Therapeutic derivatives of interleukin-22

Abstract

The present invention relates to novel derivatives of interleukin-22 (IL-22), in particular derivatives comprising a fatty acid covalently linked 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 to derivatives comprising a fatty acid covalently linked to an IL-22 protein. The invention also encompasses methods for their production and their use in therapy, including the treatment, prevention and amelioration of metabolic, liver, pulmonary, intestinal, renal and skin diseases, disorders and conditions. BACKGROUND

[0003] 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 heterodimeric receptor composed of the broadly expressed IL-10 receptor B subunit (IL-10RA2) and the IL-22 receptor A subunit (IL-22RA1) with epithelial restricted expression. It is a unique cytokine in that it is released from immune cells but selectively targets epithelial cells. Thus, the signaling pathway induced by IL-22 can have relevance in different tissues (targets include skin, intestine, lung, liver, kidney, pancreas and thymus) but IL-22 activates it in an epithelium-specific manner. The soluble binding protein IL-22BP neutralizes IL-22 and thus modulates its effects.

[0004] IL-22 is released in response to signals reflecting chemical or mechanical damage, for example activation of the aryl hydrocarbon receptor in response to environmental toxins or tryptophan intermediates, and activation of pattern recognition receptors such as toll-like receptor 4 in response to proteins, fragments and debris from dying cells or invading pathogens. Its release is further stimulated by certain cytokines, in particular IL-23 and to a lesser extent IL-1 beta. Thus, IL-22 is also secreted in response to cues reflecting pathogen infection and immune activation.

[0005] The effects of IL-22 are the result of the coordinated participation of multiple activities / pathways. IL-22 acts on epithelial barrier tissues and organs at the time of injury to protect cells and maintain barrier function (e.g. by activating anti-apoptotic gene programs). It also accelerates repair (e.g. by inducing proliferation of mature cells and activation of stem cells), prevents fibrosis (e.g. by reducing epithelial mesenchymal transition, antagonizing NLRP3 inflammasome and inducing liver 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 range of medical conditions, including conditions typically observed in diabetic or overweight mammals, such as hyperglycemia, hyperlipidemia and hyperinsulinemia.

[0006] However, IL-22 is normally rapidly cleared from the body by the kidneys, which limits its use in clinical practice. Therefore, known methods for prolonging the half-life of circulating IL-22 seek to artificially increase the size of IL-22 to above 70 kDa to avoid kidney clearance. Linking IL-22 to an Fc antibody fragment is currently the best solution to achieve this effect; both Genentech and Generon Shanghai have long-acting IL-22-Fc fusions in clinical development. PEGylation of IL-22 (polyethylene glycolation) is another known way to avoid kidney clearance.

[0007] However, these existing solutions are not without drawbacks. Existing data suggest that PEG is itself immunogenic, and vacuoles containing PEG are observed in cells with PEGylated biologies. Reduced activity and heterogeneity are also disadvantages of PEGylation. Although the Fc fusion technology is well known, the addition of an Fc antibody fragment represents a major change to the structure of IL-22, which can affect its properties beyond the half-life extension. As Fc fusions increase the size of the protein from approximately 17 kDa to approximately 85 kDa, properties such as diffusion rates, distribution, and receptor engagement kinetics can be affected. For example, some Fc fusions are absorbed slowly and / or are too large to be administered by certain routes. Genentech and Generon have also reported moderate and reversible skin reactions as dose-limiting adverse effects of IL-22-Fc fusions. Furthermore, potency can be affected by steric hindrance caused by large fusion partners.

[0008] Therefore, there remains a need in the art for new IL-22 biocompatibility modulators that can improve the circulating half-life compared to the native molecule and show optimized pharmacokinetic and pharmacodynamic properties. Ideally, it should preserve the potency and other properties of the native molecule and should also avoid the toxicity, immunogenicity, and any other adverse effects exhibited by known derivatives. SUMMARY

[0009] In a first aspect, there is provided a derivative of IL-22 comprising a fatty acid covalently linked to the IL-22 protein.

[0010] In embodiments of the application, the fatty acid is covalently linked to the IL-22 protein via a linker.

[0011] The fatty acid can have the formula I:

[0012] HOOC-(CH2) x -CO-*,

[0013] wherein x is an integer in the range of 10 to 18, optionally 12 to 18, 14 to 16, or 16 to 18, and * denotes a point of attachment to the IL-22 protein or linker. It can be a fatty diacid, for example a C12, C14, C16, C18, or C20 diacid. Advantageously, the fatty acid is a C16 or C18 diacid, and most advantageously a C18 diacid.

[0014] The IL-22 protein can be native mature human IL-22 (hereinafter “hIL-22”) or a variant thereof. The variant can be a substitution of hIL-22, optionally substituted at position 1, 21, 35, 64, 113, and / or 114. It can comprise a substitution 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 comprises a Cys residue at position 1 of hIL-22.

[0015] The variant can be an extension of hIL-22. It can comprise an N-terminal peptide, for example an N-terminal trimer. Advantageously, the variant comprises an N-terminal G-P-G.

[0016] The linker can comprise one or more amino acids, optionally including glutamic acid (Glu) and / or lysine (Lys). The linker can comprise oxyethylene glycine units or a plurality of linked oxyethylene glycine units, optionally 2 to 5 such units, advantageously 2 units. The linker can comprise one or more oligo(ethylene glycol) (OEG) residues. It can comprise an ethylenediamine (C2DA) group and / or an acetamide (Ac) group. Advantageously, the linker comprises a combination of all of the above elements. In particular, the linker can be γGlu-OEG-OEG-C2DA-Ac, γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc, or γGlu-OEG-OEG-εLys-αA.

[0017] The linker can be a Cys-reactive linker that links to a Cys residue in hIL-22 or a variant thereof. It can be linked at position -7, -5, 1, 6, 33, 113, 114, or 153 of hIL-22 (wherein positions -7, -5, etc. are as defined herein). For example, the linker can be linked to a Cys residue substituted at position 1, 6, 33, 113, or 114 of hIL-22. It can be linked to a Cys residue at -position 5, -7, or 153 relative to hIL-22. Advantageously, the linker is linked to a Cys residue substituted at position 1 of hIL-22.

[0018] In one embodiment, the derivative comprises a C14, C16, C18, or C20 diacid covalently linked to a hIL-22 variant via a linker, wherein the variant comprises an N-terminal G-P-G and a Cys residue at position 1 of hIL-22, and the linker is optionally linked to the Cys residue. Exemplary derivatives of the present application are the derivatives identified herein as Derivatives 1-10.

[0019] In a second aspect, there is provided a method for making a derivative of the first aspect, comprising covalently linking a fatty acid to an IL-22 protein.

[0020] In a third aspect, there is provided a pharmaceutical composition comprising a derivative of the first aspect and a pharmaceutically acceptable vehicle.

[0021] 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.

[0022] 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 metabolic, liver, pulmonary, intestinal, renal, or skin disease, disorder, or condition.

[0023] The metabolic disease, disorder, or condition can be obesity, type 1 diabetes, type 2 diabetes, hyperlipidemia, hyperglycemia, or hyperinsulinemia.

[0024] The liver disease, disorder, or condition can be non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, alcoholic hepatitis, acute liver failure, chronic liver failure, acute-on-chronic liver failure (ACLF), acute liver injury, acetaminophen-induced liver toxicity, sclerosing cholangitis, biliary cirrhosis, or a pathological condition resulting from surgery or transplantation.

[0025] The pulmonary disease, disorder, or condition can be chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, chemical injury, viral infection, bacterial infection, or fungal infection.

[0026] The intestinal disease, disorder, or condition can be inflammatory bowel disease (IBD), ulcerative colitis, Crohn’s disease, graft versus host disease (GvHD), chemical injury, viral infection, or bacterial infection.

[0027] The renal disease, disorder, or condition can be acute kidney disease or chronic kidney disease.

[0028] The skin disease, disorder, or condition can be a wound, an inflammatory disease, or GvHD. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 (A) C18 diacid, (B) C16 diacid, and (C) C14 diacid, each linked to a linker comprising a Cys-reactive unit. These combinations of fatty acids and linkers were used in the derivatives of the application identified herein as Derivatives 1-10.

[0031] Figure 2 The structure of a derivative of the application identified herein as Derivative 1 is illustrated.

[0032] Figure 3 The structure of a derivative of the application identified herein as Derivative 6 is illustrated.

[0033] Figure 4 The structure of a derivative of the application identified herein as Derivative 10 is illustrated.

[0034] Figure 5 The effect of daily dosing of hIL-22 and a comparative IL-22 variant having only backbone variations (identified herein as Comparative 3) on blood glucose (mean ± SEM) in an 8-day study in a diabetic mouse model is illustrated.

[0035] Figure 6 The effect of daily dosing of a derivative of the application (identified herein as Derivative 1) on (A) blood glucose and (B) food intake (mean ± SEM; * indicates p<0.05 using unpaired t-test) compared to an IL-22-Fc fusion (specifically a human Fc fused to hIL-22 N-terminus, hereinafter referred to as “hFc-hIL-22”) in a 16-day study in a diabetic mouse model is illustrated.

[0036] Figures 7A to 7C The effect of daily dosing of Derivative 1 and hFc-hIL-22 on three different target engagement biomarkers (mean ± SEM; *** indicates (A) p<0.0002, (B) p<0.0003 or (C) p<0.0026 using unpaired t-test) in a 16-day study in a diabetic mouse model is illustrated.

[0037] Figure 8 The dose-response curve of daily dosing of a derivative of the application (identified herein as Derivative 6) (three different doses) on blood glucose (mean ± SEM) compared to Derivative 1 and hFc-hIL-22 in a 13-day study in a diabetic mouse model is illustrated.

[0038] Figure 9A and Figure 9BFigure 9 illustrates the effect of Derivatives 1 and 6 in preventing liver injury in a paracetamol (APAP)-induced liver injury mouse model, as evidenced by plasma levels of two different liver enzymes. * indicates p<0.05 and ** indicates p<0.01 compared to vehicle + APAP using Dunnett’s test one-way linear model.

[0039] Figure 10 illustrates the effect of Derivatives 1 and 6 in (A) preventing apoptosis and (B) on cell proliferation in an APAP-induced liver injury mouse model. NS indicates not significant.

[0040] Figure 11 illustrates the effect of Derivative 6 compared to prednisolone in preventing and / or reducing (A) lung inflammation and (B) and (C) lung fibrosis in a bleomycin-induced lung injury rat model.

[0041] Figure 12 Figure 12 illustrates the effect of Derivative 6 in preventing colonic inflammation in a dextran sulfate sodium (DSS)-induced colitis mouse model. **** indicates p<0.0001 compared to vehicle (containing DSS).

[0042] Figure 13 Figure 13 illustrates the effect of Derivative 6 compared to hFc-hIL-22 in preventing mucosal epithelial damage in a DSS-induced colitis mouse model. Magnification 4x, scale bar = 500 pm.

[0043] Figure 14 Figure 14 illustrates plasma regenerating islet-derived protein 3 gamma (Reg3g) levels in a DSS-induced colitis mouse model as a measure of target engagement (Reg3g is a target engagement marker for IL-22).

[0044] Figure 15A and Figure 15B Figure 15 illustrates the effect of Derivative 1 in preventing liver injury in a Concanavalin A (ConA)-induced liver injury mouse model, as evidenced by serum levels of two different liver enzymes.

[0045] Figure 16 Figure 16 illustrates the effect of Derivative 6 on body weight in diet-induced obese mice compared to hFc-hIL-22 and semaglutide, a known fatty acid conjugated GLP-1 derivative. DETAILED DESCRIPTION

[0046] In the following, Greek letters are denoted by symbols rather than written names. For example, a = alpha, e = epsilon, g = gamma and m = mu. Amino acid residues can be identified by their full name, three letter code or one letter code, all of which are fully equivalent.

[0047] As used herein, the term "derivative of IL-22" refers to an IL-22 protein with a covalently attached fatty acid. The term includes derivatives in which the fatty acid is covalently attached directly to the IL-22 protein and those in which the covalent attachment is through a linker.

[0048] Covalent attachment of a fatty acid is a well-established technique for extending the half-life of peptides and proteins and is a way of directing a fatty acid from a peptide or protein. This is known from marketed products for type 1 and type 2 diabetes, such as insulin (detemir) and (degludec), and the glucagon-like peptide-1 (GLP-1) derivative (liraglutide) and (semaglutide).

[0049] The fatty acid attachment enables it to bind to albumin, preventing renal excretion and providing some steric protection against proteolysis. Advantageously, it provides minimal modification to IL-22 compared to Fc fusion or pegylation. In this respect, while Fc fusion and pegylation are intended to increase the size of IL-22 beyond the threshold for renal clearance, the derivative comprising a fatty acid covalently attached to the IL-22 protein retains a similar small size to the IL-22 protein. Therefore, since the fatty acid attachment is a minimal modification, it is believed that the resulting derivative retains similar properties to the native, including distribution, diffusion rate and receptor engagement (binding, activation and trafficking), and minimizes immunogenicity risk.

[0050] As mentioned above, fatty acid attachment has proven therapeutic effect 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 counter-intuitive to the inventors that a fatty acid could be covalently attached to IL-22 while retaining therapeutic effect. It is particularly surprising that this minimal modification to IL-22 could result in high potency (close to hIL-22) as well as extremely long circulating half-life.

[0051] Accordingly, in a first aspect, the present application relates to a derivative of IL-22 comprising a fatty acid covalently linked to the IL-22 protein. The fatty acid can be covalently linked to the IL-22 protein directly or through a linker, which itself can be designed with various subgroups. As used herein, the term "IL-22 protein" can refer to the native IL-22 protein, e.g. hIL-22 or a variant thereof. As further defined herein, a "variant" can be a protein having a similar amino acid sequence to the native protein.

[0052] In nature, the human IL-22 protein is synthesized with a signal peptide of 33 amino acids for secretion. The mature human IL-22 protein (i.e. hIL-22) is 146 amino acids long and has 80.8% sequence identity to murine IL-22 (the latter being 147 amino acids long). The amino acid sequence of hIL-22 is identified herein as SEQ ID NO. 1. Like other IL-10 family members, the IL-22 structure contains six alpha-helices (referred to as helices A to F).

[0053] The derivative of the present application can thus have the native amino acid sequence of hIL-22. Alternatively, it can have one or more amino acid sequence variations within the native sequence. It can additionally or alternatively comprise one or more amino acid sequence variations relative to the native sequence (i.e. outside the native sequence). Thus, in one embodiment, the derivative comprises a fatty acid covalently linked to hIL-22 or a variant thereof.

[0054] Expressions such as "within", "relative to", "corresponding to" and "equivalent to" are used herein to characterise the changes in the IL-22 protein and / or covalent linkage site of the fatty acid by reference to the sequence of the native protein (e.g. hIL-22). In SEQ ID NO. 1, the first amino acid residue of hIL-22 (alanine (Ala)) is designated as position 1.

[0055] Accordingly, a variation within the hIL-22 sequence is a variation of any one of residues numbered 1-146 in SEQ ID NO. 1. For example, substitution of Glu for the native Asp at residue 10 in hIL-22 is denoted herein as "D10E". If the derivative also has a fatty acid covalently linked at position 10, then this is referred to herein as a linkage at residue "10E".

[0056] However, the variations relative to the sequence of hIL-22 are variations outside of residues 1-146 in SEQ ID NO. 1. For example, Derivative 2 as defined herein comprises an N-terminal peptide of 15 amino acids in length. The residues in the N-terminal peptide are numbered as negative numbers starting with the residue connected to residue 1 in hIL-22, i.e. the first residue in the N-terminal peptide connected to residue 1 in hIL-22 is denoted "-1". Thus, since Derivative 2 has a covalently attached fatty acid at the 7th 个 residue of the N-terminal peptide starting at position -1, this is Cys, the covalently attached site of Derivative 2 is referred to herein as "-7C". However, according to WIPO Standard ST.25, the numbering used in the sequence listing of Derivative 2 naturally starts with 1; thus, position 1 in the sequence listing of Derivative 2 is actually residue -7 as referred to herein.

[0057] Two, three, four, five or more variations can be made within the native sequence to form a derivative of the application. For example, more than 10, 15, 20, 25, 50, 75, 100 or even more than 125 variations can be made in this regard. Any of residues 1-146 in the native sequence can be varied. 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, 77, 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 in hIL-22. Variations at residues 1, 21, 35, 64, 113, and / or 114 are particularly advantageous.

[0058] Variations within the native sequence are typically amino acid substitutions. As used herein, the term "substitution" can mean that an amino acid in the native protein is replaced with another amino acid. The substitution can be a conservative or non-conservative replacement. Exemplary substitutions are 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, A33C, 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, L92R, S93Y, N94C, N94Q, R95K, R95Q, L96E, S97K, T98C, T98N, T98S, C99V, H100S, E102S, G103D, D104Y, D105Y, L106E, L106Q, H107L, H107N, I108L, 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 is 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 the present application do not adversely affect IL-22 activity.

[0059] Particular 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, E54D, 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, 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;(xxxvii) A33C, N35Q, and N64Q; (xxxviii) A1H, P2H, I3H, S4H, S5H, C7G, R8G, L9S, D10S, K11G, N13G, F14S, Q15E, Q16V, P17L, 18F, Y19Q, N21Q, R22S, F24H, M25E, L26S, A27L, E29P, A30Q, L32R, A33N, D34F, N35H, 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, E84R, V86A, F88N, A90P, R91Y, L92R, S93Y, N94Q, R95K, L96E, S97K, T98N, C99V, H100S, G103D, D104Y, D105Y, L106E, 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 and all combinations of substitutions are contemplated and form part of the application.

[0060] The derivatives of the first aspect can typically comprise amino acid substitutions, whereby the natural residue is optionally replaced with Cys at any of the positions identified above, for example 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 comprised in the derivatives of the first aspect comprises a Cys residue at position 1 of hIL-22. The A1C substitution in combination with the substitution of the two glycosylation sites at positions 35 and 64 is particularly advantageous as it results in faster absorption without adversely affecting potency or half-life (see derivatives 6 and 10 in Examples 1 and 2).

[0061] Alternatively or additionally, the variation within the native sequence can be an amino acid insertion. Up to 5, 10, 15, 20, 25, 30, 35, 40, 45 or even up to 50 amino acids can be inserted in the native sequence. Trimers, pentamers, septamers, octamers, nonamers and tetradecamers are particularly advantageous in this respect. Exemplary sequences are shown in Table 1. The insertion can be made at any position in the native sequence, but insertion in helix A (e.g. at residue 30), in the loop-like CD (e.g. at residue 75), in helix D (e.g. at residue 85) and / or in helix F (e.g. at residue 124) is preferred.

[0062]

[0063] Table 1 : Exemplary amino acid insertion sequences

[0064] The sequence variation, if present, relative to the amino acid sequence of hIL-22 typically comprises an extension, e.g. the addition of a peptide at the N-terminus. The peptide can 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, 28-mers are particularly advantageous in this respect. Exemplary sequences are shown in Table 2. Suitably, the IL-22 protein comprised in the derivative of the first aspect comprises N-terminal G-P-G. In a particularly preferred embodiment, the derivative of the first aspect comprises a Cys residue at position 1 of hIL-22 (SEQ ID NO. 1) and N-terminal G-P-G. This has been found to result in a derivative with very good half-life and potency (see derivatives 1, 3 and 5 in Examples 1 and 2).

[0065]

[0066] Table 2: Sequences of exemplary N-terminal peptides

[0067] The sequence variation, if present, relative to the amino acid sequence of hIL-22 can comprise the addition of a peptide at the C-terminus. The peptide can consist of up to 5, 10, 15, 20, 25, 30, 35, 40, 45 or even up to 50 amino acids. Septamers are particularly advantageous in this respect, optionally having the amino acid sequence G-S-G-S-G-S-C (SEQ ID NO. 15).

[0068] In addition to the native or variant hIL-22 amino acid sequence as described herein, the derivative of the application can comprise an N-terminal peptide and a C-terminal peptide. Any combination of the N-terminal and C-terminal peptides described herein is envisaged and explicitly comprised in the application.

[0069] It will be appreciated that the application extends to any derivative of IL-22 comprising a fatty acid covalently linked to hIL-22 or a variant thereof. A "variant" can be a protein having at least 10% sequence identity to hIL-22. In one embodiment, the variant has at least 20% or even at least 30% sequence identity to hIL-22. The variant can "essentially" have the "amino acid sequence" of hIL-22, which can mean a sequence having at least 40% sequence identity to the amino acid sequence of hIL-22. Thus, in one embodiment, the derivative of the first aspect 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 to hIL-22. Exemplary IL-22 protein variants incorporated into particular derivatives of the application disclosed in the experimental section are shown in SEQ ID NO. 16-21.

[0070] The skilled person will understand how to calculate the percentage identity between two amino acid sequences. First an alignment of the two sequences is prepared and then the sequence identity value is calculated. The percentage identity of two sequences can take different values depending on: (i) the method used to align the sequences, for example ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs) or structural alignment from 3D comparison; (ii) the parameters used by the alignment method, for example local versus global alignment, the scoring matrix used (for example, BLOSUM62, PAM250, Gonnet, etc.) and the gap penalty, for example the function form and the constant.

[0071] After alignment, there are many different ways to calculate the percentage identity between two sequences. For example, the identity number can be divided by: (i) the shortest sequence length; (ii) the alignment length; (iii) the average length of the sequences; (iv) the number of non-gap positions; or (iv) the number of equivalent positions not including gaps. Furthermore, it will be appreciated that the percentage identity is also clearly dependent on the length. Thus, the shorter the pair of sequences, the higher the sequence identity that can be expected to occur by chance.

[0072] Accordingly, it will be appreciated that the accurate alignment of amino acid sequences is a complex process. The popular multiple alignment program ClustalW [48, 49] is the preferred way of generating a multiple alignment of proteins according to the present application. The parameters suitable for use with ClustalW are as follows: for protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, Matrix = Gonnet. For DNA and protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will appreciate that these and other parameters can need to be varied for optimal sequence alignment.

[0073] Preferably, the percentage identity between two amino acid sequences can then be calculated from the alignment such as (N / T)*100, where N is the number of positions in which the sequences share the same residue and T is the total number of positions compared. Thus, the most preferred method for calculating the percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using suitable parameter sets, for example as described above; (ii) inserting the values of N and T into the following formula: sequence identity = (N / T)*100.

[0074] Those skilled in the art will be aware of alternative methods for identifying similar sequences.

[0075] Suitably, the derivative of the first aspect comprises 200 or fewer amino acids. For example, the derivative comprises fewer than 190, fewer than 180, fewer than 170, fewer than 160 or even fewer than 150 amino acids. Suitably, the derivative will comprise at least 146 amino acids, however, this is the number of amino acids in hIL-22. It can comprise at least 150 amino acids, at least 160 amino acids, at least 170 amino acids or even at least 180 amino acids. The derivative of the present application can comprise a protein of any length within the ranges described above, but it will typically be between 146 and 180 amino acids in length.

[0076] Whether having a native or variant amino acid sequence, the derivative of the present application comprises a fatty acid covalently linked to the IL-22 protein. The fatty acid is typically covalently linked to the IL-22 protein via a linker. The fatty acid and linker are suitably linked to each other by an amide bond, and the linker is covalently linked to the IL-22 protein. The fatty acid and linker can thus be present as side chains on the IL-22 protein. It is surprising to the inventors that the covalently linked fatty acid does not adversely affect IL-22 activity. It is particularly surprising that the fatty acid linkage is associated with other advantages, such as extending the half-life.

[0077] The fatty acid can be any suitable fatty acid. In particular, the fatty acid can be of formula I:

[0078] HOOC-(CH2) x -CO-,

[0079] wherein x is an integer in the range of 10 to 18, optionally 12 to 18, 14 to 16, or 16 to 18, and * denotes the point of attachment to the IL-22 protein or linker. It can be a fatty diacid, for example a C12, C14, C16, C18, or C20 diacid. Advantageously, the fatty acid is a C16 or C18 diacid, and most advantageously a C18 diacid.

[0080] For example, -(CH2) x in formula I can be a straight chain alkylene where x is 10. This fatty acid can conveniently be referred to as a C12 diacid, i.e. a fatty dicarboxylic acid having 12 carbon atoms. Alternatively, -(CH2) x in formula I can be a straight chain alkylene where x is 12. This fatty acid can conveniently be referred to as a C14 diacid, i.e. a fatty dicarboxylic acid having 14 carbon atoms. In a similar manner, -(CH2) x in formula I can be a straight chain alkylene where x is 14 (C16 diacid), 16 (C18 diacid), or 18 (C20 diacid). Suitably, the derivative of the first aspect comprises a C14, C16, C18, or C20 diacid; more suitably a C16 or C18 diacid, and even more suitably a C18 diacid.

[0081] The diacid can be capable of forming a non-covalent association with albumin, thereby facilitating circulation of the derivative in the bloodstream. Shorter diacids (e.g. C16 diacids) have a lower affinity for albumin and therefore have a shorter half-life than longer diacids (e.g. C18 diacids). However, the shorter diacids are still long-acting derivatives with an expected half-life in humans of more than one day.

[0082] The fatty acid linkage itself also stabilises the IL-22 protein against proteolytic degradation. The resulting half-life is typically similar to that of the IL-22-Fc fusion (i.e. greatly improved compared to hIL-22).

[0083] The derivative of the first aspect can comprise a particular combination of fatty acid and IL-22 protein. For example, a C14, C16, C18, or C20 diacid can be linked to an IL-22 protein comprising a Cys residue at position 1 of hIL-22 and / or an N-terminal G-P-G. In one example, the derivative of the first aspect comprises a C18 diacid and the IL-22 protein comprises both a Cys residue at position 1 of hIL-22 and an N-terminal G-P-G.

[0084] As noted above, the fatty acid is suitably linked to a linker, which is linked to the IL-22 protein. The linker can comprise several linker elements, including one or more amino acids, such as one or more Glu and / or Lys residues. The linker can comprise an oxyethylene glycine unit or multiple linked oxyethylene glycine units, optionally 2 to 5 such units, advantageously 2 units. One or more OEG residues, C2DA and / or Ac groups are alternatively or additionally comprised. The linker can comprise a Cys reactive unit. As used herein, a "Cys reactive unit" can refer to a functional unit capable of reacting with the sulfur atom of Cys to produce a carbon-sulfur covalent bond. The Cys reactive unit can have any of several forms, but suitably comprises a carbon atom linked to a leaving group that is replaced by the sulfur atom of Cys during carbon-sulfur bond formation. The leaving group can be a halogen, optionally a bromine atom. The bromine leaving group can be alpha to an acetamide functional group; advantageously, it is a bromo-acetamide functional group. The leaving group can also be a functionalized hydroxyl in the form of a mesylate or tosylate, or a non-functionalized hydroxyl. Further, the leaving group can be a maleimide or other functional group. 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 can be used.

[0085] The fatty acid or linker can be linked 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. The native residue is typically replaced with Cys or Lys to enable linkage of the fatty acid or linker. Alternatively, the fatty acid or linker can be linked to a native Cys or Lys residue. Suitably, the fatty acid or linker is linked to a Cys residue substituted at position 1, 6, 33, 113, or 114 of hIL-22, or to a Cys residue at position -5, -7, or 153 relative to hIL-22. In particular, the fatty acid or linker can be linked to a Cys residue substituted at position 1 of hIL-22.

[0086] The attachment of the fatty acid or linker to the IL-22 protein is a covalent attachment. For example, a Cys-reactive fatty acid or linker can be used to attach the fatty acid or linker to a Cys residue in the IL-22 protein. The fatty acid or linker can be covalently attached to the sulfur atom of the Cys residue via a thioether linkage. Alternatively, a Lys-reactive fatty acid or linker can be used to attach the fatty acid or linker to a Lys residue in the IL-22 protein. The fatty acid or linker can alternatively be covalently attached to a free amine (-NH2) group at the N-terminus of the IL-22 protein (irrespective of the amino acid in position 1). The attachment can be carried out as for the Cys attachment, although sub-stoichiometric amounts of the fatty acid or linker containing the appropriate N-reactive species are used. The fatty acid or linker can be in the form of an aldehyde (N-reactive species) and covalently attached to the free amine using the classical known reductive amination.

[0087] Thus, the derivatives of the first aspect suitably comprise a C14, C16, C18 or C20 diacid attached via a linker to a hIL-22 variant, wherein the variant comprises an N-terminal G-P-G and a Cys residue at position 1 of hIL-22, and the linker is optionally attached to the Cys residue.

[0088] Exemplary derivatives of the first aspect comprise an IL-22 protein as shown in any one of SEQ ID NOs. 16-21. Particularly advantageous derivatives are shown in Table 3, in which Figures 1 to 4 are illustrated and exemplified herein.

[0089]

[0090] Table 3: Exemplary derivatives of IL-22

[0091] Figure 1 A illustrates a C18 diacid attached to a linker comprising a Cys-reactive unit. This is the fatty acid and linker (pendant) used in derivatives 1, 2 and 6 to 9. Figure 1 B illustrates a C16 diacid attached to a linker comprising a Cys-reactive unit. This is the fatty acid and linker (pendant) used in derivatives 3, 4 and 10. Figure 1 C illustrates a C14 diacid attached to a linker comprising a Cys-reactive unit. This is the fatty acid and linker (pendant) used in derivative 5.

[0092] Derivatives 1, 6 and 10 are respectively illustrated in Figures 2 to 4 .

[0093] The derivatives of the present application can exist in different stereoisomeric forms and the present application relates to all such forms.

[0094] According to a second aspect of the application, there is provided a method of making a derivative of the first aspect, comprising covalently linking a fatty acid to an IL-22 protein.

[0095] The method can be used to produce any of the different derivatives of IL-22 described or envisaged herein, but it is particularly advantageous when the fatty acid is covalently linked to a variant IL-22 protein. Thus, in one embodiment, the IL-22 protein used in the second aspect is a substituted form of hIL-22, optionally substituted at position 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.

[0096] The fatty acid can be obtained by any means known in the art, including recombinantly. Suitable fatty acids are commercially available, or are readily derivable from available starting materials using standard chemistry.

[0097] The IL-22 protein can be obtained by any means known in the art, including recombinantly. Production of recombinant hIL-22 has been described previously and is well known in the art. The desired variant IL-22 protein can be produced in a similar manner. The skilled worker in the field will be able to readily identify a suitable nucleic acid sequence encoding the desired variant IL-22 protein. The skilled worker will thus be able to readily perform this part of the application based on existing knowledge in the art. Suitably, the IL-22 protein is produced in a mammalian system using standard techniques, for example in Chinese hamster ovary (CHO) cells. A polyhistidine tag (His-tag) can be used to aid affinity purification of the recombinant protein.

[0098] In this regard, the IL-22 protein used in the present application can be prepared using a post-expression cleavable His tag, i.e. less than 10, preferably 6 histidine residues added to the N- or C-terminus, which can be purified by affinity to a nickel column. The His tag is attached to the N- or C-terminus of the protein via a linker which can be digested by a known protease, leaving the free IL-22 protein. The cleavable His tag can have the amino acid sequence HHHHHHGGSSGSGSEVLFQ (SEQ ID NO. 25) and the protease cleavable linker can be a tobacco etch virus (TEV) linker, the consensus sequence of the natural cleavage site of which is ENLYFQ\S (SEQ ID NO. 26), where the ‘\’ represents a cleaved peptide bond or a human rhinovirus 14 3C (HRV14-3C) protease cleavable linker with the EVLFQ consensus cleavage site. Cleavage can be achieved by incubating approximately 10 pg of protease with 2.5 pg of protein and 10 mM 2-mercaptoethanol at room temperature for 4 h.

[0099] To further illustrate the present application, a representative method of protein preparation is provided as follows. This method involves preparing plasmid DNA encoding the 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 culture medium before being increased in growth by the addition of known enhancers. The secreted IL-22 protein can then be harvested by known centrifugation and sterile filtration methods, followed by purification of the protein on a nickel column. After concentration and buffer exchange, the His tag is removed using the HRV14-3C protease, followed by alkylation with a fatty acid (described further below) and final purification and buffer exchange. The final product is analysed using SDS-PAGE, size exclusion chromatography or liquid chromatography and tandem mass spectrometry (LC-MS-MS), whether or not deglycosylation is performed, which can be used to ensure the quality of the final product.

[0100] The fatty acid can be covalently attached to the IL-22 protein directly or using a linker as described in the first aspect. The linker can be obtained by any means known in the art. If employed, a representative method of preparing the fatty acid and linker is as follows (using the C16 diacid used in derivative 10 as an example, but similar methods can be used to prepare any derivative).

[0101] A solution of N-(benzyloxycarbonyloxy)succinimide (100 g, 401 mmol) in dichloromethane (500 ml) was added to a solution of ethylenediamine (189 ml, 2.81 mol) in dichloromethane (750 ml). After 30 minutes the suspension was filtered, washed and concentrated in vacuo. The residue was diluted with toluene (750 ml), washed and extracted with dichloromethane (4 x 200 ml), dried over anhydrous sodium sulphate, filtered, concentrated in vacuo and diluted with hexane (200 ml). A solution of 4M hydrogen chloride in ether (100 ml, 400 mmol) was added to the solution, the resulting suspension was concentrated in vacuo and diluted with hexane (1 1). The precipitated solid was filtered, washed with hexane and dried in vacuo to give (2-aminoethyl)benzylcarbamate hydrochloride as a white powder.

[0102] 2-chlorotrityl resin 100-200 was loaded with 2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)- ethoxy]-ethoxy}-acetic acid (Fmoc-Ado-OH, 17.5 g, 45.4 mmol). The Fmoc group was 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) was added to the resin and the mixture shaken for one hour. The resin was filtered and washed. The Fmoc group was removed by treatment with 20% piperidine as before. The resin was washed as before.

[0103] A solution of (S)-2-(fluoren-9-ylmethoxycarbonylamino)-pentanedioic acid 1-tert-butyl ester (Fmoc-Glu-Ot Bu, 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) was added to the resin and the mixture shaken for one hour. The resin was filtered and washed as before. The Fmoc group was removed by treatment with 20% piperidine as before. The resin was washed as before.

[0104] 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 a mixture of N,N-dimethylformamide / dichloromethane (4:1, 200 ml) was added to the resin. The resin was shaken for one hour, filtered and washed with N,N-dimethylformamide (3 x 250 ml), dichloromethane (2 x 250 ml), methanol (2 x 250 ml) and dichloromethane (6 x 250 ml). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (250 ml) for 18 hours. The resin was filtered off and washed with dichloromethane (2 x 250 ml), a mixture of 2-propanol / dichloromethane (1 :1, 2 x 250 ml), 2-propanol (250 ml) and dichloromethane (3 x 250 ml).

[0105] The solutions were combined, the solvent was evaporated and the crude product was purified by flash column chromatography. The pure (S)-22-(tert-butoxycarbonyl)-41,41-dimethyl- 10,19,24,39-tetraoxo-3,6,12,15,40-pentaoxa-9,18,23-triazatetracontane acid was dried in vacuo to give a pale yellow thick yellow oil.

[0106] Subsequently 2-(7-aza-lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU, 11.4 g, 30.1 mmol) and triethylamine (8.77 ml, 62.9 mmol) were 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-triazatetracontane acid (22.4 g, 27.4 mmol) in dry dichloromethane (110 ml). Triethylamine (72 ml, 41.0 mmol) was added to a suspension of (2-amino- ethyl)-carbamic acid benzyl ester hydrochloride (6.94 g, 30.1 mmol) in dry dichloromethane (165 ml) and the resulting mixture was added to the above solution. The mixture was stirred at room temperature overnight and then evaporated to dryness. The residue was redissolved and washed; dried over anhydrous sodium sulphate and evaporated by column chromatography (silica gel 60, 0.040 to 0.060 mm; eluent: dichloromethane / methanol 95:5) to give 15-[(S) 3-(2-{2-[(2-{2-[(2-benzyloxy- carbonylamino-ethylcarbamoyl)-methoxy]-ethoxy}ethyl-carbamoyl)methoxy]ethoxy}- ethylcarbamoyl)-l-tert-butoxycarbonylpropylcarbamoyl]-pentadecanoic acid tert-butyl ester as a pale yellow thick oil.

[0107] Palladium on carbon (10%, 1.27g, 1.20mmol) was added to a solution of the above compound (23.8g, 24.0mmol) in methanol (350ml) and the resulting mixture hydrogenated at atmospheric pressure for four hours. The catalyst was filtered off and the filtrate evaporated to dryness. The residue was evaporated from dichloromethane several times to remove methanol residues and dried under vacuum to give (S)-1-amino-25-tert-butoxycarbonyl)-4,13,22,27-tetraoxo-6,9,15,18-tetraoxa-3,12,21,26-tetraazatetracontan-42-oic acid tert-butyl ester as a colourless thick oil.

[0108] N,N-diisopropylethylamine (4.98ml, 28.6mmol) was added to a solution of the above amine (20.5g, 23.8mmol) in dry dichloromethane (290ml) under argon at -30°C. Bromoacetyl bromide (2.48ml, 28.6mmol) was added dropwise and the resulting solution stirred at -30°C for a further three hours. The cooling bath was removed, the mixture stirred at room temperature for one hour and the solvent removed in vacuo. The residue was redissolved in ethyl acetate (450ml) and washed with a 5% aqueous solution of citric acid (300ml). The phases were separated over one hour. The organic layer was allowed to separate overnight to give three phases. The clear aqueous layer was removed and the remaining two phases shaken with a saturated aqueous solution of potassium bromide (100ml). The phases were allowed to separate overnight, the aqueous phase removed and the organic phase dried over anhydrous sodium sulphate. The solvent was removed in vacuo and the residue purified by flash column chromatography: dichloromethane / methanol 95:5) to give (S)-1-bromo-28-tert-butoxycarbonyl)-2,7,16,25,30-pentaoxo-9,12,18,21-tetraoxa-3,6,15,24,29-pentaazapentapentacontan-45-oic acid tert-butyl ester as a colourless solid.

[0109] The above compound (19.5g, 19.8mmol) was dissolved in trifluoroacetic acid (120ml) and the resulting solution stirred at room temperature for 1.5 hours. The trifluoroacetic acid was removed in vacuo and the residue evaporated from dichloromethane (6 x 200ml). Diethyl ether (200ml) was added to the oily residue and the mixture stirred overnight to give a suspension. The solid product was filtered off, washed with diethyl ether and hexane and dried under vacuum to give the desired product 15-{(S)-1-carboxy 3-[2-(2-{[2-(2-{[2-(2-bromoacetylamino)ethylcarbamoyl]methoxy}-ethoxyethyl- carbamoyl]methoxy}ethoxy-ethylcarbamoyl]propylcarbamoyl}pentadecanoic acid as a white powder.

[0110] The fatty acid or linker can be covalently attached to the IL-22 protein using standard procedures in the art. If a linker is used, the IL-22 protein can be enabled to covalently attach to the fatty acid. As a non-limiting example, a Cys-reactive fatty acid or linker can react with the sulfur atom of a Cys residue in the IL-22 protein, thereby forming a thioether bond. Suitable conditions for the covalent attachment step can be, for example, as follows: Tris aqueous solution is added to a solution of IL-22 protein (70 mg) in Tris and NaCl buffer (1.35 mg / ml) to adjust the pH to 8. Bis(p-sulfonatophenyl)-phenylphosphine disodium dihydrate (BSPP) salt (12 mg) dissolved in water is added and gently stirred at room temperature for four hours. 15-{(S)-1 -Carboxy-3-[2-(2-{[2-(2-{[2-(2-bromoacetylamino)- ethylcarbamoyl]ethoxy}ethoxy)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]propyl- carbamoyl}pentadecanoic acid (19 mg, 0.022 mmol) in ethanol (0.5 ml) is added and the mixture is gently stirred overnight. MiliQ water (150 ml) is added to reduce the conductivity to 2.5 mS / cm. The mixture is then purified on a MonoQ 10 / 100 GL column using anion exchange using a binding buffer (20 mM Tris, pH 8.0), an elution buffer (20 mM Tris, 500 mM NaCl, pH 8.0), a flow rate of 6 ml over 60 column volumes and a gradient of 0-80% elution buffer.

[0111] The derivatives of the present application can be purified using any suitable procedure known in the art, such as chromatography, electrophoresis, differential solubility, or extraction.

[0112] As described herein, the inventors surprisingly found that fatty acids can be covalently attached to IL-22 proteins while retaining biological activity. It is particularly surprising that such minimal modification to IL-22 can result in both high potency (approaching hIL-22) as well as extremely long circulating half-life. This particular combination of properties can be highly desirable.

[0113] The potency of the derivatives can be determined in an in vitro assay 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 a phospho-STAT3 (pSTAT3) response reporter. Alternatively, HepG2 cells endogenously expressing the IL-22 receptor can be used. Activation of the receptor leads to activation of the STAT3 signaling pathway, which can be measured, for example, using a luciferase reporter with a STAT3-induced promoter or by measuring pSTAT3. Non-limiting examples of such assays are described in Example 2. In vivo potency can be determined in animal models or in clinical trials, as known in the art.

[0114] The median effective concentration (EC 50 ) value is commonly used as a measure of drug potency. Because this represents the concentration of drug required to produce 50% of the maximum effect, the lower the EC 50 value, the better the potency. Suitably, the potency (EC 50 value) of a derivative of the application is 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, measured using IL-22 receptor-mediated STAT3 activation in cells, for example as described in Example 2. Suitably, the potency (EC 50 value) of a derivative of the application is less than 15 nM, less than 12 nM, less than 10 nM, less than 7 nM or even less than 5 nM, measured by measuring pSTAT3 in cells, for example as described in Example 2.

[0115] Advantageously, the potency of the derivatives of IL-22 can be higher than the potency of IL-22-Fc fusions. For example, Genentech reported a 34-fold reduction in in vitro potency of their IL-22-Fc fusion UTTR1147A compared to hIL-22 (Stefanich et al., Biochem Pharmacol, 2018, 152:224-235). In contrast, covalent attachment of a fatty acid to hIL-22 has been shown to result in only a seven-fold reduction in potency (see Derivative 1 in the Examples). While both IL-22-Fc fusions and derivatives of the application are comparable in their improved half-life and at least in some cases biological function compared to hIL-22, the derivatives of the application can have the additional advantage of minimal loss of potency.

[0116] The circulating elimination half-life (T 1 / 2). Suitable methods are described in Example 1. As a non-limiting example, the derivative of the first aspect has a circulating half-life of at least one hour, at least three hours, at least five hours, or even at least eight hours after subcutaneous or intravenous administration to mice. The derivative can have a circulating half-life of at least three hours, at least five hours, at least eight hours, at least 10 hours, or even at least 13 hours after subcutaneous or intravenous administration to rats. The derivative can have a circulating half-life of at least 25 hours, at least 40 hours, at least 70 hours, or even at least 100 hours after subcutaneous or intravenous administration to a minipig (all e.g. as determined as described in Example 1).

[0117] As exemplified herein, the inventors have also found that the derivatives of the application are rapidly absorbed in vivo. Advantageously, the derivatives can be absorbed faster than the IL-22-Fc fusion. Mean absorption time is an accurate parameter to measure uptake as it is independent of dose and maximum plasma concentration after drug administration. It can be calculated from the mean residence time, i.e. the time a drug spends in the body after absorption is complete before elimination. The derivatives of the application suitably have a mean absorption time of less than 100 h, less than 90 h, less than 80 h, less than 70 h, or even less than 60 h (e.g. as determined as described in Example 1).

[0118] The derivatives of the application also have good biophysical properties, such as high physical stability and / or solubility, which can be measured using standard methods in the art.

[0119] According to a third aspect of the application, there is thus provided a pharmaceutical composition comprising a derivative of the first aspect and a pharmaceutically acceptable vehicle.

[0120] The pharmaceutical composition of the third aspect can comprise any of the different derivatives of IL-22 described or envisaged herein. Suitably, it comprises one of the IL-22 derivatives identified herein as Derivatives 1 to 10.

[0121] The derivative of the first aspect or the pharmaceutical composition of the third aspect will suitably exhibit an increased circulating elimination half-life compared to hIL-22. Advantageously, it will exhibit an increase in circulating elimination half-life of at least 50%, at least 75%, at least 100% or more compared to hIL-22.

[0122] The pharmaceutical composition of the third aspect can be prepared by combining a therapeutically effective amount of a derivative of the first aspect with a pharmaceutically acceptable vehicle. The formulation of pharmaceutical active ingredients with various excipients is known in the art.

[0123] A "therapeutically effective amount" of the derivative of the first aspect is any amount of the derivative that, when administered to a subject, is needed to treat a disease, disorder, or condition or to produce a desired effect.

[0124] For example, the therapeutically effective amount of the derivative used can be from about 0.001 mg to about 1000 mg, and preferably from about 0.01 mg to about 500 mg. Preferably, the amount of the derivative is from about 0.1 mg to about 100 mg, and most preferably from about 0.5 mg to about 50 mg. As a guide, the dose of the derivative used in the mice of Example 3 described herein was 0.5 mg / kg (subcutaneously administered).

[0125] A "pharmaceutically acceptable vehicle" as referred to herein is any known compound or combination of known compounds known to those skilled in the art to be useful in formulating a pharmaceutical composition.

[0126] In one embodiment, the pharmaceutically acceptable vehicle can be a solid; optionally, the composition can be in the form of a powder for re-suspension. The solid pharmaceutically acceptable vehicle can include one or more substances which can also act as flavouring agents, lubricants, solubilising agents, suspending agents, dyes, fillers, glidants, inert binders, preservatives or dyes. The vehicle can also be an encapsulating material. In a powder, the vehicle is a finely divided solid with which the finely divided derivative according to the application is mixed. The powder preferably contains up to 99% of the derivative. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose and ion exchange resins.

[0127] In another embodiment, the pharmaceutical vehicle can be a gel, and the composition can be in the form of a cream or the like.

[0128] However, the pharmaceutical vehicle can be a liquid; optionally, the pharmaceutical composition is in the form of a solution. Liquid vehicles are used to prepare solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The derivative according to the application can be dissolved or suspended in a pharmaceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture of both or a pharmaceutically acceptable oil or fat. The liquid vehicle can contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickening agents, colorants, viscosity regulators, stabilizers or tonicity regulators. Suitable examples of liquid vehicles for parenteral administration include water (partly containing the above-mentioned additives, such as cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including mono- and polyhydric alcohols, such as glycols), and derivatives thereof, and oils (such as, for example, fractionated coconut oil and peanut oil). For parenteral administration, the vehicle can also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are used in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0129] Thus, the process for preparing the pharmaceutical composition of the application can comprise the usual steps that are standard in the art.

[0130] Thus, according to a fourth aspect of the application, there is provided a derivative of the first aspect or a pharmaceutical composition of the third aspect for use in therapy. There is also provided a method of treating a subject with a derivative of the application or a pharmaceutical composition comprising the derivative. Any of the different derivatives of IL-22 described or envisaged herein are expressly included in these aspects of the application.

[0131] As used herein, terms such as "treatment" and "therapy" expressly include the treatment, amelioration or prevention of a disease, disorder or condition.

[0132] The derivative of IL-22 or the pharmaceutical composition comprising the derivative can be administered directly to the subject to be treated. The derivative or the pharmaceutical composition can be administered by any means, including by inhalation, by injection, topical or ocular administration. When administered by inhalation, it can be administered through the nose or the mouth. Preferably, the derivative or the pharmaceutical composition is administered by injection, typically subcutaneously or intravenously. Thus, due to its smaller size and higher potency, the derivative has a clear advantage over Fc-fusions in terms of administration flexibility (e.g. by injection, by inhalation, topical application or ocular delivery). It will be appreciated that administration of the derivative of the application to the subject to be treated will result in an increased circulation time compared to hIL-22 and this will contribute to the treatment of the disease, disorder or condition. As mentioned above, "treatment" also includes amelioration and prevention of a disease, disorder or condition.

[0133] Liquid pharmaceutical compositions are sterile solutions or suspensions, which can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, and especially subcutaneous or intravenous injection. The derivatives can be prepared as sterile solid compositions which can be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

[0134] Forms useful for inhalation include sterile solutions, emulsions, and suspensions. Alternatively, the derivatives can be delivered by or in the form of a fine powder or an aerosol. Nasal inhalants can suitably take the form of a fine powder or aerosol nasal spray or modified or delivery device.

[0135] Topical formulations include solutions, creams, foams, gels, lotions, ointments, pastes, tinctures, and powders. They can be epicutaneous formulations, i.e., applied directly to the skin, or applied to the mucosa.

[0136] Formulations for ophthalmic application are generally solutions, suspensions, and ointments for topical application, e.g., in the form of eye drops. Alternatively, sterile solutions or suspensions can be used by intraocular injection. The derivatives can be prepared as sterile solid compositions which can be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. The formulations can be used for subconjunctival, intravitreal, retrobulbar, or intracameral injection.

[0137] The derivatives or pharmaceutical compositions of the present application can be administered to any subject in need thereof. As used herein, a "subject" can be a vertebrate, a mammal, or a domestic animal. Thus, the derivatives and compositions according to the present application are useful for the treatment of any mammal, e.g., farm animals (e.g., horses), pets, or for other veterinary applications. Most preferably, the subject is a human. The derivatives and compositions need not only be administered to those subjects already showing signs of a disease, disorder, or condition. Rather, they can be administered as a purely prophylactic measure to subjects who are apparently healthy, in order to prevent the possibility of such a disease, disorder, or condition in the future.

[0138] It will be appreciated that the derivatives and compositions of IL-22 according to the present application can be used in monotherapy (i.e., the sole use of the derivative or composition), which is useful for the treatment of a disease, disorder, or condition. Alternatively, the derivatives and compositions according to the present application can be used as an adjunct to, or in combination with, known therapies for the treatment of a disease, disorder, or condition.

[0139] It will be appreciated that the amount of IL-22 derivative required will be determined by its biological activity, half-life and bioavailability, which in turn will depend on the mode of administration, the physicochemical characteristics of the derivative and composition and whether it is used as a monotherapy or in a combination therapy. The frequency of administration will also be influenced by the half-life of the derivative in the body of the subject being treated. The optimal dosage to be administered can be determined by one skilled in the art and will vary with the particular derivative used, the strength of the pharmaceutical composition, the mode of administration, and the progression of the disease, disorder, or condition. Other factors depending on the particular subject being treated, including the subject's age, weight, gender, diet, and time of administration, will result in the need to adjust the dosages.

[0140] Generally, a daily dosage of between 0.001 pg / kg body weight and 10 mg / kg body weight of the IL-22 derivative according to the present application can be used to treat a disease, disorder, or condition, depending on which derivative or composition is used. More preferably, the daily dosage is between 0.01 pg / kg body weight and 1 mg / kg body weight, more preferably between 0.1 pg / kg and 500 pg / kg body weight, and most preferably between about 0.1 pg / kg and 100 pg / kg body weight.

[0141] The IL-22 derivative or composition can be administered before, during, or after the onset of the disease, disorder, or condition. The daily dosage can be given as a single administration (e.g., a single daily injection). Alternatively, the derivative or composition can need to be administered two or more times within a day. For example, the derivative can be administered in two daily doses (or more daily doses, depending on the severity of the disease, disorder, or condition being treated) between 0.07 pg and 700 mg (i.e., assuming a body weight of 70 kg). A patient receiving treatment can take the first dose upon waking and then a second dose in the evening (if a two-dose regimen is employed) or every 3 or 4 hours thereafter. Alternatively, the dose can be given once a week, once every two weeks, or once a month, or more frequently (e.g., twice or three times a week). Known procedures, such as those commonly used by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), can be used to develop a particular formulation of the derivative and composition according to the present application and an accurate treatment regimen (e.g., the daily dosage of the dose and the frequency of administration).

[0142] Many studies have demonstrated the key role of IL-22 in various epithelial injury models, notably in the lung, liver, intestine, kidney, skin, pancreas and thymus. Mechanistically, several researchers have well-documented in their studies that several pathways, such as anti-apoptosis, proliferation, innate immunity, anti-oxidative stress, anti-fibrosis and stem / progenitor cell recruitment, can modulate the effects of IL-22. Key mechanistic findings have been further confirmed in vitro using human cell lines or in human ex vivo models, such as primary human intestinal organoids. Thus, the strong role of IL-22 in preventing cell death, ensuring regeneration and controlling epithelial injury inflammation has been well-documented.

[0143] Many studies were performed by analyzing genetic models of injury (IL-22 knock-out or transgenic overexpression) that were subjected to injury. In these studies, the absence of IL-22 or the overexpression of IL-22 would occur at the time of injury. In other studies, IL-22 was neutralized by antibodies at the time of injury, and in some cases, IL-22 was neutralized after the acute injury phase (e.g. sub-acute or fully into the regeneration phase). Other studies were closer to the therapeutic scenario by observing the effects of exogenously administered IL-22. It is important to note that, upon a thorough review of the existing literature, different models, whether knock-out, overexpression, IL-22 neutralization before or after injury or exogenous protein administration, all paint the same picture of IL-22 protecting the injured organ and driving regeneration. This demonstrates the broad applicability of IL-22 therapeutic potential and the broad time window, and also shows the reason for the need for an IL-22 protein that is more long-acting than hIL-22.

[0144] Thus, according to a fifth aspect of the application, 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 metabolic, liver, pulmonary, intestinal, kidney or skin disease, disorder or condition. Any of the different derivatives of IL-22 described or envisaged herein are expressly included in this aspect of the application.

[0145] The metabolic disease, disorder or condition can be obesity, type 1 diabetes, type 2 diabetes, hyperlipidemia, hyperglycemia or hyperinsulinemia.

[0146] The liver disease, disorder or condition can be NAFLD, NASH, liver cirrhosis, alcoholic hepatitis, acute liver failure, chronic liver failure, ACLF, acetaminophen-induced liver toxicity, acute liver injury, sclerosing cholangitis, biliary cirrhosis or pathological conditions resulting from surgery or transplantation.

[0147] The pulmonary disease, disorder or condition can be COPD, cystic fibrosis, bronchiectasis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, chemical injury, viral infection, bacterial infection or fungal infection.

[0148] The intestinal disease, disorder or condition can be IBD, ulcerative colitis, Crohn's disease, GvHD, chemical injury, viral infection or bacterial infection.

[0149] The renal disease, disorder or condition can be acute kidney disease or chronic kidney disease.

[0150] The skin disease, disorder or condition can be a wound, an inflammatory disease or GvHD.

[0151] Also provided is a method of treating a subject having a condition responsive to IL-22 treatment (e.g. one or more of the diseases, disorders or conditions described above) with a derivative of IL-22 or a pharmaceutical composition comprising the same.

[0152] The derivative of IL-22 has all the features specified for the first aspect of the application. The pharmaceutical composition has all the features specified for the third aspect of the application. The method of treating a subject having a condition responsive to IL-22 treatment (e.g. one or more of the diseases, disorders or conditions described above) has all the features specified for the fourth aspect of the application.

[0153] There is no restriction as to which patient should be administered which derivative of IL-22 or composition as described herein. Rather, it is intended that any derivative and composition described herein is administered to any patient as described herein.

[0154] All features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, can be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0155] For a better understanding of the application, and to show how embodiments of the application can be implemented, reference will now be made, by way of example, to the following examples which are not intended to limit the application in any way.

[0156] Examples

[0157] The materials and methods used in the studies described in the examples are as follows, unless otherwise stated.

[0158] Derivatives

[0159] Table 4 provides an overview of the derivatives of IL-22 and comparators represented in the data set.

[0160] Derivatives of IL-22 have various backbones, fatty acid types, and covalent attachment sites, and thus represent the diversity of derivatives encompassed by the present invention. The linker used in all cases is γGlu-OEG-OEG-C2DA-Ac. In all cases, the linker is attached to residue 1C, with the exception of derivatives 2(-7C), 4(-7C), 8(6C), and 9(33C). While derivative 7 exemplifies covalent attachment at 1C, it lacks the G-P-G N-terminal peptide present in all other derivatives having a fatty acid covalently attached at 1C.

[0161] Comparators are hIL-22, hFc-hIL-22 (a recombinant fusion protein), and hIL-22 variants (i.e., hIL-22 having only one or more backbone variations).

[0162]

[0163]

[0164] Table 4: Overview of key derivatives and comparators represented in the data set

[0165] Quality control analysis of the derivatives produced for the examples was performed as follows.

[0166] The intact mass of the protein in the sample after deglycosylation was determined by adding 20 μΐ of 1 mg / ml sample to 2 μΐ of N-glycosidase F at room temperature for 48 h. The sample was then diluted to 0.2 mg / ml with PBS pH 7.4 and analyzed using Synapt G2 coupled to Waters Synapt G2 and Waters MassLynx 4.1. A 10-90 column Acquity UPLC protein BEH C4 1.7 μιη 1 x 100 mm was used with the following mobile phases: A: 0.1% formic acid in water; and B: acetonitrile, 0.09% formic acid. The flow rate was 120 μΐ / min, UV 214 nm (20 pts / s), and the gradient was as shown in Table 5.

[0167] Time (min) Flow rate (ml / min) A％ Initial 0.12 90 1 0.12 90 17 0.12 10 18 0.12 0 19 0.12 0 20 0.12 90 25 0.12 90

[0168] Table 5: Gradient (% and min) used for quality control of IL-22 derivatives. Results are shown in Table 6.

[0169]

[0170] Table 6: Measured mass and retention time of key derivatives of IL-22

[0171] Thus, the quality control data confirm that the intended derivatives have indeed been produced.

[0172] The following is an exemplary protocol, which is intended to illustrate the claimed invention only. The exact number of animals and time course used in the study can vary as known to one skilled in the art.

[0173] Example 1 - Pharmacokinetic study

[0174] Methods

[0175] Selected derivatives were subjected to pharmacokinetic studies in mice (n=27), rats (n=4-8) and minipigs (n=2-5). The derivatives of IL-22 were tested together with hIL-22, hFc-hIL-22 and / or hIL-22 variants as comparators.

[0176] (i) Mice and rats

[0177] Thirty 8-week old C57B1 / 6 male mice and five Sprague Dawley male rats were obtained from Taconic Biosciences. The mice were housed in groups of ten. The animals were acclimatized for one week prior to the experiment. Body weight was measured prior to dosing, which is important for pharmacokinetic calculations. The animals were kept awake and had access to food and water throughout the experiment.

[0178] All derivatives and comparators were prepared as 0.3 mg / ml solutions in PBS, pH 7.4 for use in mice and as 0.5 mg / ml solutions for use in rats. A dose of 2.0 mg / kg was tested in mice. A dose of 1 mg / kg was tested in rats.

[0179] The derivatives and comparators were administered subcutaneously to the animals. Blood samples were collected at specific time points after dosing.

[0180] Sparse sampling was used for mice; thus, 27 mice were dosed with IL-22 derivatives or comparators and blood samples were collected from three different mice at each of the following time points: 5 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, 120 min, 150 min, 3 h, 4 h, 6 h, 8 h, 16 h, 24 h, 32 h and 48 h. Thus, only two samples were collected from each mouse during the study. After the last sample was taken, the mice were euthanized by cervical dislocation.

[0181] Five rats were dosed with IL-22 derivatives or comparators and three blood samples were taken at each of the following time points: 5 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, 120 min, 150 min, 3 h, 4 h, 6 h, 8 h and 24 h. During the study, 17 samples were taken from each rat. After the last sample was taken, the rats were euthanized with carbon dioxide.

[0182] Blood samples (100 μl) were taken from mice and rats by tongue bleeding and transferred to EDTA tubes (VetMed 200 K3E, Sarstedt nr 09.1293.100). The blood was centrifuged at 8000 G, 4°C for five minutes within 20 minutes of withdrawal. The plasma samples (40 to 50 μl) were transferred to semi-micronic tubes.

[0183] (ii) Minipigs

[0184] Nine-month old female minipigs with a body weight of approximately 15 kg were obtained from Ellegaard Gd-Animal Production A / S, Dalmose, Denmark. The minipigs were housed in groups of three in pens (2.5 m x 1.5 m) with wood shavings as bedding. The minipigs had free access to water and were fed twice daily with a commercial minipig diet (Special Diets Services, Witham, UK). The minipigs were acclimatized for approximately 18 days prior to surgery (catheter insertion) during which time they were group-housed and trained to accept subcutaneous dosing and catheter blood sampling. Three to five days prior to surgery, the minipigs were housed individually. Six days prior to dosing, all minipigs were inserted with two central venous catheters (Cook Medical, C-TPNS-6.5-90-REDO, silicone, French size 6.5, 106 cm long TPN type) which allowed for a recovery time of at least five days prior to study start (dosing). All derivatives and comparators 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).

[0185] The minipigs were lightly anaesthetized with Propofol during dosing. Intravenous injections were administered to the minipigs through the long central catheter. After administration, the catheter was flushed with 10 ml sterile saline. Subcutaneous injections were performed using a 25G needle at a depth of 5 mm. The needle was kept in the skin for 10 s after injection to avoid backflow.

[0186]

[0187] ​​Blood samples were collected from miniature pigs at the following time points following intravenous administration: 1.5h, 2h, 3h, 4h, 6h, 8h, 10h, 12h, 24h, 28h, 48h, 72h, 96h, 144h, 168h, 192h, 216h, 240h, 264h, 312h, 336h, 360h, 384h, 408h, 432h, and 480h. Blood samples were collected at the following time points after subcutaneous administration: 1.5h, 2h, 3h, 4h, 5h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 46h, 52h, 72h, 96h, 144h, 168h, 192h, 216h, 240h, 264h, 312h, 336h, 360h, 384h, 408h, 432h, and 480h.

[0188] Collect a 1 ml blood sample from a miniature pig into an EDTA tube (1.3 ml tube containing K3EDTA, yielding 1.6 mg K3EDTA / ml blood (Sarstedt, Germany)). Incubate the sample on wet ice for up to 30 min until centrifuged (10 min, 4 °C, 2000 G). Transfer 200 μl of plasma to a Micronic tube for measuring IL-22 derivatives or comparisons and store at -20 °C until analysis.

[0189] (iii) Sample processing

[0190] Plasma levels of IL-22 derivatives or comparatives were assessed using internally developed luminescent oxygen channels as described above. The assay was performed using a Perkin Elmer Envision reader (Poulsen et al., J Biomol Screen, 2007, 12(2):240-7). During the assay, a concentration-dependent bead-analyte-immune complex was generated, producing light output, which was measured. Antibody conjugation to the beads, antibody biotinylation, and LOCI assay procedures were performed as previously described (Petersen et al., J Pharmaceut Biomed, 2010, 51(1):217-24). Calibrators and quality control (QC) samples were generated in the same matrix as the study samples. The assay precision (%CV) for all test samples was evaluated and showed less than 20%.

[0191] The assay uses receptor beads conjugated with anti-human IL-22 monoclonal antibody (R&D Systems MAB7822) and biotinylated monoclonal antibody (R&D Systems BAM7821; raised against human IL-22) and universal streptavidin-coated donor beads. The lower limit of quantification (LLOQ) for human IL-22 in rat plasma was 4 pM. However, each derivative or comparator was measured against a calibrator for the same derivative. The cross-reactivity of each derivative or comparator for hIL-22 was measured and used to adjust the assay sensitivity.

[0192] The plasma concentration-time curves in minipigs were measured using non-compartmental analysis (NCA) in Phoenix WinNonlin Professional 6.4 (Pharsight Inc). The calculations used a single concentration, weighted 1 / (Y*Y) and used linear-log trapezium. Intravenous dosing was used because the circulating elimination half-life (T 1 / 2 ) was the primary screening parameter. Clearance and volume of distribution were secondary parameters of interest, hence the reason for frequent blood sampling on study day 1.

[0193] The only parameter measured for the evaluation of the pharmacokinetics in mice and rats was the circulating elimination half-life (T 1 / 2 ). In minipigs, other parameters measured were the maximum (peak) plasma concentration (C max ) after administration of the drug, the time to reach C max (T max ), the area under the plasma drug concentration-time curve normalized for the dose of drug administered (AUC; which reflects the actual exposure of the body to the drug after administration of a certain dose of drug) (AUC / D), the mean residence time (MRT; i.e. the time the drug spends in the body after absorption is complete before elimination) the mean absorption time (MAT) for the dose administered and the systemic availability (i.e. bioavailability) (F). MAT was calculated as the MRT after subcutaneous administration (MRT SC ) minus the MRT after intravenous administration (MRT IV ).

[0194] Results

[0195] Table 7 shows the results obtained in mice, Table 8 shows the results obtained in rats and Tables 9 and 10 show the results obtained in minipigs. ND = not determined. IV = intravenous administration. SC = subcutaneous administration.

[0196]

[0197] Table 7: Pharmacokinetic data obtained in mice

[0198] As shown in Table 7, hIL-22 variants with backbone variations only (Comparative 1 and 3) have short circulating half-lives, regardless of the route of administration. Extension with Fc fusion (hFc-hIL-22) significantly increased the half-life. Covalent attachment of fatty acids (C18 diacid; Derivatives 1 and 6) resulted in moderate circulating half-lives in mice. The circulating time of IL-22 derivatives was longer in mice when administered subcutaneously compared to intravenously.

[0199]

[0200] Table 8: Pharmacokinetic data obtained in rats

[0201] As shown in Table 8, hIL-22 variant with backbone variations only (Comparative 1) has a short circulating half-life. Covalent attachment of fatty acids (Derivatives 1, 3 and 6) resulted in increased circulating half-lives in rats, regardless of the fatty acid employed (C16 versus C18 diacid) and route of administration. The circulating time of IL-22 derivatives was generally longer when administered subcutaneously compared to intravenously.

[0202]

[0203] Table 9: Pharmacokinetic data obtained in minipigs

[0204] As shown in Table 9, hIL-22 variants with backbone variations only (Comparative 1 and 2) have short circulating half-lives, comparable to hIL-22. Comparative Fc fusion (hFc-hIL-22) and all derivatives of IL-22 (Derivatives 1, 6 and 10) have significantly increased circulating half-lives. The circulating half-lives of derivatives of IL-22 in minipigs when administered intravenously exceed 50 hours, comparable to the comparative IL-22-Fc fusion.

[0205]

[0206] Table 10: Pharmacokinetic data obtained in minipigs

[0207] As shown in Table 10, the derivative of IL-22 (Derivative 6) demonstrated faster MAT compared to the comparative Fc fusion (hFc-IL-22). MAT is simply a comparison of T max More accurate measure of drug uptake as it also takes into account the difference in C max (T max affected by dose and C max ). This study used minipigs instead of mice or rats because it is more similar to humans.

[0208] Conclusion

[0209] The known fatty acid alkylated GLP-1 derivative semaglutide has a half-life in minipigs of 46 hours (Lau et al., J Med Chem, 2015, 58(18):7370-80) and a half-life in humans of 160 hours, corresponding to a once weekly dosing profile with a peak to trough ratio of 2. The Fc-fusion GLP-1 derivative dulaglutide has a similar half-life.

[0210] The demonstrated derivatives of IL-22 have a half-life in minipigs of at least 40 hours upon subcutaneous administration and more than 50 hours upon intravenous administration, thus hypothesizing that they correspond to a once weekly dosing profile with a peak to trough ratio of 2 in humans.

[0211] Thus, the data show that the derivatives of the present application improve the circulating half-life of IL-22 and exhibit optimized pharmacokinetic and pharmacodynamic properties, thus providing new and improved treatments for various indications, including metabolic, liver, pulmonary, intestinal, renal, ocular, thymic, pancreatic and skin diseases, disorders and conditions.

[0212] Example 2 - In vitro potency studies

[0213] Methods

[0214] Two in vitro assays were employed to study potency.

[0215] The first one is a reporter gene assay in BHK cells that have been triple transfected with IL-22Ra, IL-10Rb and luciferase with a STAT3 induced promoter. This is a highly sensitive, high throughput assay that measures IL-22 receptor mediated STAT3 activation.

[0216] A stable reporter BHK cell line was generated using the following plasmids: (i) hIL-10Rb in pcDNA3,1 hygro(+), (ii) IL22R in pcDNA3,1 (Zeocin) and (iii) 2xKZdel2 in pGL4.20. Thus, this cell line expresses human IL-10Rb, human IL-22Ra and luciferase reporter protein under the control of a pSTAT3 driven promoter.

[0217] On day 0 of the assay protocol, cells were seeded in 96 well plates (Corning #3842, black, clear bottom) in base medium (500ml: DMEM + Glutamax (Gibco, Cat. No: 31966-021), 10% (w / v) foetal calf serum (FCS; containing albumin) (50ml) and 1% (w / v) penicillin-streptomycin (P / S) (5ml)) at 15,000 to 20,000 cells / well. On day 1, the culture medium was removed by inverting the plate. Fresh base medium was added at 50μ1 per well and the cells were incubated for 60 minutes.

[0218] Derivatives of IL-22 were tested alongside hIL-22 and hIL-22 variants with only backbone variations as comparators. The number of animals ‘n’ used to test each derivative or comparator ranged from 1 to 36.

[0219] Accordingly, 50μ1 of diluted derivative or comparator (diluted in base medium) was added to each well and the plate was left to stand for four hours. The derivatives and comparators were therefore diluted 2-fold as they were diluted into the 50μ1 of medium already present in the well. The stimulation was terminated after four hours by the addition of 100μ1 Steadylite plus reagent (Perkin Elmer Cat. No 6066759). The plate was sealed with TopSeal A, shaken at 450rpm for 15 minutes and then read using a Mithras or similar system no later than 12 hours later.

[0220] Data analysis was performed using Graphpad Prism. The half maximal effective concentration (EC50) of each derivative or comparator was assessed as a measure of its potency. The EC50 was determined using a log(inhibitor) versus response - variable slope (4p). As a standard, the Hill slope was constrained to 1. 50 ) as a measure of its potency. The EC 50 50 was determined using a log(inhibitor) versus response - variable slope (4p). As a standard, the Hill slope was constrained to 1.

[0221] A second in vitro potency assay measured pSTAT3 in HepG2 cells, a human liver-derived cell line that endogenously expresses IL-22Ra and IL-10Rb.

[0222] On day 1, HepG2 cells were seeded at 25,000 to 30,000 cells / well in 96-well plates (Biocoat #35-4407 Becton Dickinson). The cell culture medium used for seeding and passage was DMEM (1x) + 25 mM (4.5 g / L) glucose, β-pyruvate (Gibco, catalog 61965-026) + 10% (w / v) FCS + 1% (w / v) P / S. On day 2, the cells were ready for assays. The cells were starved with 0.1% (w / v) FCS (i.e., a very low albumin concentration) in DMEM (Gibco, catalog 61965-026) – 50 μL was added to each well and incubated for 60 minutes.

[0223] The test was repeated using seven concentrations of each derivative or comparative as standards (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 incubated for 15 minutes. Thus, the derivative and comparative were 2-fold diluted, as they were diluted into the existing 50 μl of culture medium in the wells. To induce cell lysis, the culture medium was removed from the cells, and 50 μl of freshly prepared 1× lysis buffer (SureFire lysis buffer from the kit) was added to each well. The plate was stirred at 350 rpm for 10 minutes at room temperature.

[0224] follow The STAT3 (p-Tyr705) assay protocol (Perkin Elmer catalog number TGRS3S(500-10K-50K)) was used to measure IL-22-induced STAT3 phosphorylation. For this assay, 4 μl of lysate was transferred to a 384-well proxiplate for measurement (with 4 μl of positive and negative controls added). The acceptor mixture was prepared shortly before use (by diluting the activation buffer 5-fold in reaction buffer and the acceptor beads 50-fold in dilution buffer). 5 μl of the acceptor mixture was added to each well, the plate was sealed with Topseal A adhesive film, and incubated at room temperature for two hours. The donor mixture was prepared shortly before use (by diluting the donor beads 20-fold in dilution buffer). 2 μl of the donor mixture was added to the well under low light. The plate was again sealed with Topseal A adhesive film and incubated at room temperature for two hours. The plate was read using an Alpha Technology-compatible plate reader.

[0225] Data analysis was performed using Graphpad Prism. First, a non-linear regression was performed in Prism using a log(inhibitor) vs. response - variable slope (4p) analysis. The Hill slope was constrained to 1. Y= top from the control compound (His tagged hIL-22 or hIL-22) was then used for normalization in Prism. 0% was set as the minimum in each data set and 100% was set as Y= top of the above non-linear regression (for the control). The non-linear regression was repeated as described above and the activity / weight percentage and EC 50 values for the test derivatives were read in the top and EC 50 values, respectively.

[0226] Results

[0227] Table 11 shows the EC 50 values for key derivatives and comparators measured in the BHK cell reporter assay for IL-22 receptor mediated STAT3 activation.

[0228] ID EC 50 (nM) hIL-22 0.07 Comparative 1 0.06 Comparative 5 0.19 Comparative 14 0.09 Derivative 1 0.48 Derivative 3 0.30 Derivative 4 0.18 Derivative 6 0.61 Derivative 7 0.09 Derivative 8 1.24 Derivative 9 0.28 Derivative 10 0.37

[0229] Table 11: EC 50 values for key derivatives and comparators in the BHK cell assay

[0230] As the BHK cell assay contains a large amount of albumin, the measured EC 50 values for the test derivatives incorporate the effect of albumin binding.

[0231] Comparator 4, an IL-22 variant with only a scaffold variation, was shown to be equivalent to hIL-22. Derivative 3 has the same scaffold as comparator 4 but is covalently linked to a medium affinity albumin binder (C16 diacid) which exhibited a four-fold reduction in potency compared to hIL-22. Derivative 1 which also has the same scaffold but is covalently linked to a high affinity albumin binder (C18 diacid) exhibited only a seven-fold reduction in potency compared to hIL-22.

[0232] A scan of alkylating positions and scaffold variations was performed in the 35Q, 64Q background (i.e. two of the three IL-22 glycosylation sites are mutated) by comparing the results of derivatives 6 to 9. The covalent attachment sites in these derivatives were selected based on an analysis of the IL-22 structure which identified positions that were expected to be exposed to the surface and not involved in receptor binding. The results obtained with these derivatives indicated that Cys substitution and fatty acid covalent attachment at several (selected) positions was tolerable which was unexpected to the inventors.

[0233] Table 12 shows the EC50values of key derivatives and comparators measured in the pSTAT3 HepG2 cell assay 50 .

[0234] ID EC 50 (nM) hIL-22 3.88 Comparative 1 4.73 Comparative 4 12.11 Derivative 1 10.13 Derivative 2 6.98 Derivative 6 14.86

[0235] Table 12: EC50values of key derivatives and comparators in the HepG2 cell assay 50 values

[0236] In the HepG2 cell assay with endogenous receptor expression levels, little signal amplification and no albumin, the potency of Derivative 1 was reduced 2.5-fold compared to hIL-22 (similar to Comparator 4, an hIL-22 variant with the same backbone as Derivative 1 but without fatty acids).

[0237] Table 13 collates the results of the BHK and HepG2 cell assays to assess the covalent attachment of fatty acids in the N-terminal extension and mutation of glycosylation sites. ND = not determined.

[0238]

[0239] Table 13: EC50values of key derivatives and hIL-22 in the BHK and HepG2 cell assays 50 values

[0240] Derivative 6 differs from Derivative 1 by the additional N35Q and N64Q substitutions (two of the three glycosylation sites are mutated), but it is equivalent (tending to slightly lower potency for Derivative 6).

[0241] Surprisingly, Derivatives 2 and 4, which have a 15-mer N-terminal extension with a Cys residue (-7C) for fatty acid attachment in the extension, show good tolerability.

[0242] Conclusion

[0243] The observed reduction in potency by covalent attachment of fatty acids in the tested derivatives of the application is mainly driven by albumin binding, while backbone substitutions contribute little. This is demonstrated by the surprising equivalence of Comparator 4 and hIL-22. In contrast, and as mentioned before, Genentech reported a 34-fold reduction in potency in vitro for their Fc fusion of IL-22.

[0244] In the HepG2 cell assay with very low levels of albumin, Derivative 1 (derivative of IL-22 which showed a seven-fold reduction in potency in the BHK assay (binding to albumin)) shows only a 2.5-fold reduction in potency compared to hIL-22.

[0245] The equivalence of derivatives 1 and 6 (Table 13) shows that the 35Q and 64Q mutations are surprisingly tolerated without affecting potency.

[0246] Thus, the derivatives of IL-22 retain high potency in the presence of albumin and are almost equivalent to hIL-22 in the absence of albumin. Cys substitution and fatty acid covalent attachment are tolerated at several positions.

[0247] Thus, the data show that the derivatives of the application exhibit good bioavailability and potency, thus providing new and improved treatments for various indications, including metabolic, liver, pulmonary, intestinal, renal and skin diseases, disorders and conditions.

[0248] Example 3 - In vivo efficacy study in diabetes

[0249] This study aimed to explore the effect of once-daily dosing of the derivatives of the application for 8 to 16 days in a mouse model of diabetes. The study was performed in a therapeutic (rather than prophylactic) mode, meaning that the diabetes pathology was developed before starting the dosing. Since the mouse model has fatty liver (leptin receptor knockout), it can also be used as a metabolic model of liver disease.

[0250] Methods

[0251] Male C57BKS db / db mice, 7 to 8 weeks old, were obtained from Charles River Laboratories (Day -10) and acclimated for at least one week before the start of the experiment. One week after arrival (Day -3), the mice were randomized and housed in groups of 10 (or individually for food intake studies). Blood glucose and food intake were measured on Day -3 and each day from Day 1 to 16 of the study.

[0252] A derivative of IL-22 (derivative 1) was tested along with an Fc fusion of IL-22 (hFc-hIL-22) as a comparator and vehicle alone as a negative control. In diabetic db / db mice (n = 6 to 10 in each group), each was administered s.c. at a once-daily dose of 0.1, 0.25, 0.5, or 1.0 mg / kg on each day from Day 1 to 16. Food intake was reduced after dosing of the derivative / comparator / control.

[0253] Blood glucose was measured daily during the duration of the study. At termination, ocular blood samples were collected in anesthetized mice. 500 μΐ of blood was collected into EDTA tubes. The samples were kept on ice and centrifuged at 6000 G for five minutes at 4°C within 20 minutes. The plasma was separated into 0.75 ml micronic tubes and immediately frozen for later measurement of component concentrations.

[0254] In addition to measuring derivative or comparator concentrations, target engagement biomarkers (hepatogenic acute phase proteins, haptoglobin and serum amyloid P component (SAP) as well as intestinal-derived peptide YY (PYY)) were measured at the end of the study. Haptoglobin was measured using a commercial kit on a COBAS instrument (Roche Diagnostics) according to the manufacturer's instructions. PYY was measured with a commercial ELISA assay (ALPCO) recognizing mouse and rat PYY according to the manufacturer's instructions. SAP was measured using a commercial ELISA assay (R&D Systems) recognizing mouse Pentraxin 2 / SAP according to the manufacturer's instructions.

[0255] Results

[0256] Blood glucose levels throughout the study are shown in Figure 5 and Figure 6 A.

[0257] It can be seen from Figure 5 that hIL-22 and the hIL-22 variant with only backbone variations (Comparator 3) failed to lower blood glucose over the course of the study compared to vehicle control.

[0258] It can be seen from Figure 6 A that, although target engagement of hFc-hIL-22 was higher, reflecting the higher steady state exposure levels in the particular study, Derivative 1 and hFc-hIL-22 both lowered blood glucose to normal levels in a comparable manner over the last days of the study, with a slightly higher efficacy of Derivative 1. Reduced food intake was observed in treated animals compared to vehicle control (see Figure 6 B). Thus, the tested derivatives standardized blood glucose in the db / db model in a similar manner as hFc-hIL-22; as described above, no such effect was observed using hIL-22 or Comparator 3.

[0259] The levels of the target engagement biomarkers haptoglobin, SAP and PYY measured at the end of the study are shown in Figures 7A to 7C . It can be seen from the figures that all three target engagement biomarkers were upregulated by the tested derivatives and hFc-hIL-22, with hFc-hIL-22 upregulating them particularly more than Derivative 1.

[0260] Figure 8 Dose response data are shown for Derivative 6, another derivative of the invention which is identical to Derivative 1 but with additional substitutions at two glycosylation sites. All three doses tested (0.1, 0.25 and 0.5 m / kg) were effective in lowering blood glucose over time, and this was shown to be particularly true with increasing concentrations.

[0261] Conclusions

[0262] The tested derivatives and hFc-hIL-22 both normalized the glycemic criteria in the db / db model, thus demonstrating an in vivo therapeutic effect. Importantly, this effect was not observed when hIL-22 was dosed, indicating that the chronic exposure obtained with the long-acting derivatives and Fc-fusion is necessary for the therapeutic effect. Although the mode of action of the anti-diabetic effect is not fully elucidated, the effect of IL-22 on the liver (hepatic gluconeogenesis and lipogenesis) is considered a major contribution.

[0263] Treatment with the derivatives of the application also showed a reduction in food intake, thus demonstrating efficacy as an obesity treatment.

[0264] It was also observed that target engagement biomarkers were upregulated by the derivatives and hFc-hIL-22. It is known that specific biomarkers measured in db / db mice can be translated to humans.

[0265] It is noted that the circulating half-life (T 1 / 2 ) of subcutaneously administered hFc-hIL-22 is higher than that of Derivative 1, especially in mice (T 1 / 2 20 and 8 hours, respectively; see Table 7). Thus, the exposure of hFc-hIL-22 is higher at steady state. This is further confirmed by the observation that target engagement biomarkers (haptoglobin, SAP and PYY) are higher in the hFc-hIL-22 group than in the Derivative 1 group (Figure 7), indicating that target engagement itself is higher in the shown experiment. Thus, despite the higher exposure and target engagement, the efficacy of the Fc-fusion (hFc-hIL-22) is poorer compared to the derivative of the application (Derivative 1) in the last three days of the 16-day dosing study.

[0266] Thus, the data show that the derivatives of the application exhibit good therapeutic efficacy in a mouse model of diabetes and liver disease. Since it is known that specific biomarkers measured in db / db mice can be translated to humans, it is reasonable to predict that this therapeutic efficacy will also translate.

[0267] Example 4 - In vivo efficacy study (i) in terms of liver damage

[0268] This study was aimed at exploring the effect of dosing the derivatives of the application in a mouse model of liver damage. The study was performed in a prophylactic mode, meaning that liver damage was induced only after the start of dosing.

[0269] Methods

[0270] Ten-week-old C57B1 / 6Rj mice were obtained and acclimatized for one week prior to study initiation. Liver injury was induced with a single intraperitoneal dose (300 mg / kg, 20 ml / kg) of APAP. Test derivatives of IL-22 (Derivatives 1 and 6) were administered subcutaneously at a dose of 1.5 mg / kg two hours prior to APAP dosing along with vehicle controls (n=5 to 10). The study was terminated 24 hours after APAP dosing. Peripheral blood was collected for measurement of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0271] Blood samples were collected in heparinized tubes, plasma was isolated and stored at -80°C until analysis. ALT and AST were measured on a COBAS c501 automated analyzer using commercial kits (Roche Diagnostics) according to the manufacturer’s instructions.

[0272] Livers were formalin-fixed and paraffin-embedded for histological analysis.

[0273] Proliferation was measured by ki67 immunohistochemistry (IHC) staining. IHC positive staining was quantified by image analysis using VIS software (Visiopharm, Denmark).

[0274] Apoptosis was measured in a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Briefly, slides with paraffin-embedded sections were deparaffinized in xylene and rehydrated in a series of graded ethanol. Slides were pre-treated with proteinase K and endogenous peroxidase activity was blocked with hydrogen peroxide. TUNEL mixture (In Situ Cell Death Detection Kit, POD, Roche) was added to the slides, followed by amplification with horseradish peroxidase (HRP) and visualization by diaminobenzidine (DAB) (Chromogen). Finally, slides were counterstained in hematoxylin and coverslipped.

[0275] Results

[0276] Plasma ALT and AST levels at the end of the study are shown in Figure 9A and Figure 9B respectively. A significant reduction in the amount of ALT and AST was shown in mice treated with Derivative 1 or 6 prior to liver injury compared to vehicle / APAP controls.

[0277] The number of TUNEL and ki67 positive cells at the end of the study are shown in Figure 10A and Figure 10BThe amount of TUNEL positive cells was (significantly) reduced in mice treated with derivative 1 or 6 prior to liver injury compared to vehicle / APAP controls. The amount of ki67 positive cells was comparable in the APAP treated groups.

[0278] Conclusions

[0279] ALT and AST are liver enzymes used as indicators of liver injury. Therefore, it is shown that derivatives 1 and 6 can protect the liver from APAP-induced damage.

[0280] The results of the TUNEL assay show that derivatives 1 and 6 prevent apoptosis caused by liver injury compared to vehicle / APAP controls. However, cell proliferation is not affected by these derivatives of IL-22. Since proliferation is physiologically upregulated as a response to injury (as seen in the controls), the results indicate a proliferative effect of derivatives 1 and 6, since proliferation is not reduced after injury (the ratio of proliferation to injury is increased).

[0281] Therefore, the data show that the derivatives of the application exhibit good efficacy in protecting against liver injury in a mouse model. It is known that specific biomarkers measured in mice can be translated to humans, so it is reasonable to predict that the protective effect observed will also be translated.

[0282] Example 5 - In vivo efficacy study in terms of lung injury

[0283] This study aimed to explore the effect of administering a derivative of the application in a rat model of lung injury. The study was performed in a preventive and therapeutic mode, which means that administration was started before induction of lung injury and then continued.

[0284] Methods

[0285] To induce lung injury, 100 μΙ of bleomycin was administered as a single dose to the lungs of male Sprague Dawley rats by oropharyngeal aspiration on day 1 (groups 2 to 6). Saline was administered as a negative control (group 1).

[0286] Animals of groups 3, 4 and 5 were dosed with derivative 6 at 0.5, 1.5 or 4.5 mg / kg respectively once a day (by subcutaneous injection) from day -1 to day 3. Animals of group 6 were dosed with prednisolone at 10 mg / kg once a day (by oral gavage) from day -1 to day 3.

[0287] To measure soluble collagen in the bronchoalveolar lavage fluid (BALF) from rats, the lungs were lavaged (3 x 4 ml) with sterile PBS (without calcium and magnesium) containing added protease inhibitor cocktail and the lavage fluid from each animal was put into one tube. Soluble collagen in the BALF supernatant was measured using the Sircol S1000 (Biocolor) Collagen Assay.

[0288] All animals were submitted for necropsy on Day 4 (terminal euthanasia). The right lung of all animals was collected for histopathology and inflated with 10% neutral buffered formalin (NBF) and then immersed in fixative in NBF. Three parallel longitudinal sections were trimmed from the right caudal lobe and fixed in cassette 01. The right apical, middle and accessory lobes were also opened longitudinally and fixed in cassette 02.

[0289] Two slides were prepared from each cassette; one slide was stained with hematoxylin and eosin (H&E) and the other slide was stained with hematoxylin and picrosirius red (H&PSR).

[0290] A random number was then assigned to each slide using a random number generator. The key to identification was recorded in a Microsoft Excel spreadsheet and a copy was provided to the study pathologist after slide evaluation. Thus, the six sections of each lung were read blindly.

[0291] The veterinary pathologist then scored the severity of inflammation for each section on each H&E stained slide (where 0 = none, 1 = minimal, 2 = mild, 3 = moderate and 4 = severe). The mean score and median score were calculated for each group. The pathologist also scored the severity of fibrosis for each section on each H&PSR stained slide (using a modified Ashcroft score from 0 = low to 8 = high). The mean score and median score were calculated for each group and subjected to non-parametric ANOVA, Kruskal-Wallis post-test analysis.

[0292] Results

[0293] A summary of the microscopic findings is shown in Table 14, which reveals the mean and median scores for inflammation and fibrosis for each group.

[0294]

[0295]

[0296] Table 14: Summary of microscopic findings in the lung injury rat model

[0297] As demonstrated by comparing Group 1 and Group 2 in Table 14, bleomycin induced lung inflammation and fibrosis in the rat model. In Groups 3 to 5, i.e. rats treated with the derivative of the application (derivative 6), the mean and median scores were lower. These lower scores were comparable to the scores observed in rats treated with prednisolone (Group 6).

[0298] The median inflammation and fibrosis scores for each animal in the study are also shown in Figure 11A and Figure 11B respectively.

[0299] As shown in Figure 11A , the group median inflammation score was increased in the bleomycin / vehicle control (Group 2) compared to the negative control (Group 1). The group median inflammation score was decreased in rats treated with derivative 6 and prednisolone (Group 6) compared to the bleomycin / vehicle control (and significantly decreased in the high dose Group 5).

[0300] As shown in Figure 11B , the group median fibrosis score was increased in the bleomycin / vehicle control (Group 2) compared to the negative control (Group 1). However, the group median fibrosis score was decreased in rats treated with derivative 6 (and significantly decreased in the high dose Group 5) compared to the bleomycin / vehicle control but not compared to the control prednisolone.

[0301] As shown in Figure 11C , the amount of soluble collagen in the BALF after bleomycin-induced lung injury was increased in the bleomycin / vehicle control (Group 2) compared to the negative control (Group 1) and it was not decreased by treatment with prednisolone (Group 6). However, a significant decrease in the amount of soluble collagen was observed in the BALF of rats treated with derivative 6 (at all doses tested) compared to the bleomycin / vehicle control. Since soluble collagen in the BALF is a read-out of fibrosis, these results confirm the histological data just reported above.

[0302] Conclusion

[0303] The results of the microscopic study show that the derivative of the application is able to prevent and / or reduce bleomycin-induced lung inflammation and fibrosis in the rat model. The effect observed in terms of inflammation is comparable to that observed with prednisolone, a corticosteroid known for its use in the treatment of lung inflammation. However, the derivative of the application has a unique effect on fibrosis, which is not present with prednisolone.

[0304] Example 6 - In vivo efficacy study in terms of colitis

[0305] This study aimed to explore the effect of dosing the derivatives of the application in a mouse model of colitis. The study was performed in both a prophylactic and a therapeutic mode, meaning that dosing was started on the same day as induction of colonic inflammation, and then continued.

[0306] Methods

[0307] The fed female C57B1 / 6JRj mice were randomized into five groups (n=8 per group) according to body weight. DSS was used to induce colitis in four of the five groups. From day 0 to day 6 of the study, these mice received DSS in their drinking water for 7 days. In the fifth group, the animals received water without DSS and thus served as healthy controls. From day 0 of the study, the DSS mice were treated once daily for 10 days with vehicle, the test derivative of IL-22 (derivative 6; at 0.35 mg / kg or 1 mg / kg intraperitoneally) or, as a comparator, the IL-22-Fc fusion (hFc-hIL-22; at 0.5 mg / kg intraperitoneally). Body weight, food and water intake were monitored daily.

[0308] On day 10 of the study, blood samples from the mice were collected in EDTA tubes and plasma was isolated and stored at -80°C until analysis. Regenerating islet-derived protein 3 gamma (Reg3g) was measured in duplicate using an ELISA kit (Cloud-Clone Corp) according to the manufacturer's instructions. Reg3g is a target engagement marker for IL-22.

[0309] At termination, the intestines were removed for stereological analysis. Therefore, the intestinal tract was flushed with ice-cold saline and its contents gently removed before sampling.

[0310] The intestines were infiltrated in formalin overnight (Tissue-Tek VIP) and subsequently embedded in paraffin blocks. Then, using the principle of systematic uniform random sampling (SURS), the formalin-fixed intestines were sampled from proximal to distal direction, resulting in a total of four sections and placed in a multi-cassette. All tissue sections were placed in such a way that individual sections could be identified at later stages. The paraffin blocks were trimmed and a 5 pm top section was cut and mounted on a Superfrost+ objective. For the large intestine, another section was cut at a distance of 500 pm from the top section, resulting in a total of eight colon sections from each animal.

[0311] Colonic inflammation volume was measured in a stereological manner, i.e. using a three-dimensional interpretation of two-dimensional cross sections of the colon. Stereological volume estimation was performed on scanned H&E stained slides using the newCAST system (Visiopharm). Total intestinal volume, volume of mucosa, volume of submucosa and muscle layer, and volume of inflamed tissue were estimated by point counting using a grid system of appropriate size, where all points hitting the structure of interest were counted. The number of points hitting the structure of interest was converted to volume according to the following mathematical relationship:

[0312] Vol ref =∑p·A(p)·t

[0313] where A(p) is the area of each point, p is the total number of points hitting the structure of interest, and t is the distance between sections. The mean inflammation volume was calculated for each group and statistically analyzed.

[0314] Colonic morphology was also assessed at termination by looking at H&E stained slides.

[0315] Results

[0316] Colonic inflammation volume is shown in Figure 12 Compared to vehicle control (also containing DSS), mice treated with either dose of derivative 6 showed prevention of inflammation. Notably, in the groups treated with derivative 6, inflammation remained at normal levels, as evidenced by the fact that the colonic inflammation volume of the treated groups was the same as that of the healthy control group (vehicle without DSS). The same was true for the group treated with hFc-hIL-22.

[0317] Representative H&E stained images of colonic morphology at termination are shown in Figure 13 After DSS treatment, mucosal epithelial damage (marked with black arrows) could be found in vehicle treated animals, but not in animals treated with either dose of derivative 6 or hFc-hIL-22. This demonstrates the protective effect on epithelial tissue.

[0318] Plasma Reg3g levels are shown in Figure 14DSS treatment induced an increase in basal Reg3g levels (compare vehicle to vehicle without DSS). No further increase was detected in the low dose (0.35 mg / kg) Derivative 6 group, but a further increase was found 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 indicate that despite the lower dose, the target is engaged to a higher extent, which can be related to the longer half-life of hFc-hIL-22 in mice (T1 / 2 of 30 hours for hFc-hIL-22, compared to 9.1 hours for Derivative 6).

[0319] Conclusions

[0320] Thus, the data show that the Derivatives of the application demonstrate good efficacy in protecting against colitis and damage to the mucosal epithelium in a mouse model. This indicates that a new and improved treatment of intestinal diseases, disorders and conditions has been found. In particular, these findings demonstrate the potential to treat diseases characterized by damage to the mucosal epithelium, such as inflammatory bowel disease.

[0321] Example 7 - In vivo efficacy study (ii) in terms of liver damage

[0322] This study aimed to explore the effect of dosing the Derivatives of the application in a second mouse model of liver damage (the first being described in Example 4 above). The study was performed in a prophylactic mode, meaning that liver damage was induced only after the start of dosing.

[0323] Methods

[0324] C57B1 6 / 6j male mice were divided into five groups (n = 8 per group). Relative to ConA treatment, a test derivative of IL-22 (Derivative 1) was dosed intraperitoneally at 1 mg / kg at -26 hours and -2 hours in two of the five groups. The other two groups received vehicle only at these time points. ConA was given as an intravenous bolus at a dose of 15 mg / kg over a 30 second period to all four groups to induce liver damage. As a healthy control, the fifth group did not receive ConA (vehicle only as above).

[0325] At 8 or 24 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 clot activator). Mice that did not receive treatment were sacrificed at the 8-hour time point (group 5). The blood in each tube was mixed with clot activator by inverting the tube several times. The tubes were kept at room temperature for 15 minutes and then centrifuged at 2000 g for 10 minutes at 4°C. Serum samples were measured for ALT and AST using an automated system (Konelab 20) according to the manufacturer’s instructions.

[0326] Results

[0327] ALT and AST plasma levels at the end of the study are shown in Figure 15A and Figure 15B At both time points tested, the amount of ALT and AST in mice treated with Derivative 1 prior to liver injury showed a decrease compared to vehicle / ConA controls.

[0328] Conclusions

[0329] ALT and AST are liver enzymes used as indicators of liver injury. Therefore, it was shown that Derivative 1 protects the liver from ConA-induced injury, just as its effect on APAP-induced injury in Example 4. It is known that specific biomarkers measured in mice can be translated to humans, so it is reasonable to predict that the protective effect observed will also be translated.

[0330] Example 8 - In vivo efficacy study in obesity and NASH

[0331] This study aimed to explore the effect of dosing the Derivatives of the application in a mouse model of obesity and NASH. The study was performed in a therapeutic (not prophylactic) mode, meaning that the obesity and NASH pathology was developed before the start of dosing.

[0332] Methods

[0333] The diet-induced obesity mouse model is based on male C57BL / 6JRj mice that were fed a high-fat diet for at least 30 weeks before the experiment. The diet is rich in fat (40%), fructose (22%) and cholesterol (2%) (Research Diets D09100310). This leads to obesity, NAFLD, and eventually NASH.

[0334] Animals were individually housed six days before the first dose of test derivative (or other) and body weight was monitored daily throughout the experiment. Mice were divided into six groups (n=12 per group). Dosing was initiated on study day 0 (indicated with a dashed line in Figure 16 ) and was administered subcutaneously once daily at the following doses.

[0335] Somatropin is a long-acting GLP-1 receptor agonist used as a positive control in the first group and investigated in combination with the test derivative of IL-22 (derivative 6) in the second group. The dose of somatropin was escalated according to the following schedule: day 0 0.6 nmol / kg - day 1 1.2 nmol / kg - day 2 2.4 nmol / kg - day 3 4.8 nmol / kg - day 4 12 nmol / kg - day 5 30 nmol / kg. In the combination group, somatropin was administered starting at day 0 after body weight loss plateaued in the somatropin treatment group and derivative 6 was administered starting at day 12 (indicated by a dashed line in Figure 16 ).

[0336] The dose of derivative 6 in the third “high dose” group was escalated according to the following schedule: day 0 0.05 mg / kg - day 1 0.1 mg / kg - day 2 0.15 mg / kg - day 3 0.2 mg / kg - day 4 0.25 mg / kg. At day 14, the dose was switched from 0.25 mg / kg to 0.1 mg / kg (indicated by a dashed line in Figure 16 ). In the fourth “low dose” group, the dose of derivative 6 was started at 0.05 mg / kg without further escalation.

[0337] In the fifth group, the dose of IL-22-Fc fusion (hFc-hIL-22) as comparator was escalated according to the following schedule: day 0 0.02 mg / kg - day 1 0.04 mg / kg - day 2 0.06 mg / kg - day 3 0.08 mg / kg - day 4 0.1 mg / kg. Based on the longer half-life and the corresponding higher target engagement of derivative 6 compared to what was seen in Example 6 (see Figure 14 ), the dose of hFc-hIL-22 was chosen to match the target engagement of the 0.25 mg / kg derivative 6 group.

[0338] Vehicle only was administered to the sixth group as negative control.

[0339] Plasma triglyceride (TG) levels were measured at baseline after start of dosing (day -2), at 2 weeks (day 14) and at 4 weeks (day 28). Specifically, tail blood samples were collected for analysis by pressing tail blood with a volume of equal to or below 200 μΐ into open Microvette (100 μΐ or 200 μΐ) tubes treated with an appropriate anticoagulant. Blood was placed at 4°C until centrifugation at 3000 g for 10 min. Plasma supernatant was transferred to a new tube and immediately frozen on dry ice and stored at -80°C. According to the manufacturer’s instructions, plasma TG levels were determined using the Triglyceride Reagent (Roche Diagnostics) on a Cobas c501® analyzer. TG levels were measured on a c501 autoanalyzer using a commercial kit (Roche Diagnostics).

[0340] Results

[0341] Body weights during the course of the experiment are shown in Figure 16

[0342] This study demonstrates the dose-dependent high efficacy of derivative 6 in reducing body weight in an obese mouse model. In addition, it also exhibits additivity with the GLP-1 receptor agonist semaglutide, which is being investigated in late-stage clinical trials for obesity treatment. The data indicate that derivative 6 has an advantage over hFc-hIL-22 in inducing weight loss. Importantly, hFc-hIL-22 has a longer half-life in mice than derivative 6 and exhibits higher target engagement even when dosed at half the dose of derivative 6. Thus, the dose of 0.1 mg / kg hFc-hIL-22 used in this study was chosen for similar target engagement as the 0.25 mg / kg derivative 6 group.

[0343] Sensitivity to weight loss induced by derivative 6 was observed here in diet- induced obese mice but not in lean mice. For example, in a 10-day DSS-induced colitis study with once-daily dosing (Example 6), the body weights of the DSS / vehicle and DSS / derivative 6 (0.35 mg / kg) groups were both 19.0 g at the start of the study. At the end of the study, the body weight in the DSS / vehicle group was 17.6 g, while the body weight in the DSS / derivative 6 (0.35 mg / kg) group was 17.4 g, which was not different (p = 0.82 in an unpaired student’s t-test). In contrast, on day 10 of this study, the body weights of the vehicle and derivative 6 (0.25 mg / kg) groups were 43.5 g and 35.2 g, respectively. Thus, a significant weight loss was observed in the derivative 6 group compared to the vehicle group (p < 0.0001 in an unpaired student’s t-test on day 10). The body weight in the vehicle group was similar to the derivative 6 group at the start of the study (44.3 g and 44.1 g, respectively).

[0344] Plasma TG levels measured at baseline (day -2), week 2 (day 14), and week 4 (day 28) after starting dosing are shown in Table 15.

[0345]

[0346] Table 15: Plasma TG levels (nmol / l) in obese and NASH mouse models

[0347] ​Delta refers to the change in TG levels (nmol / l) from baseline to the indicated treatment and time point. As can be seen from Table 15, the vehicle group had a positive (increase) in levels, but all other groups had a negative (decrease) in levels.

[0348] Derivative 6 had a higher efficacy in reducing TG levels than semaglutide, even at low doses, which resulted in less weight loss than semaglutide (e.g., TG levels (nmol / l) at week 4 for semaglutide was -0.14 ± 0.066, for derivative 6 (0.05 mg / kg) was -0.30 ± 0.034, and for derivative 6 (0.25 / 0.1 mg / kg) was -0.35 ± 0.052). The results indicate that derivative 6 has a high efficacy in TG reduction that is partially independent of weight loss effects. Furthermore, the effects of derivative 6 are completely additive to semaglutide. TG levels (nmol / l) at week 4 calculated as delta TG were reduced: -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). TG levels increased in the vehicle group over the course of the study, but were reduced compared to baseline (delta TG).

[0349] Conclusions

[0350] The studies show that the derivative of the invention (derivative 6) can induce weight loss in obese mice in a dose-dependent manner, at least to levels comparable to that observed using semaglutide, a long-acting GLP-1 receptor agonist used as a positive control. Furthermore, the combination of semaglutide and derivative 6 has an additive effect on weight loss. The efficacy of derivative 6 in inducing weight loss is higher than that observed using hFc-IL-h22 at doses selected to provide similar levels of target engagement. The weight loss induced by derivative 6 in diet-induced obese mice was not observed in lean mice treated with DSS, indicating that obese mice are more sensitive to the weight loss induced by derivative 6. Since the weight loss observed in diet-induced obese mice is the same readout used in humans, it is reasonable to predict that the observed weight loss will also translate.

[0351] Derivative 6 also showed high efficacy in reducing TG levels. Derivative 6 showed higher efficacy than semaglutide at both doses tested, and complete additivity of efficacy was observed for the combination administration. Given that derivative 6 at the 0.05 mg / kg dose had higher efficacy than semaglutide despite less weight loss, it can be concluded that the TG lowering effect of derivative 6 is at least in part independent of weight loss. Furthermore, derivative 6 was superior to the hFc-hIL-22 comparator at both tested doses. Since the TG reduction observed in diet-induced obese mice is the same readout as used in humans, it is reasonable to predict that the observed effect will translate. Thus, the results indicate that a new treatment for conditions and disorders characterized by high TG levels has been discovered.

[0352] While certain features of the application have been illustrated and described, many modifications and

Claims

1. A derivative of IL-22 comprising a fatty acid covalently linked to an IL-22 protein, wherein: (i) the IL-22 protein is a variant of native mature human IL-22, the sequence of which is set out in SEQ ID NO. 1, the sequence of the variant being set out in any one of SEQ ID NO. 16 and 18-21; (ii) the fatty acid is covalently linked via a linker to a substituted Cys residue at position 1, 6 or 33 within SEQ ID NO. 1, wherein the amino acid position is defined relative to SEQ ID NO. 1; and (iii) the fatty acid is a C12, C14, C16, C18 or C20 diacid.

2. The derivative of claim 1, wherein the fatty acid is: (i) Formula I: wherein x is an integer in the range of 10 to 18, and * indicates the point of attachment to the IL-22 protein or linker; (ii) a C16 or C18 diacid; and / or (iii) a C18 diacid.

3. The derivative of claim 2, wherein the fatty acid is: Formula I: wherein x is an integer in the range of 12 to 18, and * indicates the point of attachment to the IL-22 protein or linker.

4. The derivative of claim 3, wherein x is an integer in the range of 14 to 16.

5. The derivative of claim 3, wherein x is an integer in the range of 16 to 18.

6. The derivative of claim 1 or 2, wherein the sequence of the variant is set out in any one of SEQ ID NO. 16, 18 and 19.

7. The derivative of claim 1 or 2, wherein the linker comprises: (i) one or more amino acids; (ii) an oxyethylene glycine unit or a plurality of linked oxyethylene glycine units; (iii) one or more oligo(ethylene glycol) (OEG) residues; (iv) an ethylenediamine (C2DA) group; (v) an acetamide (Ac) group; wherein R represents the fatty acid and * represents the point of attachment of the linker to the IL-22 protein; wherein R represents the fatty acid and * represents the point of attachment of the linker to the IL-22 protein; and / or wherein R represents the fatty acid and * represents the point of attachment of the linker to the IL-22 protein. HOOC-(CH2) x -CO-* 8. The derivative of claim 7, wherein the linker comprises one or more amino acids, the amino acids comprising Glu and / or Lys.

9. The derivative of claim 7, wherein the linker comprises 2 to 5 linked oxyethylene glycine units.

10. The derivative of claim 1 or 2, wherein the derivative comprises a C14, C16, C18 or C20 diacid covalently linked via a linker to a variant of native mature human IL-22, the sequence of the variant being SEQ ID NO. 16 or 18, and the linker is linked to the Cys residue at position 1, wherein the amino acid position is defined relative to SEQ ID NO.

1.

11. The derivative of claim 1, wherein: ​ HOOC-(CH2) x -CO-* ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ (vi) ​ (vii) ​ (viii) ​ ​ ​ ​ ​ (i) the sequence of the variant is set forth in SEQ ID NO. 16, the linker is attached at position 4 of SEQ ID NO. 16, and the linker and fatty acid are linked together as shown in Formula 1A; (ii) the sequence of the variant is set forth in SEQ ID NO. 16, the linker is attached at position 4 of SEQ ID NO. 16, and the linker and fatty acid are linked together as shown in Formula 1B; (iii) the sequence of the variant is set forth in SEQ ID NO. 16, the linker is attached at position 4 of SEQ ID NO. 16, and the linker and fatty acid are linked together as shown in Formula 1C; (iv) the sequence of the variant is set forth in SEQ ID NO. 18, the linker is attached at position 4 of SEQ ID NO. 18, and the linker and fatty acid are linked together as shown in Formula 1A; (v) the sequence of the variant is set forth in SEQ ID NO. 19, the linker is attached at position 1 of SEQ ID NO. 19, and the linker and fatty acid are linked together as shown in Formula 1A; (vi) the sequence of the variant is set forth in SEQ ID NO. 20, the linker is attached at position 7 of SEQ ID NO. 20, and the linker and fatty acid are linked together as shown in Formula 1A; (vii) the sequence of the variant is set forth in SEQ ID NO. 21, the linker is attached at position 33 of SEQ ID NO. 21, and the linker and fatty acid are linked together as shown in Formula 1A; (viii) the sequence of the variant is set forth in SEQ ID NO. 18, the linker is attached at position 4 of SEQ ID NO. 18, and the linker and fatty acid are linked together as shown in Formula 1B; wherein Formula 1A, Formula 1B, and Formula 1C are shown below:

12. The derivative of claim 1 or 2, wherein the derivative is:

13. The derivative of claim 1 or 2, wherein the derivative is:

14. The derivative of claim 1 or 2, wherein the derivative is:

15. A pharmaceutical composition comprising the derivative of any one of claims 1 to 14 and a pharmaceutically acceptable vehicle.

16. Use of the derivative of any one of claims 1 to 14 or the pharmaceutical composition of claim 15 in the manufacture of a medicament for the treatment of a metabolic, liver, pulmonary, or intestinal disease, disorder, or condition, wherein: (i) the metabolic disease, disorder, or condition is obesity, type 1 diabetes, type 2 diabetes, hyperlipidemia, or hyperglycemia; (ii) the liver disease, disorder, or condition is non-alcoholic steatohepatitis (NASH), liver cirrhosis, alcoholic hepatitis, acute liver failure, chronic liver failure, acute-on-chronic liver failure (ACLF), acetaminophen-induced liver toxicity, or acute liver injury; (iii) the pulmonary disease, disorder, or condition is chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, or idiopathic pulmonary fibrosis; (iv) the intestinal disease, disorder, or condition is ulcerative colitis.

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