or linkers can bind to Cys residues substituted at positions 1, 6, 33, 113, or 114 of hIL-22, or to Cys residues at positions -5, -7, or 153 relative to hIL-22. In particular, fatty acids or linkers can bind to Cys residues substituted at position 1 of hIL-22.

[0063] The binding of fatty acids or linkers to the IL-22 protein is covalent. For example, Cys-reactive fatty acids or linkers can be used to bind fatty acids or linkers to Cys residues in the IL-22 protein. The fatty acids or linkers may be covalently bonded to the sulfur atom of the Cys residue via a thioether bond. For example, Lys-reactive fatty acids or linkers can be used to bind fatty acids or linkers to Lys residues in the IL-22 protein. Alternatively, fatty acids or linkers may be covalently bonded to the free amine (-NH2) group at the N-terminus of the IL-22 protein (regardless of the amino acid at position 1). The binding may proceed in the same way as Cys binding, using a fatty acid or linker containing a quasi-stoichiometric amount of a suitable N-reactive species. Fatty acids or linkers may be provided in the form of an aldehyde (N-reactive species) and may be covalently bonded to the free amine using conventionally known reductive amination.

[0064] Therefore, the derivative of the first embodiment comprises a C14, C16, C18, or C20 diacid linked to a variant of hIL-22 by a linker, wherein the variant preferably contains an N-terminal GPG and a Cys residue at position 1 of hIL-22, and the linker is optionally linked to the Cys residue.

[0065] Exemplary derivatives of the first embodiment include the IL-22 protein represented by any of SEQ ID NOs: 16-21. Particularly advantageous derivatives are shown in Table 3 and illustrated in Figures 1-4, and are exemplified herein.

[0066] [Table 3]

[0067] Figure 1A shows a C18 diacid linked to a linker containing Cys-reactive units. This is the fatty acid and linker (side chain) used in derivatives 1, 2, and 6-9. Figure 1B shows a C16 diacid linked to a linker containing Cys-reactive units. This is the fatty acid and linker (side chain) used in derivatives 3, 4, and 10. Figure 1C shows a C14 diacid linked to a linker containing Cys-reactive units. This is the fatty acid and linker (side chain) used in derivative 5.

[0068] Derivatives 1, 6, and 10 are shown in Figures 2 to 4, respectively.

[0069] The derivatives of the present invention may exist in different stereoisomers, and the present invention relates to all of these.

[0070] A second aspect of the present invention provides a process for preparing a derivative of the first aspect, the process comprising covalently bonding a fatty acid to the IL-22 protein.

[0071] The process can be used to produce any of the different derivatives of IL-22 described or assumed herein, but it is particularly advantageous when a fatty acid is covalently bonded to the variant IL-22 protein. Thus, in one embodiment, the IL-22 protein used in the second embodiment is a substituted form of hIL-22, optionally substituted at positions 1, 21, 35, 64, 113 and / or 114. Exemplary substitutions include A1C, A1G, A1H, N21C, N21D, N21Q, N35C, N35D, N35H, N35Q, N64C, N64D, N64Q, N64W, Q113C, Q113R, K114C and / or K114R. Preferably, the IL-22 protein is substituted with a Cys residue at position 1.

[0072] Fatty acids can be obtained by any means known in the art, including recombinant methods. Preferred fatty acids are derived from commercially available or readily available starting materials using standard chemical synthesis.

[0073] The IL-22 protein can be obtained by any means known in the art, including recombinant methods. The production of recombinant hIL-22 has been previously described and is well known in the art. A desired variant IL-22 protein can be produced by similar methods. A skilled researcher in the art will be able to readily identify a suitable nucleic acid sequence encoding a desired variant IL-22 protein. Thus, a person skilled in the art will be able to readily carry out this part of the invention based on existing knowledge in the art. Preferably, the IL-22 protein is produced in a mammalian system, for example, in Chinese hamster ovary (CHO) cells, using standard techniques. A polyhistidine tag (His-tag) may be used to assist in the affinity purification of the recombinant protein.

[0074] In this regard, the IL-22 protein used in the present invention may be prepared by adding -N- or C-terminus fewer than 10, preferably 6, histidine residues, which can be purified by affinity to a post-expression cleavable His-tag-nickel column. The His-tag is ligated to the N-terminus or C-terminus of the protein via a linker which can be digested by a known protease to yield the free IL-22 protein. The cleavable His-tag may have the amino acid sequence HHHHHHGGSSGSGSEVLFQ (SEQ ID NO: 25), and the protease-cleavable linker may be a tobacco etch virus (TEV) linker (with a consensus sequence for the native cleavage site being ENLYFQ\S (SEQ ID NO: 26)) (where "\" indicates the peptide bond to be cleaved) or a human rhinovirus-14 3C (HRV14-3C) protease-cleavable linker having an EVLFQ consensus cleavage site. Cleavage can be achieved by incubating approximately 10 μg of protease with 2.5 μg of protein and 10 mM 2-mercaptoethanol at room temperature for 4 hours.

[0075] To further illustrate the present invention, a representative process for protein preparation is provided below. The process involves preparing plasmid DNA encoding a desired amino acid sequence of the IL-22 protein. This plasmid can be transiently transfected into a cell line, e.g., CHO-K1, which is grown in a relevant medium before growth is increased by the addition of an enhancer of known properties. The secreted IL-22 protein can then be collected by known methods of centrifugation and sterile filtration, after which the protein is purified on a nickel column. After concentration and buffer exchange, the His-tag is removed using HRV14-3C protease, followed by alkylation with fatty acids (as further described below), as well as final purification and buffer exchange. Analysis of the final product using SDS-PAGE, size exclusion chromatography, or liquid chromatography with or without deglycosylation (LC-MS-MS) may be used to ensure the quality of the final product.

[0076] The fatty acid may be covalently bonded to the IL-22 protein directly or using a linker, as described in the first embodiment. The linker may be obtained by any means known in the art. Typical methods for preparing the fatty acid and linker (if used) are as follows (exemplified by the C16 diacid used in derivative 10, but any derivative may be prepared using similar methods):

[0077] A solution of N-(benzoyloxycarbonyloxy)succinimide (100 g, 401 mmol) in dichloromethane (500 ml) is added to a solution of ethylenediamine (189 ml, 2.81 mol) in dichloromethane (750 ml). After 30 minutes, the suspension is filtered, washed, and concentrated under vacuum. The residue is diluted with toluene (750 ml), washed, extracted with dichloromethane (4 × 200 ml), dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and diluted with hexane (200 ml). A 4 M solution of hydrochloric acid in ether (100 ml, 400 mmol) is added to the solution, the resulting suspension is concentrated under vacuum, and diluted with hexane (1 l). The precipitated solid is filtered, washed with hexane, and dried under vacuum to obtain (2-aminoethyl)carbamate benzyl hydrochloride as a white powder.

[0078] {2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}acetic acid (Fmoc-Ado-OH, 17.5 g, 45.4 mmol) is loaded onto 100-200 ml of 2-chlorotrityl resin. The Fmoc group is removed, and a solution of 0-6-chloro-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU, 24.2 g, 68.1 mmol) and N,N-diisopropylethylamine (21.4 ml, 123 mmol) in N,N-dimethylformamide (140 ml) is added to the resin, and the mixture is shaken for 1 hour. The resin is filtered and washed. The Fmoc group is removed by treatment with 20% piperidine as described above. The resin is washed as described above.

[0079] A solution of (S)-2-(fluorene-9-ylmethoxycarbonylamino)-pentanedioic acid 1-tert-butyl ester (Fmoc-Glu-OtBu, 29.0 g, 68.1 mmol), TCTU (24.2 g, 68.1 mmol), and N,N-diisopropylethylamine (21.4 ml, 123 mmol) in N,N-dimethylformamide (140 ml) is added to the resin, and the mixture is shaken for 1 hour. The resin is filtered and washed as described above. The Fmoc group is removed by treatment with 20% piperidine as described above. The resin is washed as described above.

[0080] A solution of 16-tert-butoxy)-16-oxohexadecanoic acid (23.3 g, 68.1 mmol), TCTU (24.2 g, 68.1 mmol), and N,N-diisopropylethylamine (21.4 ml, 123 mmol) in an N,N-dimethylformamide / dichloromethane mixture (4:1, 200 ml) is added to the resin. The resin is shaken for 1 hour, filtered, and washed with N,N-dimethylformamide (3 × 250 ml), dichloromethane (2 × 250 ml), methanol (2 × 250 ml), and dichloromethane (6 × 250 ml). The product is cleaved from the resin by treatment with 2,2,2-trifluoroethanol (250 ml) for 18 hours. The resin is filtered and washed with dichloromethane (2 x 250 ml), 2-propanol / dichloromethane mixture (1:1, 2 x 250 ml), 2-propanol (250 ml), and dichloromethane (3 x 250 ml).

[0081] The solutions are combined, the solvent is evaporated, and the crude product is purified by flash column chromatography. Pure (S)-22-(tert-butoxycarbonyl)-41,41-dimethyl-10,19,24,39-tetraoxo-3,6,12,15,40-pentaoxa-9,18,23-triazadotetracontanoic acid is dried under vacuum to obtain a pale yellow, highly viscous yellow oil.

[0082] Subsequently, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU, 11.4 g, 30.1 mmol) and triethylamine (8.77 ml, 62.9 mmol) are added to a solution of (S)-22-(tert-butoxycarbonyl)-41,41-dimethyl-10,19,24,39-tetraoxo-3,6,12,15,40-pentaoxa-9,18,23-triazodotetracontanoic acid (22.4 g, 27.4 mmol) in dry dichloromethane (110 ml). Triethylamine (72 ml, 41.0 mmol) is added to a suspension of (2-amino-ethyl)-carbamate benzyl hydrochloride (6.94 g, 30.1 mmol) in dry dichloromethane (165 ml), and the resulting mixture is added to the above solution. The mixture is stirred at room temperature overnight, and then evaporated until dry. The residue is redissolved and washed; dried over anhydrous sodium sulfate and evaporated by column chromatography (silica gel 60, 0.040-0.060 mm; eluent: dichloromethane / methanol 95:5) to obtain 15-[(S)3-(2-{2-[(2-{2-[(2-benzyloxycarbonylaminoethylcarbamoyl)-methoxy]-ethoxy}ethyl-carbamoyl)methoxy]ethoxy)-ethylcarbamoyl)-1-tert-butoxycarbonylpropylcarbamoyl]-pentadecanoate tert-butyl ester as a pale yellow, highly viscous oil.

[0083] Palladium-supported carbon (10%, 1.27 g, 1.20 mmol) is added to a solution of the above compound (23.8 g, 24.0 mmol) in methanol (350 ml), and the resulting mixture is hydrogenated at standard pressure for 4 hours. The catalyst is filtered, and the filtrate is evaporated until dry. The residue is evaporated several times from dichloromethane to remove methanol residue, and dried in vacuum to produce tert-butyl(S)-1-amino-25-tert-butoxycarbonyl)-4,13,22,27-tetraoxo-6,9,15,18-tetraoxa-3,12,21,26-tetraazadotetracontane-42-oate as a highly viscous, colorless oil.

[0084] N,N-diisopropylethylamine (4.98 ml, 28.6 mmol) is added to a solution of the above amine (20.5 g, 23.8 mmol) in dry dichloromethane (290 ml) under argon at -30°C. Bromoacetyl bromide (2.48 ml, 28.6 mmol) is added dropwise, and the resulting solution is stirred for a further 3 hours at -30°C. The cooling bath is removed, the mixture is stirred at room temperature for 1 hour, and the solvent is removed under vacuum. The residues are redissolved in ethyl acetate (450 ml) and washed with a 5% aqueous solution of citric acid (300 ml). The phases are separated within 1 hour. The organic layer is left overnight to separate and obtain three layers. The clear aqueous layer is removed, and the remaining two phases are shaken with saturated aqueous solution of potassium bromide (100 ml). The organic layer is left overnight to separate, the aqueous phase is removed, and the organic phase is dried over anhydrous sodium sulfate. The solvent is removed under vacuum, and the residue is purified by flash column chromatography (dichloromethane / methanol 95:5) to obtain tertbutyl(S)-1-bromo-28-tert-butoxycarbonyl)-2,7,16,25,30-pentaoxo-9,12,18,21-tetraoxa-3,6,15,24,29-pentazapentatetracontane-45-oate as a colorless solid.

[0085] Dissolve the above compound (19.5 g, 19.8 mmol) in trifluoroacetic acid (120 ml), and stir the resulting solution at room temperature for 1.5 hours. Remove the trifluoroacetic acid under vacuum, and evaporate the residue from dichloromethane (6 × 200 ml). Add diethyl ether (200 ml) to the oily residue, and stir the mixture overnight to obtain a suspension. Filter the solid product, wash with diethyl ether and hexane, and dry under vacuum to obtain the desired product 15-{(S)-1-carboxy3-[2-(2-{[2-(2-{[2-(2-bromoacetylamino)ethylcarbamoyl]methoxy}-ethoxyethyl-carbamoyl]methoxy}ethoxyl-ethylcarbamoyl]propylcarbamoyl}pentadecanoic acid as a white powder.

[0086] The covalent bonding of fatty acids or linkers to the IL-22 protein can be carried out using standard procedures in the art. Therefore, linkers, when used, enable the covalent bonding of the IL-22 protein to fatty acids. As a non-limiting example, a Cys-reactive fatty acid or linker may be reacted with the sulfur atom of a Cys residue in the IL-22 protein, thereby forming a thioether bond. Suitable conditions for the covalent bonding step can be exemplified as follows: Add Tris in water to IL-22 protein (70 mg) in Tris and NaCl buffer (1.35 mg / ml) to adjust the pH to 8. Add bis(p-sulfonatophenyl)-phenylphosphine dihydrate dipotassium (BSPP) salt (12 mg) dissolved in water and stir gently at room temperature for 4 hours. Add 15-{(S)-1-carboxy-3-[2-(2-{[2-(2-{[2-(2-(2-bromoacetylamino)-ethylcarbamoyl]ethoxy}ethoxy)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]propyl-carbamoyl}pentadecanoic acid (19 mg, 0.022 mmol) to ethanol (0.5 ml) and stir the mixture overnight. Add MiliQ water (150 ml) to reduce the conductivity to 2.5 mS / cm. Then purify the mixture by anion exchange on a MonoQ 10 / 100 GL column using binding buffer (20 mM Tris, pH 8.0), elution buffer (20 mM Tris, 500 mM NaCl, pH 8.0), flow 6 ml and column volume with a gradient of elution buffer from 0 to 80%.

[0087] The derivatives of the present invention can be purified using any preferred procedure known in the art, such as chromatography, electrophoresis, solubility differential, or extraction.

[0088] As described herein, the inventors have surprisingly found that fatty acids were able to covalently bind to the IL-22 protein while maintaining its biological activity. Particularly surprising was that such minimal modification to IL-22 resulted in high potency (close to that of hIL-22) combined with an extremely long cyclic half-life. This particular combination of properties may be highly desirable.

[0089] The potency of the derivatives can be determined by in vitro assays using whole cells expressing the human IL-22 receptor. For example, the response of the human IL-22 receptor can be measured using baby hamster kidney (BHK) cells overexpressing IL-22R1, IL-10R2, and phospho-STAT3 (pSTAT3) reactive reporter genes. Alternatively, HepG2 cells endogenously expressing the IL-22 receptor can be used. Receptor activation leads to activation of the STAT3 signaling pathway, which can be measured, for example, by assaying pSTAT3 using a luciferase reporter gene with a STAT3-inducible promoter. Non-limiting examples of such assays are described in Example 2. In vivo potency can be determined in animal models or clinical trials, as is known in the art.

[0090] Maximum half-dose effective concentration (EC 50 The EC2 value is often used as an indicator of drug potency. It represents the concentration of the drug required to produce 50% of its maximum effect, so 50 The lower the value, the better the potency. The derivatives of the present invention preferably have potency (EC) measured using IL-22 receptor-mediated STAT3 activation in cells, with values ​​of less than 1.5 nM, less than 1.25 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, or even less than 0.1 nM. 50having a value) (determined as described in Example 2, for example). The derivative of the present invention preferably has an efficacy (EC measured by assaying pSTAT3 in cells that is less than 15 nM, less than 12 nM, less than 10 nM, less than 7 nM or even less than 5 nM 50 having a value) (determined as described in Example 2, for example).

[0091] Advantageously, the efficacy of the derivative of IL-22 may be higher than that of the IL-22-Fc fusion. For example, Genentech reported a 1 / 34 reduction in in vitro efficacy for its IL-22-Fc fusion, UTTR1147A, compared to hIL-22 (Stefanich et al., Biochem Pharmacol, 2018, 152:224-235). On the other hand, covalent attachment of a fatty acid to hIL-22 was shown to cause only a 1 / 7 reduction in efficacy (see Derivative 1 in the examples). Both the IL-22-Fc fusion and the derivative of the present invention may be equivalent in terms of their improved half-life compared to hIL-22 and biological function in at least some situations, but the derivative of the present invention may have the additional advantage of minimal loss of efficacy.

[0092] The circulating disappearance half-life (T 1 / 2The circulating half-life (HA) of the derivative can be determined in vivo by administering the derivative subcutaneously or intravenously in a suitable animal model such as a mouse, rat, or miniature pig. A preferred method is described in Example 1. As a non-limiting example, the derivative of the first embodiment has a circulating half-life of at least 1 hour, at least 3 hours, at least 5 hours, or at least 8 hours after subcutaneous or intravenous administration to a mouse. The derivative may have a circulating half-life of at least 3 hours, at least 5 hours, at least 8 hours, at least 10 hours, or at least 13 hours after subcutaneous or intravenous administration to a rat. The derivative may have a circulating half-life of at least 25 hours, at least 40 hours, at least 70 hours, or at least 100 hours after subcutaneous or intravenous administration to a miniature pig (all are determined, for example, as described in Example 1).

[0093] As illustrated herein, the inventors have also found that derivatives of the present invention are rapidly absorbed in vivo. Advantageously, the absorption of derivatives may occur faster than that of the IL-22-Fc fusion. The mean absorption time is a precise parameter for measuring uptake, because it is independent of the dose and the maximum plasma concentration after drug administration. It can be calculated based on the mean residence time, i.e., the time the drug is present in the body after absorption is complete and before excretion. Derivatives of the present invention preferably have mean absorption times of less than 100 hours, less than 90 hours, less than 80 hours, less than 70 hours, or even less than 60 hours (determined, for example, as described in Example 1).

[0094] The derivatives of the present invention also possess good biophysical properties, such as high physical stability and / or solubility, which can be measured using standard methods in the art.

[0095] Therefore, according to a third aspect of the present invention, a pharmaceutical composition is provided comprising a derivative of the first aspect and a pharmaceutically acceptable vehicle.

[0096] A third embodiment of the pharmaceutical composition may comprise any of the different derivatives of IL-22 described or assumed herein. Preferably, it comprises one of the derivatives of IL-22 identified herein as derivatives 1 to 10.

[0097] The derivatives of the first embodiment, or the pharmaceutical compositions of the third embodiment, preferably exhibit an increased cyclic elimination half-life compared to hIL-22. Advantageously, it exhibits a cyclic elimination half-life that is at least 50%, at least 75%, at least 100%, or more than 50%, 75%, 100%, or more compared to hIL-22.

[0098] A third embodiment of the pharmaceutical composition may be prepared by combining a therapeutically effective amount of the derivative of the first embodiment with a pharmaceutically acceptable vehicle. Formulation of pharmaceutically active ingredients with various excipients is known in the art.

[0099] The “therapeutic effective dose” of the derivative in the first embodiment is any amount of the derivative that, when administered to a subject, is required to treat a disease, disorder or condition or to produce a desired effect.

[0100] For example, the therapeutically effective dose of the derivative used may be about 0.001 mg to about 1000 mg, preferably about 0.01 mg to about 500 mg. The amount of the derivative is preferably about 0.1 mg to about 100 mg, most preferably about 0.5 mg to about 50 mg. As a guideline, the dose of the derivative used in mice in Example 3 described herein was 0.5 mg / kg (administered subcutaneously).

[0101] Where used herein, "pharmaceutically acceptable vehicle" is any known compound or combination of known compounds that is known to those skilled in the art to be useful in the formulation of a pharmaceutical composition.

[0102] In one embodiment, the pharmaceutically acceptable vehicle may be a solid; optionally, the composition may be in the form of a powder for resuspension. A solid pharmaceutically acceptable vehicle may contain one or more substances that can also act as flavoring agents, lubricants, solubilizers, suspending agents, colorants, fillers, flow promoters, inert binders, preservatives, or pigments. The vehicle may also be an encapsulating substance. In powder form, the vehicle is a finely ground solid mixed with a finely ground derivative according to the present invention. The powder preferably contains up to 99% derivative. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, and ion exchange resins.

[0103] In another embodiment, the pharmaceutical vehicle may be a gel, and the composition may be in the form of a cream or the like.

[0104] However, the pharmaceutical vehicle may be a liquid; optionally, the pharmaceutical composition may be in the form of a solution. Liquid vehicles are used in the preparation of solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The derivatives according to the present invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture thereof, or a pharmaceutically acceptable oil or fat. The liquid vehicle may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity modifiers, stabilizers or osmotic pressure modifiers. Suitable examples of liquid vehicles for parenteral administration include water (partially containing the above additives, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle may also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in compositions in sterile liquid form for parenteral administration. For pressurized compositions, the liquid vehicle may be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0105] Therefore, the process for preparing the pharmaceutical composition of the present invention may include common steps that are standard in the art.

[0106] Therefore, according to a fourth aspect of the present invention, a derivative of the first aspect or a pharmaceutical composition of the third aspect is provided for use in therapy. A method of treating a subject with the derivative of the present invention or a pharmaceutical composition containing the same is also provided. Any of the different derivatives of IL-22 described or assumed herein are expressly included in these aspects of the present invention.

[0107] As used herein, terms such as “treatment” and “therapy” expressly include the treatment, improvement, or prevention of a disease, disorder, or condition.

[0108] IL-22 derivatives or pharmaceutical compositions containing them may be administered directly to the subject to be treated. The derivatives or pharmaceutical compositions may be administered by any means, including inhalation, injection, topical application, or ophthalmic application. When administered by inhalation, it may be via the nose or mouth. Preferably, the derivatives or pharmaceutical compositions are administered by injection, typically subcutaneously or intravenously. Therefore, the derivatives have a clear advantage over the Fc fusion in their flexibility of administration (e.g., injection, inhalation, topical application, or ophthalmic delivery) due to their smaller size and higher potency. It is understood that administration of the derivatives of the present invention to the subject to be treated results in increased circulation time compared to hIL-22, and this aids in the treatment of diseases, disorders, or conditions. As stated above, “treatment” also includes improving and preventing diseases, disorders, or conditions.

[0109] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be used, for example, by intramuscular, intrathecal, epidural, intraperitoneal, and especially subcutaneous or intravenous injection. Derivatives can be prepared as sterile solid compositions that can be dissolved or suspended at the time of administration using sterile water, saline, or other suitable sterile injection media.

[0110] Forms useful for inhalation include sterile solutions, emulsions, and suspensions. Alternatively, derivatives may be administered in the form of a fine powder or aerosol via Dischaler® or Turbohaler®. Nasal inhalants may preferably be in the form of a fine powder or aerosol nasal spray or a modified Dischaler® or Turbohaler®.

[0111] Topical preparations include solutions, creams, foams, gels, lotions, ointments, pastes, tinctures, and powders. They may be applied transdermally, that is, directly to the skin or to mucous membranes.

[0112] Formulations for ocular administration are typically in the form of solutions, suspensions, and ointments for topical application, such as eye drops. Alternatively, sterile solutions or suspensions may be used by intraocular injection. Derivatives may be prepared as sterile solid compositions that can be dissolved or suspended at the time of administration using sterile water, saline solution, or other suitable sterile injection medium. Formulations may be for subconjunctival, intravitreous, retrobulbar, or anterior chamber injection.

[0113] The derivatives or pharmaceutical compositions for the present invention may be administered to any subject that requires them. “Subject” as used herein may be a vertebrate, mammal, or livestock. Thus, the derivatives and compositions according to the present invention may be used to treat any mammal, e.g., livestock (e.g., horses), pets, or for other veterinary purposes. Most preferably, the subject is human. The derivatives and compositions do not need to be administered only to those already showing signs of disease, disorder, or pathology. Rather, they may be administered to obviously healthy subjects as a purely prophylactic measure against the potential for such disease, disorder, or pathology in the future.

[0114] It is understood that the IL-22 derivatives and compositions according to the present invention may be used as monotherapy (i.e., the use of the derivative or composition alone) to treat a disease, disorder, or condition. Alternatively, the derivatives and compositions according to the present invention may be used as adjuncts to or in combination with known therapies for treating a disease, disorder, or condition.

[0115] The required amount of IL-22 derivative is determined by its biological activity, half-life, and bioavailability, which in turn depends on the mode of administration, the physiological and chemical properties of the derivative and composition, and whether it is used as monotherapy or in combination therapy. The frequency of administration may also be influenced by the half-life of the derivative in the subject being treated. The optimal dosage to be administered can be determined by those skilled in the art and varies depending on the strength of the particular derivative and pharmaceutical composition in use, the mode of administration, and the progression of the disease, disorder, or condition. Further factors specific to the subject being treated, including age, weight, sex, diet, and timing of administration, may necessitate adjustments to the dosage.

[0116] Typically, a daily dose of an IL-22 derivative according to the present invention, ranging from 0.001 μg / kg body weight to 10 mg / kg body weight, may be used to treat a disease, disorder, or condition, depending on which derivative or composition is used. More preferably, the daily dose is between 0.01 μg / kg body weight and 1 mg / kg body weight, more preferably between 0.1 μg / kg and 500 μg / kg body weight, and most preferably between approximately 0.1 μg / kg and 100 μg / kg body weight.

[0117] IL-22 derivatives or compositions may be administered before, during, or after the onset of a disease, disorder, or condition. The daily dose may be provided as a single dose (e.g., a single daily injection). Alternatively, the derivative or composition may require administration two or more times a day. For example, the derivative may be administered in two (or more) daily doses between 0.07 μg and 700 mg (i.e., assuming a body weight of 70 kg) depending on the severity of the disease, disorder, or condition being treated. The patient receiving treatment may take the first dose upon waking, then the second dose in the evening (in the case of a two-dose regimen) and thereafter at intervals of 3 or 4 hours. The dose may alternatively be provided once a week, every two weeks, or once a month, or more frequently, for example, two or three times a week. Known procedures, such as those conventionally used in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), can be used to generate specific formulations and precise therapeutic regimes (e.g., daily doses and frequencies of administration) of the derivatives and compositions according to the present invention.

[0118] Numerous studies have demonstrated the significant effects of IL-22 in multiple epithelial injury models, particularly in the lungs, liver, intestines, kidneys, skin, pancreas, and thymus. Mechanistically, several pathways, such as anti-apoptosis, proliferation, innate immunity, antioxidant stress, anti-fibrosis, and stem cell / progenitor cell recruitment, have been well-documented in multi-investigator studies to mediate the effects of IL-22. Further key mechanistic insights have been confirmed in vitro using human cell lines or in human ex vivo models (e.g., primary human intestinal organoids). Thus, the potent role of IL-22 in preventing cell death, ensuring regeneration, and controlling inflammation in epithelial injury is well-established.

[0119] Many studies are conducted by analyzing genetic models subjected to injury (IL-22 knockout or transgenic overexpression). In these studies, IL-22 deficiency or overexpression is present at the time of injury. In other studies, IL-22 is neutralized with antibodies at the time of injury, and in some cases, IL-22 is neutralized beyond the acute injury phase (e.g., subacute or well into the regenerative phase). Other studies are getting closer to treatment scenarios by examining the effects of exogenously administered IL-22. A comprehensive review of the available literature reveals that it is important to note that the different models, whether knockout, overexpression, pre- or post-injury IL-22 neutralization, or exogenous protein administration, represent the same entity of IL-22 that protects injured organs and promotes regeneration. This indicates a broad applicability and wide time window for IL-22 treatment potential and also explains why IL-22 proteins that act longer than hIL-22 are needed.

[0120] Accordingly, according to a fifth aspect of the present invention, a derivative of the first aspect or a pharmaceutical composition of the third aspect is provided for use in a method of treating metabolic, hepatic, pulmonary, intestinal, renal, or skin diseases, disorders, or conditions. Any of the different derivatives of IL-22 described or assumed herein are expressly included in this aspect of the present invention.

[0121] Metabolic disorders, conditions, or pathologies may include obesity, type 1 diabetes, type 2 diabetes, dyslipidemia, hyperglycemia, or hyperinsulinemia.

[0122] Liver disease, disorder, or condition may be NAFLD, NASH, cirrhosis, alcoholic hepatitis, acute liver failure, chronic liver failure, ACLF, acetaminophen-induced hepatotoxicity, acute liver injury, sclerosing cholangitis, biliary cirrhosis, or a pathological condition caused by surgery or transplantation.

[0123] Lung diseases, disorders, or conditions may include COPD, cystic fibrosis, bronchiectasis, idiopathic pulmonary fibrosis, acute distress syndrome, chemical injury, viral infection, bacterial infection, or fungal infection.

[0124] Intestinal diseases, disorders, or conditions may include IBD, ulcerative colitis, Crohn's disease, GvHD, chemical injury, viral infection, or bacterial infection.

[0125] Kidney disease, disorder, or condition may be acute kidney disease or chronic kidney disease.

[0126] Skin diseases, disorders, or conditions may include wounds, inflammatory diseases, or GvHD.

[0127] A method is also provided for treating a subject with a IL-22 derivative or a pharmaceutical composition containing an IL-22 derivative, for example, a subject having one or more of the above-mentioned diseases, disorders, or conditions that are responsive to IL-22 treatment.

[0128] A derivative of IL-22 has all the features specified in the first aspect of the present invention. A pharmaceutical composition has all the features specified in the third aspect of the present invention. A method for treating a subject having a condition responsive to IL-22 treatment, such as one or more of the above-mentioned diseases, disorders, or conditions, has all the features specified in the fourth aspect of the present invention.

[0129] There are no restrictions on which IL-22 derivatives or compositions described herein should be administered to which patients. Rather, it is intended that any of the derivatives and compositions described herein may be administered to any of the patients described herein.

[0130] All of the features described herein (including any appended claims, abstract and drawings) and / or all of the steps of any methods or processes disclosed herein may be combined in any combination with any of the above embodiments, except in any combination in which at least part of such features and / or steps are mutually exclusive.

[0131] Examples will be referenced hereafter to provide a better understanding of the present invention and to show how the same embodiments can be realized, but no limitation of the present invention is intended in any way. [Examples]

[0132] Unless otherwise specified, the substances and methods used in the studies described in the examples are as follows:

[0133] derivative Table 4 provides an overview of IL-22 derivatives and comparative products represented in the dataset.

[0134] The derivatives of IL-22 exhibit a variety of skeletons, fatty acid types, and covalent bonding sites, thus representing the diversity of derivatives encompassed in this invention. In all cases, the linker used was γGlu-OEG-OEG-C2DA-Ac. In all cases, the linker was attached to residue 1C, except for derivatives 2(-7C), 4(-7C), 8(6C), and 9(33C). Derivative 7 exemplifies covalent bonding at 1C, while it lacks the GPG N-terminal peptide present in all other derivatives with covalently bonded fatty acids at 1C.

[0135] The comparison subjects were hIL-22, hFc-hIL-22 (recombinant fusion protein), and hIL-22 variants (i.e., hIL-22 having only one or more skeletal variations).

[0136] [Table 4]

[0137] The following quality control analysis was performed on the generated derivatives in the example.

[0138] The intact mass of the protein was determined by adding 20 μl of 1 mg / ml sample to 2 μl of N-glycosidase F at room temperature for 48 hours in the deglycosylated sample. The sample was then diluted to 0.2 mg / ml at pH 7.4 with PBS and analyzed using a Synapt G2 connected to a Waters Synapt G2 equipped with a Waters MassLynx 4.1. A 10-90 Column Acquity UPLC Protein BEH C4 1.7 μm 1x100 mm was used as the following mobile phases: A: 0.1% formic acid in water; and B: acetonitrile, 0.09% formic acid. The flow rate was 120 μl / min, UV 214 nm (20 pts / s), and the gradient was as shown in Table 5.

[0139] [Table 5]

[0140] The results are shown in Table 6.

[0141] [Table 6]

[0142] Therefore, the quality control data demonstrated that the intended derivative was indeed produced.

[0143] The following is an illustrative protocol intended solely to illustrate the claimed invention. The animals used in the study and the exact time elapsed may be modified as known to those skilled in the art.

[0144] Example 1 - Pharmacokinetic Study method Pharmacokinetic studies were conducted on selected derivatives in mice (n=27), rats (n=4-8), and miniature pigs (n=2-5). IL-22 derivatives were tested in parallel with hIL-22, hFc-hIL-22, and / or hIL-22 variants as comparators.

[0145] (i) Mice and rats Thirty 8-week-old C57Bl / 6 male mice and five Sprague Dawley male rats were obtained from Taconic Biosciences. The mice were housed in groups of 10. The animals were acclimatized for one week prior to the experiment. Pre-dose body weight was measured, which is important for pharmacokinetic calculations. The animals had access to food and water and remained awake throughout the experiment.

[0146] All derivatives and comparators were prepared as a 0.3 mg / ml solution in PBS, pH 7.4, for use in mice, and as a 0.5 mg / ml solution for use in rats. A dose of 2.0 mg / kg was tested in mice, and a dose of 1 mg / kg was tested in rats.

[0147] Derivatives and comparators were administered subcutaneously to animals. Blood samples were collected at specific time points after administration.

[0148] Spurge sampling was used in mice; therefore, 27 mice were administered IL-22 derivatives or comparators, and blood samples were taken from three different mice at each of the following time points: 5 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 150 minutes, 3 hours, 4 hours, 6 hours, 8 hours, 16 hours, 24 hours, 32 hours, and 48 hours. Thus, only two samples were taken from each mouse during the course of the study. After the last sample was taken, the mice were euthanized by cervical dislocation.

[0149] Five rats were administered IL-22 derivatives or comparators, and three blood samples were taken at each of the following time points: 5 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 150 minutes, 3 hours, 4 hours, 6 hours, 8 hours, and 24 hours. A total of 17 samples were taken from each rat during the course of the study. After the last sample was taken, the rats were euthanized with carbon dioxide.

[0150] Blood samples (100 μl) were collected from mice and rats by tongue blood collection and transferred to EDTA tubes (Microvette® VetMed 200 K3E, Sarstedt nr 09.1293.100). Within 20 minutes of collection, the blood was centrifuged at 8000G at 4°C for 5 minutes. Plasma samples (40-50 μl) were transferred to half-micronic tubes.

[0151] (ii) Miniature pig Nine-month-old female Gottingen minipigs weighing approximately 15 kg were obtained from Ellegaard Gottingen Minipigs A / S. An acclimatization period of approximately 18 days was provided before surgery (catheter insertion), during which the minipigs were socialized and trained for subcutaneous drug administration and blood sampling via catheter. Three to five days before surgery, the minipigs were kept individually. Six days before drug administration, two central venous catheters (Cook Medical, C-TPNS-6.5-90-REDO, silicon, size 6.5 French, 106 cm long type TPN) were inserted into all minipigs, thereby providing a recovery period after surgery at least five days before the start of the study (drug administration).

[0152] All derivatives and comparative compounds were prepared as solutions in PBS, pH 7.4. The doses used were 0.1 mg / kg (intravenous administration) or 0.2 mg / kg (subcutaneous administration).

[0153] The miniature pigs were lightly anesthetized with propofol during administration. Intravenous injection was administered to the miniature pigs via a long central catheter. After administration, the catheter was flushed with 10 ml of sterile saline. Subcutaneous injection was performed to a depth of 5 mm using a 25 G needle. The needle was held in the skin for 10 seconds after injection to avoid reflux.

[0154] Blood samples were taken from miniature pigs at the following time points after intravenous administration: 1.5 hours, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 28 hours, 48 ​​hours, 72 hours, 96 hours, 144 hours, 168 hours, 192 hours, 216 hours, 240 hours, 264 hours, 312 hours, 336 hours, 360 hours, 384 hours, 408 hours, 432 hours, and 480 hours. Blood samples were collected at the following time points after subcutaneous administration: 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 46 hours, 52 hours, 72 hours, 96 hours, 144 hours, 168 hours, 192 hours, 216 hours, 240 hours, 264 hours, 312 hours, 336 hours, 360 hours, 384 hours, 408 hours, 432 hours, and 480 hours.

[0155] Blood samples (1 ml) were collected from miniature pigs into EDTA tubes (1.3 ml tubes containing K3EDTA (Sarstedt, Germany) to yield 1.6 mg K3EDTA / ml blood). Samples were maintained on moist ice for up to 30 minutes before centrifugation (10 minutes, 4°C, 2000 G). 200 μl of plasma was transferred to micronic tubes for measurement of IL-22 derivatives or comparators and stored at -20°C until analysis.

[0156] (iii) Sample processing Plasma levels of IL-22 derivatives or comparators were measured using the previously described in-house developed luminescent oxygen channeling (LOCI®) assay (Poulsen et al., J Biomol Screen, 2007, 12(2):240-7). During the assay, concentration-dependent bead-analyte-immunocomplexes were formed, generating a photooutput which was measured using a Perkin Elmer Envision reader. Antibody coupling to beads, antibody biotinylation, and the LOCI assay procedure were performed as previously described (Petersen et al., J Pharmaceut Biomed, 2010, 51(1):217-24). Calibrator and quality control (QC) samples were generated using the same matrix as the study samples. Assay precision (%CV) was assayed and shown to be less than 20% for all tested samples.

[0157] The assay used acceptor beads conjugated with an anti-human IL-22 monoclonal antibody (R&D Systems MAB7822) along with donor beads coated with a biotinylated monoclonal antibody (R&D Systems BAM7821; generated against human IL-22) and unbranded streptavidin. The lower limit of quantification (LLOQ) for human IL-22 in rat plasma was 4 pM. However, each derivative or comparator was measured against the same derivative calibrator series. Cross-reactivity of each derivative or comparator against hIL-22 was measured and used to adjust assay sensitivity.

[0158] Plasma concentration-time profiles were measured in miniature pigs using non-compartmental analysis (NCA) with Phoenix WinNonlin Professional 6.4 (Pharsight Inc). Individual concentrations were used, metered by 1 / (Y*Y), and calculations were performed using a linear logarithmic trapezoid. Circulation elimination half-life (T) 1 / 2Intravenous administration was used because ) was the primary screening parameter. Clearance and volume of distribution were secondary parameters to be considered, and therefore the reason for the multiple blood samples on day 1 of the study.

[0159] The only parameter measured to evaluate pharmacokinetics in mice and rats was the circulating elimination half-life (T). 1 / 2 In miniature pigs, the additional parameter measured was the maximum (peak) plasma concentration (C) after drug administration. max ), C max Time to reach (T max The metrics included the area under the plasma drug concentration-time curve (AUC; reflecting the actual physical exposure to the drug after administration of the drug dose) (AUC / D), normalized for the drug dose, mean residence time (MRT; i.e., the time the drug is present in the body after absorption is complete and before excretion), mean absorption time (MAT), and systemic availability (i.e., bioavailability; F) of the administered dose. MAT was calculated as MRT after subcutaneous administration (MRT SC ) after intravenous administration (MRT) IV It is calculated by subtracting ).

[0160] result Table 7 shows results obtained in mice, Table 8 shows results obtained in rats, and Tables 9 and 10 show results obtained in miniature pigs. ND = Undetermined. IV = Intravenous administration. SC = Subcutaneous administration.

[0161] [Table 7]

[0162] As shown in Table 7, hIL-22 variants with only skeletal variations (comparatives 1 and 3) had short circulating half-lives regardless of the route of administration. The extension in the Fc fusion (hFc-hIL-22) significantly increased the half-life. Covalent bonding of fatty acids (C18 diacid; derivatives 1 and 6) resulted in intermediate circulating half-lives in mice. IL-22 derivatives circulated longer in mice when administered subcutaneously compared to intravenously.

[0163] [Table 8]

[0164] As shown in Table 8, hIL-22 variants with only skeletal variations (comparative 1) had short circulating half-lives. Covalent bonding of fatty acids (derivatives 1, 3, and 6) resulted in increased circulating half-lives in rats, regardless of the fatty acids used (C16 vs. C18 diacitates) and route of administration. IL-22 derivatives typically circulated longer when administered subcutaneously compared to intravenously.

[0165] [Table 9]

[0166] As shown in Table 9, hIL-22 variants with only skeletal variations (comparatives 1 and 2) had short circulating half-lives comparable to hIL-22. The comparative Fc fusion (hFc-hIL-22) and all IL-22 derivatives (derivatives 1, 6, and 10) had significantly increased circulating half-lives. When administered intravenously in miniature pigs, the IL-22 derivatives had circulating half-lives exceeding 50 hours, which was comparable to the comparative IL-22-Fc fusion.

[0167] [Table 10]

[0168] As shown in Table 10, a faster MAT was demonstrated for the IL-22 derivative (derivative 6) compared to the comparative Fc fusion (hFc-IL-22). MAT is T max It is a more accurate indicator of drug uptake than simply comparing them, because it is C max This is because we also take into account the differences (T max The dosage and C max (Influenced by both). Miniature pigs were used in this study, rather than mice or rats, due to their similarity to humans.

[0169] conclusion Semaglutide, a known fatty acid alkylated GLP-1 derivative, has a half-life of 46 hours in miniature pigs (Lau et al., J Med Chem, 2015, 58(18):7370-80) and 160 hours in humans, which corresponds to a once-weekly dosing profile with a peak-to-trough ratio of 2. Dulaglutide, an Fc-fusion GLP-1 derivative, has a similar half-life.

[0170] Therefore, the half-lives demonstrated by IL-22 derivatives in miniature pigs—at least 40 hours when administered subcutaneously and over 50 hours when administered intravenously—are expected to correspond to a once-weekly dosing profile in humans with a peak-to-trough ratio of 2.

[0171] Therefore, the data demonstrate that the derivatives of the present invention increase the circulating half-life of IL-22, exhibit optimized pharmacokinetic and pharmacodynamic properties, and provide novel and improved treatments for a diverse range of indications, including diseases, disorders, and conditions of metabolism, the liver, lungs, intestines, kidneys, eyes, thymus, pancreas, and skin.

[0172] Example 2 - In vitro efficacy study method Two in vitro assays were used to study the efficacy.

[0173] The first was a reporter gene assay in BHK cells triple-transfected with IL-22Ra, IL-10Rb, and luciferase along with a STAT3-inducing promoter. This was a highly sensitive, high-throughput assay for measuring IL-22 receptor-mediated STAT3 activation.

[0174] Stable reporter BHK cell lines were generated using the following plasmids: (i) hIL-10Rb in pcDNA3,1hygro(+), (ii) IL22R in pcDNA3,1(Zeocin), and (iii) 2xKZdel2 in pGL4.20. Thus, the cell lines expressed human IL-10Rb, human IL-22Ra, and the luciferase reporter under the control of the pSTAT3-driven promoter.

[0175] On day 0 of the assay protocol, cells were seeded at 15,000–20,000 cells / well in 96-well plates (Corning #3842, black, clear bottom) in basal medium (500 ml: DMEM + Glutamax (Gibco, cat. number: 31966-021), 10% (w / v) fetal bovine serum (FCS; containing albumin) (50 ml), and 1% (w / v) penicillin-streptomycin (P / S) (5 ml)). On day 1, the medium was removed by inverting the plate. Fresh basal medium was added at 50 μl per well, and the cells were incubated for 60 minutes.

[0176] Derivatives of IL-22 were tested in parallel with hIL-22 and hIL-22 variants that only had skeletal variations, as comparators. The number of animals (n) used to test each derivative or comparator ranged from 1 to 36.

[0177] Therefore, 50 μl of diluted derivative or comparator (diluted in basal medium) was added to each well, and the plate was left for 4 hours. Thus, the derivative and comparator were 2-fold diluted, as they had already been diluted in 50 μl of medium in the wells. Stimulation was terminated after 4 hours by adding 100 μl of Steadylite+ reagent (Perkin Elmer cat number 6066759). The plate was sealed with TopSeal A, shaken at 450 rpm for 15 minutes, and then read using a Mithras or similar system before 12 hours.

[0178] Data analysis was performed using Graphpad Prism. The maximum half-volume effective concentration (EC) of each derivative or comparator was determined. 50 ) was evaluated as an indicator of its effectiveness. EC 50 The reaction was determined using Log(compound) versus reaction-variable slope(4p). The hill slope was constrained to 1 as the standard.

[0179] The second in vitro efficacy assay measured pSTAT3 in HepG2 cells—human liver-derived cell lines—that endogenously express IL-22Ra and IL-10Rb.

[0180] On day 1, HepG2 cells were seeded at 25,000–30,000 cells / well in 96-well plates (Biocoat #35-4407 Becton Dickinson). The cells used for seeding and subculturing were in DMEM (1x) + 25 mM (4.5 g / l) glucose, -pirubate (Gibco, cat. no. 61965-026) + 10% (w / v) FCS + 1% (w / v) P / S. On day 2, the cells were ready for assay. 50 μl of cells starved in DMEM (Gibco, cat. no. 61965-026) with 0.1% (w / v) FCS (i.e., extremely low albumin concentration) was added to each well and left for 60 minutes.

[0181] Using technical duplicates, each derivative or comparative standard was tested at seven different concentrations (0.001, 0.01, 0.1, 1, 10, 100, 1000 nM). Therefore, 50 μl of diluted derivative or comparative (diluted in 0.1% (w / v) FCS in DMEM) was added to each well, and the plate was left to stand for 15 minutes. Thus, the derivatives and comparatives were 2-fold diluted, as they had already been diluted in 50 μl of medium in each well. To lyse the cells, the medium was removed from the cells, and 50 μl of freshly prepared 1x lysis buffer (SureFire lysis buffer from the kit) was added to each well. The plate was agitated at 350 rpm for 10 minutes at room temperature.

[0182] IL-22-induced phosphorylation of STAT3 was measured according to the AlphaScreen® SureFire® STAT3 (p-Tyr705) assay protocol (Perkin Elmer cat. number TGRS3S (500-10K-50K)). For this purpose, 4 μl of lysate was transferred to a 384-well proxy plate for the assay (4 μl of positive and negative control were added). Immediately before use, the acceptor mix was prepared (by 5-fold dilution of activation buffer in reaction buffer and 50-fold dilution of acceptor beads in the diluted buffer). 5 μl of acceptor mix was added to each well, the plate was sealed with Topseal A adhesive film, and incubated at room temperature for 2 hours. Immediately before use, the donor mix was prepared (by 20-fold dilution of donor beads in dilution buffer). 2 μl of donor mix was added to the wells under dim light. The plate was resealed with Topseal A adhesive film and incubated at room temperature for 2 hours. The plate was read using an Alpha Technology compatible plate reader.

[0183] Data analysis was performed using Graphpad Prism. First, nonlinear regression was performed in Prism using Log(compound) vs. reaction-variable slope (4p) analysis. The Hill slope was constrained to 1. Next, the highest value Y from the control compound (either His-tagged hIL-22 or hIL-22) was used for normalization in Prism. In each dataset, 0% was set as the minimum value and 100% was set as the highest value Y from the above nonlinear regression (for the control). The nonlinear regression was repeated as described above to obtain the % activity / wt and EC of the tested derivatives. 50 The highest value and EC, respectively. 50 This was observed in the results below.

[0184] result Table 11 shows the EC values ​​of key derivatives and comparative products measured in the BHK cell reporter gene assay for IL-22 receptor-mediated STAT3 activation. 50 This indicates that.

[0185] [Table 11]

[0186] The BHK cell assay contained a high amount of albumin, so the measured EC 50 The study incorporated the effect of albumin binding when testing derivatives.

[0187] Comparator 4, an IL-22 variant with only a skeletal variation, was shown to have equivalent potency to hIL-22. Derivative 3, which has the same skeletal structure as comparator 4 but is covalently bonded to a medium-affinity albumin binder (C16 diacid), showed a one-quarter decrease in potency compared to hIL-22. In this case, derivative 1, which also has the same skeletal structure but is covalently bonded to a high-affinity albumin binder (C18 diacid), showed only a one-seventh decrease in potency compared to hIL-22.

[0188] Alkylation site and skeletal variation scans were performed using offsets in the 35Q and 64Q backgrounds (i.e., two of the three mutated IL-22 glycosylation sites) by comparing the results for derivatives 6–9. The covalent sites in these derivatives were selected based on an analysis of the IL-22 structure to identify positions that were predicted to be surface-exposed and not involved in receptor binding. The results obtained for these derivatives demonstrated that Cys substitutions and fatty acid covalent bonds can be tolerated at several (selected) positions, which was surprising to the inventors.

[0189] Table 12 shows the EC values ​​of key derivatives and comparative products measured in the HepG2 cell assay for pSTAT3. 50 This indicates that.

[0190] [Table 12]

[0191] In a HepG2 cell assay with endogenous receptor expression levels, minimal signal amplification, and no albumin, derivative 1 exhibited 1 / 2.5 the potency of hIL-22 (similar to comparative 4, an hIL-22 variant with the same skeleton as derivative 1 but lacking fatty acids).

[0192] Table 13 presents the results of both BHK and HepG2 cell assays to evaluate mutations in fatty acid covalent bonds and glycosylation sites during N-terminal extension. ND = Undetermined.

[0193] [Table 13]

[0194] Derivative 6 differs from derivative 1 due to the additional N35Q and N64Q substitutions (two of the three mutated glycosylation sites), but they have equivalent potency (derivative 6 tends to have slightly reduced potency).

[0195] Derivatives 2 and 4 have a 15-mer N-terminal extension, and the extension contains a Cys residue (-7C) for fatty acid binding, which has been shown to be surprisingly well-tolerated.

[0196] conclusion The decrease in potency observed in the fatty acid covalent bond in the tested derivatives of the present invention was primarily facilitated by albumin binding, with little contribution from skeletal substitution. This was demonstrated by the remarkable equivalence of comparative 4 and hIL-22. In comparison, as previously mentioned, Genentech reported a 1 / 34 decrease in in vitro potency for its Fc fusion of IL-22.

[0197] In a HepG2 cell assay at extremely low albumin levels, derivative 1 (an IL-22 derivative that showed a 1 / 7 reduction in potency in the BHK assay (with albumin binding)) showed only a 1 / 2.5 reduction in potency compared to hIL-22.

[0198] The equivalent potency of derivatives 1 and 6 (Table 13) showed that the 35Q and 64Q mutations were surprisingly tolerated without affecting potency.

[0199] Therefore, derivatives of IL-22 maintain high potency in the presence of albumin and exhibit nearly equivalent potency to hIL-22 in the absence of albumin. Cys substitutions and fatty acid covalent bonds are permissible at several positions.

[0200] Therefore, the data show that the derivatives of the present invention exhibit good bioavailability and efficacy, providing novel and improved treatments for a diverse range of indications, including metabolic, hepatic, pulmonary, intestinal, renal, and skin diseases, disorders, and conditions.

[0201] Example 3 - In vivo efficacy study in diabetes This study was designed to investigate the effects of once-daily administration of the derivative of the present invention for 8–16 days in a diabetic mouse model. The study was conducted in a treatment (not prophylactic) manner, meaning that diabetic pathology had developed before the start of administration. Since the mouse model has a fatty liver (leptin receptor knockout), it also serves as a metabolic model of liver disease.

[0202] method Male C57BKS db / db mice, 7-8 weeks old, were obtained from Charles River Laboratories (-10 days) and acclimatized for at least one week prior to the start of the experiment. One week after arrival (-3 days), the mice were randomized and housed in groups of 10 (or individually in the case of food intake studies). Blood glucose and food intake were measured on -3 days and at each of the 1-16 days of the study.

[0203] A derivative of IL-22 (derivative 1) was tested in parallel with an IL-22 Fc fusion (hFc-hIL-22) as a comparator and a vehicle alone as a negative control. Each drug was administered subcutaneously to diabetic db / db mice (n=6-10 in each group) at doses of 0.1, 0.25, 0.5, or 1.0 mg / kg once daily on days 1-16, respectively. Food intake was reduced after derivative / comparative / control administration.

[0204] Blood glucose levels were measured daily throughout the study period. Eye blood samples were collected from anesthetized mice at the end of the study. 500 μl of blood was collected in an EDTA tube. The samples were kept on ice and centrifuged at 6000 G for 5 minutes at 4°C within 20 minutes. The plasma was divided into 0.75 ml micronic tubes and immediately frozen for later measurement of component concentrations.

[0205] In addition to measuring derivative or comparator concentrations, plasma levels of target engagement biomarkers (liver-derived acute-phase protein, haptoglobin and serum amyloid P component (SAP), and intestinal peptide YY (PYY)) were measured at the end of the study. Haptoglobin was measured using a commercially available kit with a COBAS instrument (Roche Diagnostics) according to the manufacturer's instructions. PYY was measured using a commercially available ELISA assay (ALPCO) that recognizes mouse and rat PYY according to the manufacturer's instructions. SAP was measured using a commercially available ELISA assay (R&D Systems) that recognizes mouse pentraxin 2 / SAP according to the manufacturer's instructions.

[0206] result Blood glucose levels throughout the study period are shown in Figures 5 and 6A.

[0207] As can be seen in Figure 5, hIL-22 and the hIL-22 variant with only a skeletal variation (comparison 3) did not reduce blood glucose levels throughout the study compared to the vehicle control.

[0208] As can be seen from Figure 6A, both derivative 1 and hFc-hIL-22 reduced blood glucose to normal levels equally, with derivative 1 showing slightly higher efficacy on the final day of the study, despite hFc-hIL-22's higher target engagement reflecting higher steady-state exposure levels in the specific study. A decrease in food intake was observed in the treated animals compared to the vehicle control (see Figure 6B). Thus, the derivatives tested normalized blood glucose in the db / db model as well as hFc-hIL-22; as mentioned above, such an effect was not observed with hIL-22 or comparator 3.

[0209] The levels of the target engagement biomarkers haptoglobin, SAP, and PYY measured at the end of the study are shown in Figures 7A and 7C, respectively. As can be seen in the graph, all three target engagement biomarkers were upregulated by the tested derivatives and hFc-hIL-22, and were upregulated more by hFc-hIL-22 than by derivative 1.

[0210] Figure 8 shows dose-response data for derivative 6 (another derivative of the present invention, which is identical to derivative 1 except for additional substitutions at two glycosylation sites). All three doses tested (0.1, 0.25, and 0.5 m / kg) were effective in reducing blood glucose over time, with the decrease becoming progressively more pronounced with increasing concentration.

[0211] conclusion Both the tested derivatives and hFc-hIL-22 normalized blood glucose in a db / db model, thereby demonstrating in vivo therapeutic effects. Importantly, such effects were not observed with hIL-22 administration alone, demonstrating that chronic exposure obtained with the long-acting derivatives and Fc fusions is necessary for therapeutic effects. Although the mechanism of action for the antidiabetic effect is not yet fully understood, the effects of IL-22 on the liver (hepatic gluconeogenesis and lipid synthesis) are thought to be a major factor.

[0212] Food intake has also been shown to decrease with treatment using the derivatives of the present invention, thus demonstrating its effectiveness as a treatment for obesity.

[0213] Target engagement biomarkers were also observed to be upregulated by derivatives and hFc-hIL-22. Certain biomarkers measured in db / db mice are known to be convertible to humans.

[0214] The circulating half-life (T) of subcutaneously administered hFc-hIL-22 1 / 2 It is important to note that the duration of action of derivative 1 is longer than that of derivative 1, particularly in mice (20 hours and 8 hours respectively). 1 / 2(See Table 7). Therefore, exposure to hFc-hIL-22 is high at steady state. This is further supported by the observation that target engagement biomarkers (haptoglobin, SAP, and PYY) were higher in the hFc-hIL-22 group than in the derivative 1 group (Figure 7), demonstrating higher target engagement itself in the experiments shown. Thus, despite higher exposure and target engagement, the efficacy of the Fc fusion (hFc-hIL-22) was inferior to that of the derivative of the present invention (derivative 1) during the last three days of the 16-day drug-administered study.

[0215] Therefore, the data demonstrate that the derivatives of the present invention exhibit good therapeutic efficacy in mouse models of diabetes and liver disease. Since certain biomarkers measured in db / db mice are known to be convertible to humans, it is reasonable to predict that such therapeutic efficacy can be similarly converted.

[0216] Example 4 - In vivo efficacy study in liver injury (i) This study was designed to investigate the effects of administering the derivative of the present invention in a mouse model of liver injury. The study was conducted in a prophylactic manner, meaning that liver injury was induced only after the start of drug administration.

[0217] method Ten-week-old C57Bl / 6Rj mice were obtained and acclimatized for one week prior to the start of the study. Liver injury was induced with a single intraperitoneal dose of APAP (300 mg / kg, 20 ml / kg). Test derivatives of IL-22 (derivatives 1 and 6) were administered subcutaneously at 1.5 mg / kg two hours before APAP administration, in parallel with vehicle controls (n=5-10). The study was completed 24 hours after APAP administration. Final bleeding was ensured for measurement of plasma alanine transaminase (ALT) and aspartate transaminase (AST).

[0218] Blood samples were collected in heparinized tubes, plasma was separated, and stored at -80°C until analysis. ALT and AST were measured using a commercially available kit (Roche Diagnostics) on a COBAS c501 automated analyzer according to the manufacturer's instructions.

[0219] The liver was subjected to formalin fixation and paraffin embedding for histological analysis.

[0220] Growth was measured via ki67 immunohistochemistry (IHC) staining. IHC-positive staining was quantified by image analysis using VIS software (Visiopharm, Denmark).

[0221] Apoptosis was measured using the terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) assay. Briefly, slides with paraffin-embedded sections were deparaffinized in xylene and rehydrated with a series of stepwise ethanols. Slides were pretreated with proteinase K, and endogenous peroxidase activity was blocked with hydrogen peroxide. The TUNEL mixture (in situ cell death detection kit, POD, Roche) was added to the slides and subsequently amplified with horseradish peroxidase (HRP), and visualized with diaminobenzidine (DAB) (Chromogen). Finally, the slides were counterstained in hematoxylin and covered with coverslips.

[0222] result Plasma levels of ALT and AST at the end of the study are shown in Figures 9A and 9B, respectively. ALT and AST levels were shown to be significantly reduced before liver injury in mice treated with derivative 1 or 6 compared to vehicle / APAP controls.

[0223] The number of TUNEL-positive and ki67-positive cells at the end of the study are shown in Figures 10A and 10B, respectively. The amount of TUNEL-positive cells was shown to be significantly reduced before liver injury in mice treated with derivative 1 or 6 compared to vehicle / APAP controls. The amount of ki67-positive cells was similar between APAP-treated groups.

[0224] conclusion ALT and AST are liver enzymes used as indicators of liver damage. Therefore, derivatives 1 and 6 have been shown to protect the liver against APAP-induced injury.

[0225] The TUNEL assay results showed that derivatives 1 and 6 protected against apoptosis induced by liver injury compared to the vehicle / APAP control. However, cell proliferation was not affected by these derivatives of IL-22. Since proliferation is physiologically upregulated as a response to injury (as seen in the control), the results demonstrate the proliferative effect of derivatives 1 and 6, as the decrease in proliferation does not follow the decrease in injury (the ratio of proliferation to injury increases).

[0226] Therefore, the data demonstrate that the derivatives of the present invention exhibit good efficacy in protecting against liver injury in a mouse model. Since certain biomarkers measured in mice are known to be convertible to humans, it is reasonable to predict that the observed protection will be similarly convertible.

[0227] Example 5 - In vivo efficacy study in lung injury This study was designed to investigate the effects of administering derivatives of the present invention in a rat model of lung injury. The study was conducted in both prophylactic and treatment modes, meaning that administration was initiated before lung injury was induced and continued thereafter.

[0228] method To induce lung injury, 100 μl of bleomycin was administered as a single dose to the lungs of male Sprague Dawley rats via oropharyngeal inhalation on day 1 (groups 2-6). Physiological saline was administered as a negative control (group 1).

[0229] Animals in groups 3, 4, and 5 were administered derivative 6 (by subcutaneous injection) at doses of 0.5, 1.5, or 4.5 mg / kg, respectively, once daily from day 1 to day 3. Animals in group 6 were administered prednisolone (by oral administration) at a dose of 10 mg / kg once daily from day 1 to day 3.

[0230] To measure soluble collagen in bronchoalveolar lavage (BALF) from rats, lungs were washed with sterile PBS (calcium and magnesium-free) containing a cocktail of added protease inhibitors (3 × 4 ml), and the lavage fluid per animal was placed in one tube. Soluble collagen in the BALF supernatant was measured using a Sircol S1000 (Biocolor) (Charles River Laboratories) soluble collagen assay.

[0231] On day 4, all animals were provided for necropsy (final euthanasia). The right lung was collected from all animals for histopathological examination, inflated and fixed in 10% neutral buffered formalin (NBF), and then immersed and fixed in NBF. Three parallel longitudinal sections were cut from the right caudal lobe and loaded into cassette 01. The anterior, middle, and accessory lobes of the right lung were also longitudinally sectioned and loaded into cassette 02.

[0232] Two slides were prepared from each cassette; one slide was stained with hematoxylin and eosin (H&E), while the other was stained with hematoxylin and picrosilius red (H&PSR).

[0233] Next, a random number generator was used to assign random numbers to each slide. The search table was recorded in a Microsoft Excel spreadsheet, and a copy was provided to the research pathologist after the slide evaluation. As a result, six sections per lung were read blind.

[0234] Next, veterinary pathologists scored each section of each H&E-stained slide for the severity of inflammation (0=none, 1=minimal, 2=mild, 3=moderate, and 4=severe). Mean and median scores per group were calculated. Pathologists also scored each section of each H&PSR-stained slide for the severity of fibrosis (using a modified Ashcroft score from 0=low to 8=high). Mean and median scores per group were calculated and subjected to nonparametric ANOVA, Kruskal-Wallis test-based analysis.

[0235] result Table 14 provides a summary of the microscopic findings, revealing the mean and median scores for inflammation and fibrosis in each group.

[0236] [Table 14]

[0237] As is evident from a comparison of groups 1 and 2 in Table 14, bleomycin induced both pneumonia and fibrosis in the rat model. Mean and median scores were lower in groups 3–5, i.e., rats treated with derivative 6, a derivative of the present invention. These lower scores were comparable to those observed in rats treated with prednisolone (group 6).

[0238] The median inflammation and fibrosis scores for each animal in the study are also shown in Figures 11A and 11B, respectively.

[0239] As shown in Fig. 11A, the group median of the inflammation score increased in the bleomycin / vehicle control (Group 2) compared to the negative control (Group 1). The group median of the inflammation score decreased in the rats treated with Derivative 6 (significantly in the high-dose Group 5) and in the rats treated with prednisolone (Group 6) compared to the bleomycin / vehicle control.

[0240] As shown in Fig. 11B, the group median of the fibrosis score increased in the bleomycin / vehicle control (Group 2) compared to the negative control (Group 1). However, the group median of the fibrosis score decreased in the rats treated with Derivative 6 (also significantly in the high-dose Group 5) compared to the bleomycin / vehicle control, but did not decrease compared to the control prednisolone.

[0241] As shown in Fig. 11C, the amount of soluble collagen in the BALF after bleomycin-induced lung injury increased in the bleomycin / vehicle control (Group 2) compared to the negative control (Group 1), which did not decrease with treatment with prednisolone (Group 6). However, in the BALF from rats treated with Derivative 6 (across all doses tested), a significantly decreased amount of soluble collagen was observed compared to the bleomycin / vehicle control. Since soluble collagen in the BALF is a readout for fibrosis, these results immediately confirm the histological data reported above.

[0242] Conclusion The results of the microscopic studies showed that the derivatives of the present invention can prevent and / or reduce bleomycin-induced lung inflammation and fibrosis in a rat model. The effect seen with respect to inflammation was equivalent to that observed with prednisolone, a corticosteroid known to treat lung inflammation. However, the derivatives of the present invention had a unique effect on fibrosis that was not seen with prednisolone.

[0243] Example 6 - In Vivo Efficacy Study in Colitis This study was designed to investigate the effects of dosing of the derivatives of the present invention in a murine model of colitis. The study was conducted in both preventive and treatment modalities, meaning that dosing started on the same day as colitis was induced and continued thereafter.

[0244] Methods Female C57Bl / 6JRj mice fed a diet were randomized based on body weight into five groups (n = 8 per group). Colitis was induced using DSS in four of the five groups. These mice ingested DSS in their drinking water for 7 days from study day 0 to day 6. In the fifth group, the animals ingested water without DSS and thus functioned as healthy controls. From study day 0, DSS mice were treated once daily for 10 days with vehicle, a test derivative of IL-22 (derivative 6; dosed intraperitoneally at 0.35 mg / kg or 1 mg / kg) or an IL-22-Fc fusion as a comparator (hFc-hIL-22; dosed intraperitoneally at 0.5 mg / kg). Body weight, food, and water intake were monitored daily.

[0245] On study day 10, blood samples were collected from the mice into EDTA tubes, plasma was separated, and stored at -80 °C until analysis. Regenerating islet-derived protein 3 gamma (Reg3g) was measured in duplicate according to the manufacturer's instructions using an ELISA kit (Cloud-Clone Corp). Reg3g is a target engagement marker for IL-22.

[0246] At the end, the intestines were removed for stereological analysis. Thus, the intestines were flushed with ice-cold saline, their contents gently removed, and then sampled.

[0247] The intestines were immersed overnight in formalin (Tissue-Tek VIP) and then embedded in paraffin blocks. The formalin-fixed intestines were then sampled proximal to distal using the principle of systematic homogeneous random sampling (SURS), yielding a total of four slabs, which were placed in multicassettes. All tissue slabs were arranged in a manner that would allow for the identification of individual slabs at a later stage. The paraffin blocks were trimmed, and 5 μm upper sections were cut and placed on a Superfrost+ objective lens. For larger intestines, another section 500 μm long was cut up to the upper section, thereby yielding a total of eight colon sections from each animal.

[0248] Colitis volume was measured stereoscopically, i.e., using a three-dimensional interpretation of a two-dimensional cross-section of the colon. Stereoscopic volume estimation was performed on scanned H&E-stained slides using the newCAST system (Visiopharm). Total bowel volume, mucosal volume, submucosa and muscularis fasciae volume, and inflammatory tissue volume were estimated by point counting using an appropriately sized grid system, where all points corresponding to the target structure were counted. The number of points corresponding to the target structure was converted to volume according to the following mathematical relationship:

[0249]

number

[0250] In the formula, A(p) is the area per point, p is the total number of points corresponding to the target structure, and t is the distance between cross-sections. The average inflammation volume per group was calculated and subjected to statistical analysis.

[0251] Colon morphology was also evaluated by observing H&E-stained slides at the end of the procedure.

[0252] result Colitis volume is shown in Figure 12. Inflammation was shown to be prevented at all doses in mice treated with derivative 6 compared to vehicle controls (which also contained DSS). In particular, inflammation was maintained at normal levels in the group treated with derivative 6, as is evident from the same colitis volume in the group treated with healthy controls (vehicle without DSS). The same was true for the group treated with hFc-hIL-22.

[0253] Representative H&E stained images of the colon morphology at the end of treatment are shown in Figure 13. After DSS treatment, mucosal epithelial wounds can be observed in animals treated with the vehicle (indicated by black arrows), but not in animals treated with any dose of derivative 6 or with hFc-hIL-22. This demonstrates a protective effect on epithelial tissue.

[0254] Plasma Reg3g levels are shown in Figure 14. DSS treatment induced an increase in basal Reg3g levels (comparison between vehicle and vehicle without DSS). No further increase was detectable in the low-dose (0.35 mg / kg) derivative 6 group, but it was detectable in the higher-dose (1 mg / kg) derivative 6 group and the hFc-hIL-22 group. The higher Reg3g levels in the hFc-hIL-22 (0.5 mg / kg) group compared to the derivative 6 (1 mg / kg) group showed higher target engagement despite the lower dose, which was likely associated with the longer half-life of hFc-hIL-22 in mice (T1 / 2 of 30 hours for hFc-hIL-22 vs. 9.1 hours for derivative 6).

[0255] conclusion Therefore, the data demonstrate that the derivatives of the present invention exhibit good efficacy in protecting against colitis and mucosal epithelial wounds in a mouse model. This indicates the discovery of novel and improved treatments for intestinal diseases, disorders, and pathologies. In particular, the findings demonstrate the potential for treating diseases characterized by mucosal epithelial damage, such as inflammatory bowel disease.

[0256] Example 7 - In vivo efficacy study in liver injury (ii) This study was designed to investigate the effects of administering the derivative of the present invention in a second mouse model of liver injury (the first being described above in Example 4). The study was conducted in a prophylactic manner, meaning that liver injury was induced only after the start of administration.

[0257] method Male C57Bl6 / 6j mice were divided into five groups (n=8 per group). Two of the five groups received intraperitoneal administration of a test derivative of IL-22 (derivative 1) at a dose of 1 mg / kg at -26 hours and -2 hours before ConA treatment. The other two groups received only vehicle at these time points. Liver injury was induced in all four groups by administering ConA as an intravenous bolus at a dose of 15 mg / kg over 30 seconds. The fifth group was a healthy control and did not receive ConA (only vehicle, as described above).

[0258] Eight or twenty-four hours after ConA injection, mice were placed under isoflurane anesthesia, and the maximum volume of blood was collected by cardiac puncture (using polypropylene serum gel tubes containing a coagulation activator). Untreated mice (group 5) were sacrificed at eight hours. Blood was mixed with the coagulation activator by inverting the tubes several times within each tube. The tubes were kept at room temperature for 15 minutes, then centrifuged at 2000 g for 10 minutes at 4°C. ALT and AST in serum samples were measured using an automated system (Konelab 20) according to the manufacturer's instructions.

[0259] result Plasma levels of ALT and AST at the end of the study are shown in Figures 15A and 15B, respectively. ALT and AST levels were shown to be reduced at both time points tested in mice treated with derivative 1 prior to liver injury, compared to the vehicle / ConA control.

[0260] conclusion ALT and AST are liver enzymes used as indicators of liver injury. Thus, Derivative 1 was shown to protect the liver against injury induced by ConA, just as it did against injury induced by APAP in Example 4. Since specific biomarkers measured in mice are known to be translatable to humans, it is reasonable to predict that the observed protection would be similarly translatable.

[0261] Example 8 - In Vivo Efficacy Studies in Obesity and NASH This study was designed to investigate the effect of dosing of the derivatives of the present invention in an obesity and NASH mouse model. The study was conducted in a treatment (not preventive) manner, meaning that obesity and NASH pathology had developed prior to the start of dosing.

[0262] Methods The diet-induced obesity mouse model was based on male C57BL / 6JRj mice that had been fed a high-fat diet for at least 30 weeks prior to the experiment. The diet was high in fat (40%), fructose (22%), and cholesterol (2%) (Research Diets D09100310). This resulted in obesity, NAFLD, and ultimately NASH.

[0263] Animals were housed alone for 6 days prior to the first dosing of the test derivative (or other), and body weight was monitored daily throughout the experiment. The mice were divided into 6 groups (n = 12 per group). Dosing was started on day 0 of the study (indicated by the dotted line in Figure 16) and administered subcutaneously once daily at the following doses.

[0264] Semaglutide, a long-acting GLP-1 receptor agonist, was used as a positive control in Group 1, and in Group 2, it was investigated in combination with a test derivative of IL-22 (derivative 6). Semaglutide dosage was gradually increased according to the following schedule: 0.6 nmol / kg Day 0 - 1.2 nmol / kg Day 1 - 2.4 nmol / kg Day 2 - 4.8 nmol / kg Day 3 - 12 nmol / kg Day 4 - 30 nmol / kg Day 5. In the combination group, semaglutide dosage was started on Day 0, and after weight loss plateaued in the semaglutide-treated group, derivative 6 dosage was started on Day 12 (shown by the dotted line in Figure 16).

[0265] In the third "high-dose" group, the dosage of derivative 6 was gradually increased according to the following schedule: 0.05 mg / kg on day 0, 0.1 mg / kg on day 1, 0.15 mg / kg on day 2, 0.2 mg / kg on day 3, and 0.25 mg / kg on day 4. The dosage was changed from 0.25 mg / kg to 0.1 mg / kg on day 14 (shown by the dotted line in Figure 16). In the fourth "low-dose" group, the dosage of derivative 6 was started at 0.05 mg / kg and no further increases were made.

[0266] In the fifth group, the dosage of the IL-22-Fc conjugate (hFc-hIL-22) as a comparator was gradually increased according to the following schedule: 0.02 mg / kg day 0 - 0.04 mg / kg day 1 - 0.06 mg / kg day 2 - 0.08 mg / kg day 3 - 0.1 mg / kg day 4. The dose of hFc-hIL-22 was selected to match the 0.25 mg / kg derivative 6 group in terms of target engagement based on a longer half-life, and accordingly higher target engagement compared to derivative 6, as seen in Example 6 (see Figure 14).

[0267] In the sixth group, only the vehicle was administered as a negative control.

[0268] Plasma triglyceride (TG) levels were measured at baseline (-2 days), 2 weeks after the start of medication (14 days), and 4 weeks after the start of medication (28 days). Specifically, tail blood samples were collected for analysis by injecting 200 μl or less of tail blood volume into open Microvette (100 μl or 200 μl) tubes treated with an appropriate anticoagulant. The blood was left at 4°C until centrifuged at 3000 g for 10 minutes. The plasma supernatant was transferred to a new tube, immediately frozen on dry ice, and stored at -80°C. TG levels were measured using a commercially available kit (Roche Diagnostics) with a cobas® c501 analyzer according to the manufacturer's instructions.

[0269] result Figure 16 shows the body weight throughout the experiment.

[0270] The study demonstrated the dose-dependent high efficacy of derivative 6 in weight loss in an obese mouse model. Furthermore, it demonstrated additiveity to the GLP-1 receptor agonist (semaglutide), which is being investigated in late-stage clinical trials for obesity treatment. The data suggested the superiority of derivative 6 compared to hFc-hIL-22 in inducing weight loss. Importantly, hFc-hIL-22 had a longer half-life than derivative 6 in mice and demonstrated higher target engagement even when administered at half the dose of derivative 6. Therefore, the 0.1 mg / kg dose of hFc-hIL-22 used in this study was selected for similar target engagement to the 0.25 mg / kg dose of derivative 6.

[0271] Here, the sensitivity to weight loss induced by derivative 6 observed in diet-induced obese mice is not observed in lean mice. For example, in a 10-day DSS-induced colitis study with once-daily administration (Example 6), the baseline body weight was 19.0 g in both the DSS / vehicle group and the DSS / derivative 6 (0.35 mg / kg) group. At the end of the study, the body weight was 17.6 g in the DSS / vehicle group compared to 17.4 g in the DSS / derivative 6 (0.35 mg / kg) group, which differs (p=0.82 by unpaired Student's t-test). By comparison, in this study, the mice in the vehicle group and the derivative 6 (0.25 mg / kg) group had a body weight of 43.5 g and 35.2 g, respectively, on day 10. Therefore, a significant weight loss was observed in the derivative 6 group compared to the vehicle group (p<0.0001 by unpaired Student's t-test for weight on day 10). At the start of the study, weight was similar in the vehicle group and the derivative 6 group (44.3g and 44.1g, respectively).

[0272] Table 15 shows the plasma TG levels measured at baseline (-2 days), 2 weeks after the start of medication (14 days), and 4 weeks after the start of medication (28 days).

[0273] [Table 15]

[0274] Delta refers to the change in TG levels (nmol / l) from baseline for the indicated treatment and time point. As can be seen from Table 15, the level increased in the vehicle group, but decreased in all other groups.

[0275] Derivative 6 demonstrated higher efficacy than semaglutide in lowering TG levels, even at lower doses, and was less likely to cause weight loss than semaglutide (for example, the delta TG levels (nmol / l) at week 4 were -0.14±0.066 for semaglutide, -0.30±0.034 for derivative 6 (0.05 mg / kg), and -0.35±0.052 for derivative 6 (0.25 / 0.1 mg / kg)). The results indicate that derivative 6 had high efficacy in lowering TG, partially independent of weight loss effects. Furthermore, there was complete additiveity in the effects of derivative 6 in addition to semaglutide. At week 4, the decrease in TG levels (nmol / l), calculated as delta TG, was -0.30±0.034 for derivative 6 (0.05 mg / kg), -0.14±0.066 for semaglutide, and -0.53±0.042 for semaglutide + derivative 6 (0.05 mg / kg). Despite the increasing TG levels in the vehicle group throughout the study, the result was a decrease in TG compared to baseline (delta TG).

[0276] conclusion The study demonstrated that the derivative of the present invention (derivative 6) can induce dose-dependent weight loss in obese mice to at least the same level as that observed with semaglutide-long-acting GLP-1 receptor agonist, which was used as a positive control. Furthermore, an additive effect on weight loss was observed using the combination of semaglutide and derivative 6. The efficacy of derivative 6 in inducing weight loss was higher than that observed with hFc-IL-h22 using doses selected to provide a similar level of targeted engagement. Weight loss induced by derivative 6 in diet-induced obese mice was not observed in lean mice treated with DSS, demonstrating that obese mice are more sensitive to derivative 6-induced weight loss. Since the weight loss observed in diet-induced obese mice is the same reading as that which would be used in humans, it is reasonable to predict that the observed weight loss would be similarly convertible.

[0277] Derivative 6 also demonstrated high efficacy in reducing TG levels. Derivative 6 showed higher efficacy than semaglutide at both doses tested, and full additiveity to efficacy in combination dosing was observed. Considering that derivative 6 at a dose of 0.05 mg / kg had higher efficacy than semaglutide despite less weight loss, it can be concluded that the TG-reducing effect of derivative 6 was at least partially independent of weight loss. Furthermore, derivative 6 was superior to the hFc-hIL-22 comparator at both doses tested. Since the TG reduction observed in diet-induced obese mice was the same reading as that which would be used in humans, it is reasonable to predict that the observed effect would translate similarly. Thus, the results indicate the discovery of a new treatment for disorders and conditions characterized by high TG levels.

[0278] While certain features of the present invention are illustrated and described herein, many modifications and equivalents will become apparent to those skilled in the art. Therefore, the claims are intended to encompass all such modifications and equivalents that fall within the true spirit of the invention.

Claims

1. A derivative of IL-22 comprising a fatty acid covalently attached to the IL-22 protein, (i) the IL-22 protein is native mature human IL-22 (hIL-22; SEQ ID NO:1) or a variant thereof, wherein the variant comprises one, two, or three amino acid substitutions within SEQ ID NO:1, and / or the variant comprises an N-terminal peptide; (ii) the fatty acid is covalently attached via a linker to a substituted Cys residue in SEQ ID NO:1 or covalently attached via a linker to a Cys residue in an N-terminal peptide; A derivative, wherein the fatty acid is a C12, C14, C16, C18 or C20 diacid.

2. The fatty acid is (i) Formula I: HOOC-(CH ２ ) ｘ－ CO- ＊ , wherein x is an integer ranging from 10 to 18, optionally 12 to 18, 14 to 16 or 16 to 18, and * indicates the point of attachment of said IL-22 protein or linker; (ii) a C16 or C18 diacid; and / or (iii) C18 diacid The derivative according to claim 1 ,

3. The variant is (i) comprising two, three, four, five or more variations in SEQ ID NO:1, said variations being independently selected from amino acid substitutions and amino acid insertions; (ii) is substituted at positions 1, 21, 35, 64, 113 and / or 114 of hIL-22; (iii) comprising a substitution in hIL-22 selected from the group consisting of A1C, A1G, A1H, N21C, N21D, N21Q, N35C, N35D, N35H, N35Q, N64C, N64D, N64Q, N64W, Q113C, Q113R, K114C, and K114R; (iv) having a sequence as set forth in any of SEQ ID NOs: 16 to 21, and / or (v) containing one, two, or three amino acid substitutions selected from A1C, H6C, A33C, N35Q, or N64Q (amino acid positions are defined relative to SEQ ID NO:1); A derivative according to claim 1 or 2.

4. The variant, (i) positions 1, 21, 35, 64, 113, and / or 114 of hIL-22, or (ii) Position 1 of hIL-22 The derivative according to any one of claims 1 to 3, comprising a Cys residue substituted with

5. The variant is (i) an extended form of hIL-22; (ii) comprises an N-terminal peptide; (iii) comprises an N-terminal trimer; (iv) contains an N-terminal G-P-G; and / or (v) comprising an N-terminal peptide selected from G*, G-P-G*, or G-P-G-S-G-S-G-S-C-G-S-G-S-G-S* (where * indicates the point of attachment to the N-terminus of the hIL-22 variant); The derivative according to any one of claims 1 to 4.

6. The derivative comprises a linker, the linker comprising: (i) one or more amino acids, optionally including Glu and / or Lys; (ii) an oxyethyleneglycine unit or a plurality of linked oxyethyleneglycine units, optionally 2 to 5 such units; (iii) one or more oligo(ethylene glycol) (OEG) residues; (iv) ethylenediamine (C ２ D A) group; (v) acetamide (Ac) group; (vi) 【Chemistry 1】 (wherein R represents a fatty acid and * indicates the point of attachment of the linker to the IL-22 protein). (vii) 【Chemistry 2】 where R represents a fatty acid and * indicates the point of attachment of the linker to the IL-22 protein; and / or (viii) 【Chemistry 3】 (wherein R represents a fatty acid and * indicates the point of attachment of the linker to the IL-22 protein). The derivative according to any one of claims 1 to 5, comprising:

7. The variant comprises a Cys substitution selected from A1C, H6C, and A33C; and / or the variant comprises an N-terminal peptide G-P-G-S-G-S-G-S-C-G-S-G-S-G-S* (where * indicates the point of attachment to the N-terminus of the hIL-22 variant); The derivative according to any one of claims 1 to 6.

8. The derivative of claim 7, wherein the variant comprises a Cys substitution selected from A1C, H6C, and A33C, and the linker is attached to the Cys residue.

9. The derivative described in claim 7, wherein the variant comprises an N-terminal peptide G-P-G-S-G-S-G-S-C-G-S-G-S-G-S*, and the linker is bonded to a Cys residue in the N-terminal peptide.

10. The derivative according to any one of claims 7 to 9, wherein the variant comprises one or both of N35Q and N64Q.

11. 7. The derivative of any one of claims 1 to 6, wherein the derivative comprises a C14, C16, C18 or C20 diacid covalently attached by a linker to a variant of hIL-22, the variant comprising a Cys residue at position 1 of hIL-22, and the linker is attached to the Cys residue.

12. The linker having the structure: 【Chemistry 4】 (wherein R represents a fatty acid and * indicates the point of attachment of the linker to the IL-22 protein). The derivative according to claim 11 , comprising:

13. The derivative according to claim 11 or 12, wherein the variant comprises an N-terminal G-P-G.

14. A derivative described in any one of claims 11 to 13, wherein the variant has a sequence as shown in SEQ ID NO: 16 or 18.

15. The derivative according to any one of claims 11 to 14, wherein the fatty acid is a C16 or C18 diacid.

16. (i) the variant has a sequence as set forth in SEQ ID NO:17, wherein the linker is attached to position 9 of SEQ ID NO:17, and wherein the linker and the fatty acid together form the structure shown in FIG. 1A; (ii) the variant has a sequence as set forth in SEQ ID NO: 16, wherein the linker is attached to position 4 of SEQ ID NO: 16, and wherein the linker and the fatty acid together form the structure shown in FIG. 1B; (iii) the variant has a sequence as set forth in SEQ ID NO: 17, wherein the linker is attached to position 9 of SEQ ID NO: 17, and wherein the linker and the fatty acid together form the structure shown in FIG. 1B; (iv) the variant has a sequence as set forth in SEQ ID NO: 16, wherein the linker is attached to position 4 of SEQ ID NO: 16, and wherein the linker and the fatty acid together form the structure shown in FIG. 1C; (v) the variant has a sequence as set forth in SEQ ID NO:19, wherein the linker is attached to position 1 of SEQ ID NO:19, and wherein the linker and the fatty acid together form the structure shown in FIG. 1A; (vi) the variant has a sequence as set forth in SEQ ID NO:20, wherein the linker is attached to position 7 of SEQ ID NO:20, and wherein the linker and the fatty acid together form the structure shown in FIG. 1A; (vii) the variant has a sequence as set forth in SEQ ID NO:21, wherein the linker is attached at position 33 of SEQ ID NO:21, and wherein the linker and the fatty acid together form the structure shown in FIG. 1A; or (viii) the variant has a sequence as set forth in SEQ ID NO: 18, wherein the linker is attached to position 4 of SEQ ID NO: 18, and the linker and the fatty acid together form the structure shown in FIG. 1B; Formulas 1A, 1B, and 1C are as follows: 【Chemistry 5】 The derivative according to claim 1.

17. (i) the variant has a sequence as set forth in SEQ ID NO:16, wherein the linker is attached to position 4 of SEQ ID NO:16, and wherein the linker and the fatty acid together form the structure shown in FIG. 1B; (ii) the variant has a sequence as set forth in SEQ ID NO: 17, wherein the linker is attached to position 9 of SEQ ID NO: 17, and wherein the linker and the fatty acid together form the structure shown in FIG. 1B; (iii) the variant has a sequence as set forth in SEQ ID NO:19, wherein the linker is attached to position 1 of SEQ ID NO:19, and wherein the linker and the fatty acid together form the structure shown in FIG. 1A; (iv) the variant has a sequence as set forth in SEQ ID NO:20, wherein the linker is attached to position 7 of SEQ ID NO:20, and wherein the linker and the fatty acid together form the structure shown in FIG. 1A; (v) the variant has a sequence as set forth in SEQ ID NO:21, wherein the linker is attached to position 33 of SEQ ID NO:21, and wherein the linker and the fatty acid together form the structure shown in FIG. 1A; or (vi) the variant has a sequence as set forth in SEQ ID NO: 18, the linker is attached to position 4 of SEQ ID NO: 18, and the linker and the fatty acid together form the structure shown in FIG. 1B; The derivative according to claim 16.

18. The derivative, 【Chemistry 6】 (Derivative 10) 18. The derivative according to claim 16 or 17,

19. A pharmaceutical composition comprising a derivative according to any one of claims 1 to 18 and a pharma- ceutically acceptable vehicle.

20. A derivative according to any one of claims 1 to 18 or a pharmaceutical composition according to claim 19 for use in therapy.

21. A derivative according to any one of claims 1 to 18 or a pharmaceutical composition according to claim 19 for use in a method for the treatment of a metabolic, hepatic, pulmonary, intestinal, renal or skin disease, disorder or condition.

22. (i) the metabolic disease, disorder or condition is obesity, type 1 diabetes, type 2 diabetes, dyslipidemia, hyperglycemia or hyperinsulinemia; (ii) the liver disease, disorder or condition is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, alcoholic hepatitis, acute liver failure, chronic liver failure, acute exacerbation of chronic liver failure (ACLF), acetaminophen-induced liver toxicity, acute liver injury, sclerosing cholangitis, biliary cirrhosis or a pathological condition caused by surgery or transplantation; (iii) the pulmonary disease, disorder or condition is chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, idiopathic pulmonary fibrosis, acute distress syndrome, chemical injury, viral infection, bacterial infection or fungal infection; (iv) the intestinal disease, disorder or condition is inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, graft-versus-host disease (GvHD), chemical injury, viral infection or bacterial infection; (v) the kidney disease, disorder or condition is acute kidney disease or chronic kidney disease; (vi) The skin disease, disorder or condition is a wound, an inflammatory disease or GvHD.

22. A derivative or pharmaceutical composition according to claim 21.

23. The use comprises administering to a patient in need thereof 0.001 μg / kg body weight to 10 mg / kg body weight.

23. The derivative or pharmaceutical composition according to any one of claims 20 to 22, comprising administering a weekly dose of said derivative between two doses.

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