HK40108760A - Therapeutic derivatives of interleukin-22 - Google Patents

Description

Therapeutic derivatives of interleukin-22

This application is a divisional application of Chinese Patent Application No. 202080077355.4 entitled "Therapeutic Derivatives of Interleukin-22", filed on November 9, 2020.

Invention Field

This invention relates to novel derivatives of interleukin-22 (IL-22), and more particularly to derivatives comprising fatty acids covalently linked to the IL-22 protein. The invention also covers methods of their production and their use in therapy, including the treatment, prevention, and improvement of metabolic, liver, lung, intestinal, kidney, and skin diseases, conditions, and disorders.

Background of the Invention

IL-22 is a 146-amino acid protein with a molecular weight of 17 kDa. It belongs to the IL-10 family of cytokines and selectively activates a heterodimer receptor composed of the widely expressed IL-10 receptor B subunit (IL-10RA2) and the epithelially restricted IL-22 receptor A subunit (IL-22RA1). It is a unique cytokine because, although released from immune cells, it selectively targets epithelial cells. Therefore, IL-22-induced signaling pathways may be correlated in different tissues (targets include skin, intestine, lung, liver, kidney, pancreas, and thymus), but IL-22 activates it in an epithelially specific manner. The soluble binding protein IL-22BP neutralizes IL-22 and thus regulates its function.

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

The effects of IL-22 are the result of the coordinated involvement of multiple activities/pathways. Upon injury, IL-22 acts on epithelial barrier tissues and organs to protect cells and maintain barrier function (e.g., by activating anti-apoptotic gene programs). It also accelerates repair (e.g., by inducing the proliferation of mature cells and activation of stem cells), prevents fibrosis (e.g., by reducing epithelial-mesenchymal transition, antagonizing the NLRP3 inflammasome, and inducing hepatic stellate cell senescence), and controls inflammation (e.g., by inducing antimicrobial peptides and chemotactic signaling). IL-22 has been reported to treat a range of medical conditions, including those commonly observed in diabetic or overweight mammals, such as hyperglycemia, hyperlipidemia, and hyperinsulinemia.

However, IL-22 is typically rapidly cleared from the body via the kidneys, limiting its use in clinical practice. Therefore, known methods for prolonging the half-life of circulating IL-22 attempt to artificially increase its size to over 70 kDa to avoid renal clearance. Linking IL-22 to an Fc antibody fragment is currently the best solution for achieving this effect; both Genentech and Generon Shanghai have long-acting IL-22-Fc fusions in clinical development. Modifying IL-22 with polyethylene glycol (PEGylation) is another known method to avoid renal clearance.

However, these existing solutions are not without drawbacks. Existing data indicate that PEG itself is immunogenic, and PEG-containing vacuoles have been observed in cells containing PEGylated biologics. Reduced activity and heterogeneity are also disadvantages of PEGylation. Although Fc fusion technology is well-known, the addition of an Fc antibody fragment represents a significant change in the structure of IL-22, which affects its properties beyond just an extended half-life. As Fc fusions increase the protein size from approximately 17 kDa to approximately 85 kDa, properties such as diffusion rate, distribution, and receptor engagement kinetics may be affected. For example, some Fc fusions are slowly absorbed and/or too large to be administered via 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 may be affected by steric hindrance caused by large fusion couplers.

Therefore, there remains a need in the field for novel IL-22 biocompatibility modifiers that, compared to the natural molecule, improve circulating half-life and exhibit optimized pharmacokinetic and pharmacodynamic properties. Ideally, they should retain the potency and other properties of the natural molecule while avoiding the toxicity, immunogenicity, and any other adverse reactions exhibited by known derivatives.

Summary of the Invention

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

In an embodiment of the invention, fatty acids are covalently linked to the IL-22 protein via a linker.

Fatty acids can have formula I:

HOOC-(CH ₂ ) _x -CO-*,

Where x is an integer in the range of 10 to 18, optionally 12 to 18, 14 to 16, or 16 to 18, and * indicates a connection point with the IL-22 protein or linker. It can be a fatty diacid, such as a C12, C14, C16, C18, or C20 diacid. Advantageously, the fatty acid is a C16 or C18 diacid, and most advantageously, a C18 diacid.

The IL-22 protein can be naturally occurring, mature human IL-22 (hereinafter referred to as "hIL-22") or a variant thereof. This variant can be a substituted form of hIL-22, optionally substituted at positions 1, 21, 35, 64, 113, and/or 114. It can contain substitutions of hIL-22 selected from the group consisting of: A1C, A1G, A1H, N21C, N21D, N21Q, N35C, N35D, N35H, N35Q, N64C, N64D, N64Q, N64W, Q113C, Q113R, K114C, and K114R. Advantageously, this variant contains a Cys residue at position 1 of hIL-22.

This variant can be an extended form of hIL-22. It may contain an N-terminal peptide, such as an N-terminal trimer. Advantageously, this variant contains an N-terminal G-P-G.

The linker may contain one or more amino acids, optionally including glutamic acid (Glu) and/or lysine (Lys). The linker may contain an oxyethyleneglycine unit or multiple linked oxyethyleneglycine units, optionally 2 to 5 such units, advantageously 2 units. The linker may contain one or more oligo(ethylene glycol) (OEG) residues. It may contain an ethylenediamine ( _C2DA ) group and/or an acetamide (Ac) group. Advantageously, the linker contains a combination of all the above elements. Specifically, the linker may be γGlu-OEG-OEG-C2DA-Ac, γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc, or γGlu-OEG-OEG-εLys-αA.

The linker can be a Cys reactive linker that attaches to Cys residues in hIL-22 or its variants. It can be attached to positions -7, -5, 1, 6, 33, 113, 114, or 153 of hIL-22 or its variants (where positions -7, -5, etc., are as defined herein). For example, the linker can be attached to Cys residues substituted at positions 1, 6, 33, 113, or 114 of hIL-22. It can be attached to Cys residues at positions -5, -7, or 153 relative to hIL-22. Advantageously, the linker is attached to a Cys residue substituted at position 1 of hIL-22.

In one embodiment, the derivative comprises a C14, C16, C18, or C20 diacid covalently linked to an 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 said Cys residue. Exemplary derivatives of the present invention are those identified herein as derivatives 1 to 10.

In a second aspect, a method for preparing a derivative of the first aspect is provided, comprising covalently linking a fatty acid to the IL-22 protein.

In a third aspect, a pharmaceutical composition is provided comprising a derivative of the first aspect and a pharmaceutically acceptable mediator.

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

In the fifth aspect, a derivative of the first aspect or a pharmaceutical composition of the third aspect is provided for use in a method of treating metabolic, liver, lung, intestinal, kidney, or skin diseases, symptoms, or ailments.

Metabolic diseases, conditions, or disorders can include obesity, type 1 diabetes, type 2 diabetes, hyperlipidemia, hyperglycemia, or hyperinsulinemia.

Liver diseases, conditions, or disorders can include 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 hepatotoxicity, sclerosing cholangitis, biliary cirrhosis, or pathological conditions resulting from surgery or transplantation.

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

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

Kidney disease, condition, or disorder can be acute or chronic.

Skin diseases, conditions, or disorders can be wounds, inflammatory diseases, or GvHD.

Brief description of the attached diagram

Figure 1 illustrates (A) C18 diacid, (B) C16 diacid, and (C) C14 diacid, each connected to a linker containing a Cys reactive unit. These combinations of fatty acids and linkers are used in the derivatives of the present invention identified herein as derivatives 1 to 10.

Figure 2 illustrates the structure of the derivative of the present invention identified as derivative 1 herein.

Figure 3 illustrates the structure of the derivative of the present invention identified herein as derivative 6.

Figure 4 illustrates the structure of the derivative of the present invention identified as derivative 10 in this paper.

Figure 5 illustrates the effect of daily administration of hIL-22 and a comparative IL-22 variant with only skeletal variation (identified as Comparative 3 in this paper) on blood glucose (mean ± SEM) in an 8-day study conducted in a diabetic mouse model.

Figure 6 illustrates the effects of daily administration of the derivative of the present invention (identified herein as derivative 1) on (A) blood glucose and (B) food intake compared to IL-22-Fc fusions (specifically, human Fc fused to the N-terminus of hIL-22, hereinafter referred to as “hFc-hIL-22”) in a 16-day study conducted in a diabetic mouse model (mean ± SEM; unpaired t-test used, * indicates p < 0.05).

Figures 7A to 7C illustrate the effects of daily administration of derivative 1 and hFc-hIL-22 on biomarkers of three different targets in a 16-day study conducted in a diabetic mouse model (mean ± SEM; unpaired t-tests were used, *** indicates (A) p < 0.0002, (B) p < 0.0003 or (C) p < 0.0026).

Figure 8 illustrates the dose-response curves (mean ± SEM) of daily administration of the derivative of the present invention (identified herein as derivative 6) (three different doses) to blood glucose in a 13-day study conducted in a diabetic mouse model, compared with derivative 1 and hFc-hIL-22.

Figures 9A and 9B illustrate the role of derivatives 1 and 6 in preventing liver injury in a mouse model of acetaminophen (APAP)-induced liver injury, as demonstrated by plasma levels of two different liver enzymes. A one-way linear model was constructed using the Dunnett test; * indicates p < 0.05 and ** indicates p < 0.01 compared to the mediator + APAP.

Figures 10A and 10B illustrate the effects of derivatives 1 and 6 on (A) prevention of apoptosis and (B) cell proliferation in an APAP-induced mouse model of liver injury. NS indicates no significant effect.

Figures 11A, 11B, and 11C illustrate the effects of derivative 6 compared to prednisolone in preventing and/or reducing (A) lung inflammation and (B) and (C) pulmonary fibrosis in a bleomycin-induced lung injury rat model.

Figure 12 illustrates the role of derivative 6 in preventing colitis in a mouse model of colitis induced by sodium dextran sulfate (DSS). **** indicates p < 0.0001 compared to the mediator (containing DSS).

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. Magnified 4x, scale bar = 500 μm.

Figure 14 illustrates the plasma level of regenerated islet-derived protein 3γ (Reg3g) in a DSS-induced colitis mouse model, as a measure of its target involvement (Reg3g is a target involvement marker of IL-22).

Figures 15A and 15B illustrate the role of derivative 1 in preventing liver injury in a mouse model of concanavalin A (ConA)-induced liver injury, as demonstrated by serum levels of two different liver enzymes.

Figure 16 illustrates the effect of derivative 6 on body weight in diet-induced obese mice compared to hFc-hIL-22 and the known fatty acid-conjugated GLP-1 derivative semaglutide.

Detailed Implementation

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

As used herein, the term "derivative of IL-22" refers to the IL-22 protein having covalently linked fatty acids. This term includes derivatives in which fatty acids are directly covalently linked to the IL-22 protein and those derivatives in which fatty acids are covalently linked via linkers.

Covalent linking of fatty acids is a proven technique for extending the half-life of peptides and proteins, and a way to link fatty acids from peptides or proteins. This is known from marketed products used for type 1 and type 2 diabetes, such as insulin (detemir) and (degludec), and glucagon-like peptide-1 (GLP-1) derivatives (liraglutide) and (semaglutide).

Fatty acid linkage allows it to bind to albumin, thereby preventing renal excretion and providing some steric protection against proteolytic degradation. Advantageously, it provides minimal modification to IL-22 compared to Fc fusion or PEGylation. In this respect, although Fc fusion and PEGylation aim to increase the size of IL-22 beyond the threshold of renal clearance, derivatives containing fatty acids covalently linked to the IL-22 protein maintain a small size similar to that of the IL-22 protein. Therefore, since fatty acid linkage is minimal modification, the resulting derivatives are considered to retain properties similar to the native form, including distribution, diffusion rate, and receptor involvement (binding, activation, and transport), while minimizing the risk of immunogenicity.

As mentioned above, the therapeutic effects of fatty acid linkage to insulin and GLP-1 derivatives on diabetes have been demonstrated. However, IL-22 is a very different protein in terms of its size, sequence, and biological properties. Therefore, it was counterintuitive for the inventors that fatty acids could be covalently linked to IL-22 while maintaining therapeutic efficacy. What is particularly surprising is that this minimal modification to IL-22 can result in high potency (approaching hIL-22) and an extremely long circulating half-life.

Therefore, in a first aspect, the present invention relates to derivatives of IL-22 comprising a fatty acid covalently linked to an IL-22 protein. The fatty acid may be covalently linked to the IL-22 protein directly or via a linker, which itself may be designed to have various subunits. As used herein, the term "IL-22 protein" may refer to a native IL-22 protein, such as hIL-22 or a variant thereof. As further defined herein, a "variant" may be a protein having an amino acid sequence similar to that of the native protein.

In nature, the human IL-22 protein is synthesized as a signal peptide with 33 amino acids for secretion. The mature human IL-22 protein (hIL-22) is 146 amino acids long and shares 80.8% sequence identity with mouse IL-22 (which is 147 amino acids long). The amino acid sequence of hIL-22 is identified as SEQ ID NO.1 in this paper. Like other members of the IL-10 family, the IL-22 structure contains six α-helices (referred to as helices A through F).

The derivatives of the present invention can therefore have the natural amino acid sequence of hIL-22. Alternatively, they can have one or more amino acid sequence variations within the natural sequence. They can additionally or alternatively include one or more amino acid sequence variations relative to the natural sequence (i.e., outside the natural sequence). Thus, in one embodiment, the derivative comprises a fatty acid covalently linked to hIL-22 or a variant thereof.

In this document, expressions such as “within,” “relative to,” “corresponding to,” and “equivalent to” are used to characterize fatty acid variations and/or covalent linkage sites in the IL-22 protein by referencing the sequence of a 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.

Therefore, variations within the hIL-22 sequence are variations of any one of residues 1-146 in SEQ ID NO. 1. For example, the Glu substitution of natural Asp at residue 10 in hIL-22 is referred to herein as "D10E". If the derivative also has a fatty acid covalently linked at position 10, then it is referred to herein as the link at residue "10E".

However, the sequence variation relative to hIL-22 is the variation other than 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 negatively starting from the residue linked to residue 1 in hIL-22, i.e., the first residue in the N-terminal peptide linked to residue 1 in hIL-22 is denoted as "-1". Therefore, since derivative 2 has a covalently linked fatty acid, Cys, at the ^7th residue of the N-terminal peptide starting from position -1, the covalent linking 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 from 1; therefore, position 1 in the sequence listing of derivative 2 is actually residue -7 as indicated herein.

Two, three, four, five, or more variations can be made within the natural sequence to form derivatives of the present invention. For example, more than 10, 15, 20, 25, 50, 75, 100, or even more than 125 variations can be made in this respect. Any of residues 1-146 in the natural sequence can be varied. Exemplary residues used for mutation 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 in hIL-22. 78, 79, 82, 83, 84, 86, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 126, 127, 128, 129, 130, 132, 133, 134, 135, 137, 138, 139, 141, 143, 144, 145 and/or 146. Variations at residues 1, 21, 35, 64, 113 and/or 114 are particularly advantageous.

Variations within a native sequence are typically amino acid substitutions. As used herein, the term "substitution" can mean the replacement of an amino acid in a native protein with another amino acid. Such substitutions can be conserved or non-conserved. Exemplary replacements 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, A3 3C, A33N, D34F, N35C, N35D, N35H, N35Q, N36Q, T37C, T37I, D38L, V39Q, R40W, L41Q, I42P, E44R, K45A, F47T, H48G, H48R, G49N, V50S, S51C , M52A, M52C, M52L, M52V, S53C, S53K, S53Y, E54D, E54F, R55Q, R55V, C56Q, L58K, M59I, Q61E, V62D, L63C, N64C, N64D, N64Q, N64W, F65G, L6 7Q, 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, H107 N, 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 substitutes may be freely chosen from the group consisting of: A1C, A1G, A1H, N21C, N21D, N21Q, N35C, N35D, N35H, N35Q, N64C, N64D, N64Q, N64W, Q113C, Q113R, K114C, and K114R. Surprisingly, the substitutions used in this invention do not adversely affect IL-22 activity.

The specific combinations of replacements 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, and D10 E, 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)A1C, N35Q and N64Q; (xxiii) (xxiv) H6C, N35Q and N64Q; (xxv) 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; (xxxi ii) N35Q, N64Q, and R91C; (xxxiv) N35Q, N64Q, and R110C; (xxxv) S12C, N35Q, and N64Q; (xxxvi) N35Q, S51C, and N64Q; (xxxvii) N35Q, S53C, and N64Q; (xxxviii) N35Q, T37C, and N64Q; (xxxix) N35Q, N64Q, and T98C; (xxxx) Q15C, N35Q, and N64Q; (xxxxi) N35C and N64Q; (xxxxii) H6C, N35Q, and N 64Q; (xxxxiii)A33C, N35Q and N64Q; and (xxxxiv)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, K 45A, 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 substitution combinations are contemplated and constitute a part of this invention.

The derivatives of the first aspect may typically contain amino acid substitutions, thereby optionally replacing native residues with Cys at any of the positions identified above, such as positions 1, 2, 3, 6, 11, 12, 13, 15, 21, 32, 33, 35, 37, 51, 52, 53, 63, 64, 71, 73, 91, 94, 98, 110, 113, 114 and/or 127. Advantageously, the IL-22 protein contained in the derivatives of the first aspect contains a Cys residue at position 1 of hIL-22. The combination of A1C substitution with substitutions at the two glycosylation sites at positions 35 and 64 is particularly advantageous because it results in faster absorption without adversely affecting potency or half-life (see derivatives 6 and 10 in Examples 1 and 2).

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

Table 1: Exemplary amino acid insertion sequences

Sequence variations, if present, relative to the amino acid sequence of hIL-22 typically include extensions, such as the addition of a peptide at the N-terminus. The peptide may consist of up to 5, 10, 15, 20, 25, 30, 35, 40, 45, or even up to 50 amino acids. Monomers, trimers, octamers, 13-mers, 15-mers, 16-mers, 21-mers, and 28-mers are particularly advantageous in this regard. Exemplary sequences are shown in Table 2. Suitably, the IL-22 protein contained in the derivative of the first aspect comprises an 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 an N-terminal G-P-G. This has been found to produce derivatives with very good half-life and potency (see Derivatives 1, 3, and 5 in Examples 1 and 2).

Table 2: Sequences of exemplary N-terminal peptides

Sequence variations, if present, relative to the amino acid sequence of hIL-22 may include the addition of a peptide at the C-terminus. The peptide may consist of up to 5, 10, 15, 20, 25, 30, 35, 40, 45, or even up to 50 amino acids. Heptamers are particularly advantageous in this regard, optionally having the amino acid sequence G-S-G-S-G-S-C (SEQ ID NO. 15).

In addition to the natural or variant hIL-22 amino acid sequence as described herein, derivatives of the present invention may comprise N-terminal and C-terminal peptides. Any combination of N-terminal and C-terminal peptides described herein is contemplated and is expressly included in the present invention.

It should be understood that the present invention 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 with hIL-22. In one embodiment, the variant has at least 20% or even at least 30% sequence identity with hIL-22. The variant can “substantially” have the “amino acid sequence” of hIL-22, which can refer to a sequence having at least 40% sequence identity with 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 with hIL-22. Exemplary IL-22 protein variants in specific derivatives of the invention disclosed in the experimental section are shown in SEQ ID NO. 16-21.

Skilled technicians will understand how to calculate the percentage of identity between two amino acid sequences. First, the two sequences must be aligned, and then the sequence identity value must be calculated. The percentage of identity between the two sequences may take different values, depending on: (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used in the alignment method, such as local versus global alignment, the alignment matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the vacancy penalty, such as functional forms and constants.

After alignment, there are many different ways to calculate the percentage of identity between two sequences. For example, the identity number can be divided by: (i) the length of the shortest sequence; (ii) the alignment length; (iii) the average length of the sequences; (iv) the number of non-vacant positions; or (iv) the number of equivalent positions excluding protrusions. Furthermore, it will be understood that percentage identity also significantly depends on length. Therefore, the shorter the sequence pair, the higher the expected chance of sequence identity.

Therefore, it should be understood that accurate alignment of amino acid sequences is a complex process. The popular multiplex alignment program ClustalW [48,49] is a preferred method for generating protein multiplex alignments according to the present invention. The parameters applicable to ClustalW are as follows: For protein alignment: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, matrix = Gonnet. For DNA and protein alignment: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will recognize that these and other parameters may need to be changed for optimal sequence alignment.

Preferably, the percentage of identity between two amino acid sequences can then be calculated based on an alignment such as (N/T)*100, where N is the number of positions in the sequences that share the same residues, and T is the total number of positions in the comparison that include vacancies but not overhangs. Therefore, the most preferred method for calculating the percentage of identity between two sequences includes (i) preparing the sequence alignment using the ClustalW program with, for example, a suitable set of parameters as described above; and (ii) inserting the values of N and T into the following formula: Sequence Identity = (N/T)*100.

Those skilled in the art will know alternative methods for identifying similar sequences.

Suitable, 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. Suitable, the derivative will comprise at least 146 amino acids; however, this is the number of amino acids in hIL-22. It may comprise at least 150, at least 160, at least 170, or even at least 180 amino acids. The derivatives of the present invention may comprise proteins of any length within the above range, but their length is typically 146 to 180 amino acids.

Regardless of whether they have a natural or variant amino acid sequence, the derivatives of this invention contain 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 interconnected via amide bonds, and the linker is covalently linked to the IL-22 protein. The fatty acid and linker can thus exist as side chains on the IL-22 protein. Surprisingly to the inventors, the covalently linked fatty acid does not adversely affect IL-22 activity. Particularly surprising is that the fatty acid linkage is associated with other advantages, such as extended half-life.

Fatty acids can be any suitable fatty acid. More precisely, fatty acids can be of formula I:

HOOC-(CH ₂ ) _x -CO-*,

Where x is an integer in the range of 10 to 18, optionally 12 to 18, 14 to 16, or 16 to 18, and * indicates a connection point with the IL-22 protein or linker. It can be a fatty diacid, such as a C12, C14, C16, C18, or C20 diacid. Advantageously, the fatty acid is a C16 or C18 diacid, and most advantageously, a C18 diacid.

For example, -( _CH2 ) _x- in Formula I can be a straight-chain alkylene group, where x is 10. This fatty acid can conveniently be called a C12 diacid, i.e., an aliphatic dicarboxylic acid with 12 carbon atoms. Alternatively, -( _CH2 ) _x- in Formula I can be a straight-chain alkylene group, where x is 12. This fatty acid can conveniently be called a C14 diacid, i.e., an aliphatic dicarboxylic acid with 14 carbon atoms. In a similar manner, -( _CH2 ) _x- in Formula I can be a straight-chain alkylene group, where x is 14 (C16 diacid), 16 (C18 diacid), or 18 (C20 diacid). Suitably, the derivatives of the first aspect comprise C14, C16, C18, or C20 diacids; more suitable are C16 or C18 diacids, and even more suitable are C18 diacids.

Diacids may be able to form non-covalent associations with albumin, thereby promoting the circulation of the derivative in the bloodstream. Shorter diacids (e.g., C16 diacids) have lower albumin affinity, and therefore longer diacids (e.g., C18 diacids) have shorter half-lives. However, the shorter diacids are still long-acting derivatives with an expected half-life of more than one day in humans.

The fatty acid linkage itself also stabilizes the IL-22 protein, preventing protein hydrolysis and degradation. The resulting half-life is typically similar to that of the IL-22-Fc fusion (i.e., significantly increased compared to hIL-22).

The derivative of the first aspect may comprise a specific combination of a fatty acid and the IL-22 protein. For example, a C14, C16, C18, or C20 diacid may be linked to an IL-22 protein containing a Cys residue at position 1 of hIL-22 and/or an N-terminal G-P-G. In one instance, 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.

As described above, a fatty acid is suitably linked to a linker that connects to the IL-22 protein. The linker may comprise several linker elements, including one or more amino acids, such as one or more Glu and/or Lys residues. The linker may comprise an oxyethyleneglycine unit or multiple linked oxyethyleneglycine units, optionally two to five such units, advantageously two units. Alternatively or additionally, it may comprise one or more OEG residues, _C2DA and/or Ac groups. The linker may comprise a Cys reactive unit. As used herein, “Cys reactive unit” can refer to a functional unit capable of reacting with the sulfur atom of Cys to form a carbon-sulfur covalent bond. The Cys reactive unit may have any of several forms, but suitably comprises a carbon atom linked to a leaving group, which is replaced by the sulfur atom of Cys during carbon-sulfur bond formation. The leaving group may be a halogen, optionally a bromine atom. This bromine leaving group may be an α-acetamide functional group; advantageously, it is a bromoacetamide functional group. The leaving group can also be a functionalized hydroxyl group in the form of a methanesulfonate or toluenesulfonate, or a non-functionalized hydroxyl group. Furthermore, the leaving group can be maleimide or other functional groups. Exemplary linkers include γGlu-OEG-OEG- _C2DA -Ac, γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc, and γGlu-OEG-OEG-εLys-αAc, but any suitable linker can be used.

Fatty acids or linkers can be attached to any amino acid residue in the IL-22 protein. Examples in this regard are residues -7, -5, 1, 6, 33, 113, 114, and 153 in or relative to the hIL-22 amino acid sequence. Native residues are typically substituted with Cys or Lys to allow the fatty acid or linker to attach. Alternatively, the fatty acid or linker can be attached to native Cys or Lys residues. Appropriately, the fatty acid or linker is attached to Cys residues substituted at positions 1, 6, 33, 113, or 114 of hIL-22, or to Cys residues substituted at positions -5, -7, or 153 of hIL-22. Specifically, the fatty acid or linker can be attached to Cys residues substituted at position 1 of hIL-22.

The linking of fatty acids or linkers to the IL-22 protein is covalent. For example, Cys-reactive fatty acids or linkers can be used to link fatty acids or linkers to Cys residues in the IL-22 protein. Fatty acids or linkers can be covalently linked to the sulfur atom of a Cys residue via a thioether bond. Alternatively, Lys-reactive fatty acids or linkers can be used to link fatty acids or linkers to Lys residues in the IL-22 protein. Alternatively, fatty acids or linkers can be covalently linked to the N-terminal free amine ( _-NH₂ ) group of the IL-22 protein (regardless of the amino acid at position 1). The linking can proceed similarly to Cys linkages, although sub-stoichiometric amounts of fatty acids or linkers containing suitable N-reactive substances are used. Fatty acids or linkers can be in the form of aldehydes (N-reactive substances) and covalently linked to the free amine using classically known reductive amination.

Therefore, the derivative of the first aspect suitably comprises a C14, C16, C18 or C20 diacid linked to an 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 first aspect comprise the IL-22 protein as shown in any of SEQ ID NO. 16-21. Particularly advantageous derivatives are shown in Table 3, illustrated in Figures 1 to 4, and illustrated herein by example.

Table 3: Exemplary Derivatives of IL-22

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

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

The derivatives of this invention can exist in different stereoisomers and this invention relates to all of these forms.

According to a second aspect of the invention, a method for preparing a derivative of the first aspect is provided, comprising covalently linking a fatty acid to an IL-22 protein.

This method can be used to generate any different derivatives of IL-22 described or contemplated herein, but it is particularly advantageous when fatty acids are covalently linked to variant IL-22 proteins. Therefore, in one embodiment, the IL-22 protein used for the second aspect is a substituted form of hIL-22, optionally substituted at positions 1, 21, 35, 64, 113, and/or 114. Indicative 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.

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

The IL-22 protein can be obtained by any means known in the art, including recombinant methods. The generation of recombinant hIL-22 has been previously described and is well known in the art. The desired variant IL-22 protein can be generated in a similar manner. Researchers experienced in the art will be able to readily identify suitable nucleic acid sequences encoding the desired variant IL-22 protein. Those skilled in the art will therefore be able to readily perform this part of the invention based on existing knowledge in the art. Appropriately, the IL-22 protein is generated in a mammalian system using standard techniques, such as Chinese hamster ovary (CHO) cells. Multihistidine tags (His-tags) can be used to aid in the affinity purification of the recombinant protein.

In this regard, the IL-22 protein used in this invention can be prepared using a post-expression cleavable His tag, i.e., by adding fewer than 10, preferably 6, histidine residues to the N-terminus or C-terminus, the protein being purified by affinity for a nickel column. The His tag is attached to the N-terminus or C-terminus of the protein via a linker that can be digested by a known protease, leaving 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, whose native cleavage site has the concordant sequence ENLYFQ\S (SEQ ID NO. 26), where '\' represents a cleaved peptide bond or a human rhinovirus 14 3C (HRV14-3C) protease-cleavable linker with the EVLFQ concordant cleavage site. Cleavage can be achieved by incubating approximately 10 μg of protease with 2.5 μg of protein and 10 mM 2-mercaptoethanol at room temperature for 4 h.

To further illustrate the invention, a representative method for protein preparation is provided below. 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) and allowed to grow in the appropriate medium before increasing growth by adding a known enhancer. The secreted IL-22 protein can then be harvested using known centrifugation and aseptic filtration methods, followed by protein purification on a nickel column. After concentration and buffer exchange, the His tag is removed using the HRV14-3C protease, followed by alkylation with fatty acids (described further below), and final purification and buffer exchange. The final product is analyzed using SDS-PAGE, size exclusion chromatography, or liquid chromatography-tandem mass spectrometry (LC-MS-MS), with or without deglycosylation, to ensure the quality of the final product.

Fatty acids can be directly or covalently linked to the IL-22 protein using a linker as described in the first aspect. The linker can be obtained by any means known in the art. If so, a representative method for preparing the fatty acid and linker is as follows (taking the C16 diacid used in Derivative 10 as an example, but any derivative can be prepared using a similar method).

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 under vacuum. The residue was diluted with toluene (750 mL), washed, and extracted with dichloromethane (4 × 200 mL), dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and diluted with hexane (200 mL). A solution of 4 M hydrogen chloride in diethyl ether (100 mL, 400 mmol) was added to this solution, and the resulting suspension was concentrated under vacuum and diluted with hexane (1 L). The precipitated solid was filtered, washed with hexane, and dried under vacuum to give benzyl (2-aminoethyl)carbamate hydrochloride as a white powder.

2-Chlorotriphenylmethyl resin 100-200 was loaded with 2-[2-(9H-fluorene-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid (Fmoc-Ado-OH, 17.5 g, 45.4 mmol). The Fmoc groups were removed, and a solution of 0-6-chloro-benzotriazol-1-yl)-N,N,N',N'-tetramethylureon 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 was shaken for one hour. The resin was filtered and washed. The Fmoc groups were removed as previously described by treatment with 20% piperidine. The resin was washed as previously described.

A solution of (S)-2-(fluorene-9-ylmethoxycarbonylamino)-1-tert-butyl glutarate (Fmoc-Glu-OtBu, 29.0 g, 68.1 mmol), TCTU (24.2 g, 68.1 mmol), and N,N-diisopropylethylamine (21.4 ml, 123 mmol) in N,N-dimethylformamide (140 ml) was added to the resin, and the mixture was shaken for one hour. The resin was filtered and washed as previously described. The Fmoc groups were removed by treatment with 20% piperidine as previously described. The resin was washed as previously described.

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 and 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 × 250 mL), dichloromethane (2 × 250 mL), methanol (2 × 250 mL), and dichloromethane (6 × 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 out and washed with dichloromethane (2×250ml), a 2-propanol/dichloromethane mixture (1:1, 2×250ml), 2-propanol (250ml), and dichloromethane (3×250ml).

The solutions were combined, the solvent was evaporated, and the crude product was purified by rapid column chromatography. Pure (S)-22-(tert-butoxycarbonyl)-41,41-dimethyl-10,19,24,39-tetraoxo-3,6,12,15,40-pentaoxa-9,18,23-triaza-tetracocoic acid was dried under vacuum to give a pale yellow viscous oil.

Subsequently, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylureonium 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-pentaoxo-9,18,23-triaza-tetracododecanoic acid (22.4 g, 27.4 mmol) in anhydrous dichloromethane (110 mL). Triethylamine (72 mL, 41.0 mmol) was added to a suspension of (2-amino-ethyl)-benzyl carbamate hydrochloride (6.94 g, 30.1 mmol) in anhydrous dichloromethane (165 mL), and the resulting mixture was added to the above solution. The mixture was stirred overnight at room temperature and then evaporated to dryness. The residue was reconstituted and washed; dried over anhydrous sodium sulfate 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-benzyloxycarbonylamino-ethylcarbamoyl)-methoxy]ethoxy}ethyl-carbamoyl)methoxy]ethoxy)-ethylcarbamoyl)-1-tert-butoxycarbonylpropylcarbamoyl]-tert-butyl pentadecanoate, which was a pale yellow viscous oil.

Palladium/carbon (10%, 1.27 g, 1.20 mmol) was added to a solution of the above compound (23.8 g, 24.0 mmol) in methanol (350 ml), and the resulting mixture was hydrogenated at atmospheric pressure for four hours. The catalyst was filtered off, and the filtrate was evaporated to dryness. The residue was evaporated several times from dichloromethane to remove methanol residue 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-tetraaza-tetradodecane-42-oic acid tert-butyl ester, a colorless, viscous oil.

Under argon atmosphere, at -30°C, N,N-diisopropylethylamine (4.98 mL, 28.6 mmol) was added to a solution of the above amine (20.5 g, 23.8 mmol) in anhydrous dichloromethane (290 mL). Acetyl bromide (2.48 mL, 28.6 mmol) was added dropwise, and the resulting solution was stirred at -30°C for another three hours. The cooling bath was removed, the mixture was stirred at room temperature for one hour, and the solvent was removed under vacuum. The residue was redissolved in ethyl acetate (450 mL) and washed with 5% citric acid aqueous solution (300 mL). The phases were separated over one hour. The organic layer was allowed to separate overnight, yielding three phases. The clear aqueous layer was removed, and the remaining two phases were shaken together with a saturated aqueous solution of potassium bromide (100 mL). The phases were allowed to separate overnight, the aqueous phase was removed, and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the residue was purified by rapid 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-pentazatriapentadecano-45-olate tert-butyl ester as a colorless solid.

The above compound (19.5 g, 19.8 mmol) was dissolved in trifluoroacetic acid (120 ml), and the resulting solution was stirred at room temperature for 1.5 hours. The trifluoroacetic acid was removed under vacuum, and the residue was evaporated from dichloromethane (6 × 200 ml). Diethyl ether (200 ml) was added to the oily residue, and the mixture was stirred overnight to obtain a suspension. The solid product was filtered, 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-bromoacetamido)ethylcarbamoyl]methoxy}-ethoxyethyl-carbamoyl]methoxy}ethoxy-ethylcarbamoyl]propylcarbamoyl}pentadecanoic acid, as a white powder.

Fatty acids or linkers can be covalently linked to the IL-22 protein using standard procedures in the art. If a linker is used, the IL-22 protein can be covalently linked 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 to form a thioether bond. Suitable conditions for the covalent linking step can be exemplified as follows: Tris aqueous solution is added to a Tris and NaCl buffer solution (1.35 mg/ml) containing IL-22 protein (70 mg) to adjust the pH to 8. Then, 12 mg of bis(p-sulfonylphenyl)-phenylphosphine dihydrate dihydrate (BSPP) salt dissolved in water is added, and the mixture is gently stirred at room temperature for four hours. Add 19 mg (0.022 mmol) of 15-{(S)-1-carboxy-3-[2-(2-{[2-(2-{[2-(2-bromoacetamido)-ethylcarbamoyl]ethoxy}ethoxy)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]propyl-carbamoyl}pentadecanoic acid to ethanol (0.5 ml) and gently stir the mixture overnight. Add MiliQ water (150 ml) to reduce the conductivity to 2.5 mS/cm. Then purify the mixture using anion exchange on a MonoQ 10/100GL column with binding buffer (20 mM Tris, pH 8.0), elution buffer (20 mM Tris, 500 mM NaCl, pH 8.0), 60 column volumes of elution buffer at a flow rate of 6 ml, and a gradient of 0-80%.

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

As described in this article, the inventors were surprised to find that fatty acids can be covalently linked to the IL-22 protein while maintaining its biological activity. Particularly surprising is that this minimal modification of IL-22 can result in high potency (approaching that of hIL-22) and an extremely long cycling half-life. This particular combination of properties may be highly desirable.

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

The half-maximal effective concentration ( _EC50 ) value is commonly used as a measure of drug efficacy. Because this represents the drug concentration required to produce 50% of the maximum effect, a lower _EC50 value indicates better efficacy. Suitably, the efficacy ( _EC50 value) of the derivatives of the present invention is measured using IL-22 receptor-mediated STAT3 activation in cells at levels below 1.5 nM, below 1.25 nM, below 1 nM, below 0.75 nM, below 0.5 nM, below 0.25 nM, or even below 0.1 nM (e.g., as determined in Example 2). Suitably, the efficacy ( _EC50 value) of the derivatives of the present invention is measured by determining pSTAT3 in cells at levels below 15 nM, below 12 nM, below 10 nM, below 7 nM, or even below 5 nM (e.g., as determined in Example 2).

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

The circulating elimination half-life (T _{_1/2} ) of the derivative can be determined in vivo by subcutaneous or intravenous administration of the derivative in a suitable animal model (e.g., mouse, rat, or miniature pig). 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 may 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 may 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 miniature pigs (all determined, for example, as described in Example 1).

As illustrated herein, the inventors have also found that the derivatives of the present invention are rapidly absorbed in vivo. Advantageously, the absorption of the derivatives may be faster than that of the IL-22-Fc fusion. The mean absorption time is an accurate parameter for measuring uptake because it is independent of the dose and maximum plasma concentration following drug administration. It can be calculated based on the mean residence time, i.e., the time the drug spends in the body after absorption and before elimination. The derivatives of the present invention suitably have mean absorption times 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., determined as described in Example 1).

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

Therefore, according to a third aspect of the invention, a pharmaceutical composition is provided comprising a derivative of the first aspect and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the third aspect may comprise any of the different derivatives of IL-22 described or contemplated herein. Properly, it comprises one of the IL-22 derivatives identified herein as derivatives 1 to 10.

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

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 medium. The formulation of the active pharmaceutical ingredient with various excipients is known in the art.

The "therapeutic effective amount" of a derivative of the first aspect is any amount of the derivative that is required to treat a disease, ailment, or disorder or to produce a desired effect when administered to a subject.

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 guideline, the dose of the derivative used in the mice of Example 3 described herein was 0.5 mg/kg (subcutaneous administration).

As used herein, a “pharmaceutically acceptable medium” is any known compound or combination of known compounds known to those skilled in the art as being suitable for the formulation of pharmaceutical compositions.

In one embodiment, the pharmaceutically acceptable medium can be a solid; optionally, the composition can be in powder form for resuspension. Solid pharmaceutically acceptable mediums can include one or more substances that can also be used as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, flow aids, inert binders, preservatives, or dyes. The medium can also be an encapsulating material. In the powder, the medium is a fine solid mixed with a fine derivative according to the invention. The powder preferably contains up to 99% of the derivative. Suitable solid mediums include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, and ion exchange resins.

In another embodiment, the drug carrier may be a gel, and the composition may be in the form of an ointment or the like.

However, the pharmaceutical carrier can be a liquid; optionally, the pharmaceutical composition is in solution form. Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. Derivatives according to the invention can be dissolved or suspended in pharmaceutically acceptable liquid carriers, such as water, organic solvents, mixtures of both, or pharmaceutically acceptable oils or fats. Liquid carriers may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity modifiers, stabilizers, or osmotic pressure modifiers. Suitable examples of liquid carriers for parenteral administration include water (partially containing the above-mentioned additives, such as cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric and polyhydric alcohols, such as diols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the carrier can also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid carriers can be used in sterile liquid forms of compositions for parenteral administration. The liquid medium used in pressurized compositions may be a halocarbon or other pharmaceutically acceptable propellant.

Therefore, the method for preparing the pharmaceutical composition of the present invention may include standard and commonly used steps in the art.

Therefore, according to a fourth aspect of the invention, a derivative of the first aspect or a pharmaceutical composition of the third aspect is provided for use in therapy. A method of treating a subject with a derivative of the invention or a pharmaceutical composition comprising the derivative is also provided. Any of the various derivatives of IL-22 described or contemplated herein are expressly included in these aspects of the invention.

As used in this article, terms such as “treatment” and “therapeutic” explicitly include the treatment, improvement or prevention of disease, symptom or illness.

Derivatives of IL-22 or pharmaceutical compositions comprising such derivatives can be administered directly to a subject to be treated. The derivatives or pharmaceutical compositions can be administered by any means, including by inhalation, injection, topical application, or ocular administration. When administered by inhalation, they can be administered through the nose or mouth. Preferably, the derivatives or pharmaceutical compositions are administered by injection, typically subcutaneously or intravenously. Therefore, due to their smaller size and higher potency, derivatives offer significant advantages over Fc fusions in terms of administration flexibility (e.g., by injection, inhalation, topical application, or ocular delivery). It should be understood that administering the derivatives of the present invention to a subject to be treated, compared to hIL-22, will result in an increased circulation time, and this will contribute to the treatment of a disease, condition, or disorder. As stated above, "treatment" also includes the improvement and prevention of a disease, condition, or disorder.

Liquid pharmaceutical compositions are sterile solutions or suspensions that can be administered, for example, by intramuscular, intrathecal, epidural, intraperitoneal, and especially subcutaneous or intravenous injection. Derivatives can be prepared as sterile solid compositions that dissolve or suspend when administered using sterile water, saline, or other suitable sterile injectable media.

Inhalable forms include sterile solutions, emulsions, and suspensions. Alternatively, derivatives can be administered by or in the form of fine powder or aerosol. Nasal inhalers can be suitably administered as fine powder or aerosol nasal sprays or modified forms.

Topical preparations include solutions, creams, foams, gels, lotions, ointments, pastes, tinctures, and powders. They can be epidermal preparations, i.e., applied directly to the skin, or applied to mucous membranes.

Formulations for ocular application are typically solutions, suspensions, and ointments for topical application, such as in the form of eye drops. Alternatively, sterile solutions or suspensions can be administered via intraocular injection. Derivatives can be formulated as sterile solid compositions that dissolve or suspend when administered using sterile water, saline, or other suitable sterile injectable media. These formulations can be used for subconjunctival, intravitreal, retrobulbar, or intraanterior chamber injection.

The derivatives or pharmaceutical compositions of the present invention can be administered to any subject in need. As used herein, "subject" can be a vertebrate, mammal, or domestic animal. Therefore, the derivatives and compositions according to the present invention can be used to treat any mammal, such as livestock (e.g., horses), pets, or for other veterinary applications. Most preferably, the subject is a human. The derivatives and compositions are not only intended for use on subjects who have already shown signs of disease, symptom, or illness, but can also be administered as a purely preventative measure to subjects who appear healthy to prevent the possibility of future occurrence of such disease, symptom, or illness.

It should be understood that the derivatives and compositions of IL-22 according to the present invention can be used in a single therapy (i.e., the sole use of the derivative or composition) for the treatment of a disease, condition, or ailment. Alternatively, the derivatives and compositions according to the present invention can be used as adjuvants to or in combination with known therapies for the treatment of a disease, condition, or ailment.

It should be understood that the required amount of an IL-22 derivative is determined by its biological activity, half-life, and bioavailability, which in turn depends on the administration method, the physicochemical properties of the derivative and the composition, and whether it is used as a monotherapy or in combination therapy. The frequency of administration will also be affected by the half-life of the derivative in the treated subject. The optimal dose to be administered can be determined by those skilled in the art and will vary depending on the specific derivative used, the strength of the pharmaceutical composition, the administration method, and the progression of the disease, condition, or disorder. Other factors depending on the specific subject being treated will necessitate dose adjustments, including the subject's age, weight, sex, diet, and timing of administration.

Generally, daily doses of the IL-22 derivative according to the invention, between 0.001 μg/kg body weight and 10 mg/kg body weight, can be used to treat diseases, conditions, or disorders, depending on which derivative or composition is used. More preferably, the daily dose is between 0.01 μg/kg body weight and 1 mg/kg body weight, more preferably between 0.1 μg/kg and 500 μg/kg body weight, and most preferably between about 0.1 μg/kg and 100 μg/kg body weight.

IL-22 derivatives or compositions may be administered before, during, or after the onset of a disease, condition, or ailment. The daily dose may be given as a single administration (e.g., a single daily injection). Alternatively, the derivative or composition may require two or more administrations within a single day. For example, the derivative may be administered in two daily doses (or more daily doses, depending on the severity of the disease, condition, or ailment being treated) ranging from 0.07 μg to 700 mg (i.e., assuming a body weight of 70 kg). Patients receiving treatment may take the first dose upon waking, followed by the second dose in the evening (if a two-dose regimen is used), or every 3 or 4 hours thereafter. Alternatively, the dose may be administered once weekly, once every two weeks, or once monthly, or more frequently (e.g., twice or three times weekly). Known procedures, such as those commonly used in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), can be used to formulate specific formulations and precise treatment regimens (e.g., daily doses and frequency of administration) of the derivatives and compositions according to the invention.

Numerous studies have demonstrated the crucial role of IL-22 in various epithelial injury models, particularly in the lung, liver, intestine, kidney, skin, pancreas, and thymus. Mechanistically, multiple researchers have thoroughly demonstrated that several pathways, including anti-apoptosis, proliferation, innate immunity, anti-oxidative stress, anti-fibrosis, and stem cell/progenitor cell recruitment, regulate the effects of IL-22. Key mechanistic investigations have been further confirmed in vitro using human cell lines or in ex vivo human models (e.g., primary human intestinal organoids). Therefore, the powerful role of IL-22 in preventing cell death, ensuring regeneration, and controlling inflammation in epithelial injury is well-established.

Many studies have been conducted by analyzing genetic models of injury (IL-22 knockout or transgene overexpression). In these studies, either IL-22 deficiency or IL-22 overexpression is present at the time of injury. In other studies, IL-22 is neutralized by antibodies at the time of injury, and in some cases, IL-22 is neutralized after the acute injury phase (e.g., subacute or complete regeneration). Other studies more closely approximate the therapeutic scenario by observing the effects of exogenously administered IL-22. It is noteworthy that, upon a comprehensive review of the existing literature, different models—whether knockout, overexpression, IL-22 neutralization before or after injury, or exogenous protein administration—all depict the same picture of IL-22 protecting damaged organs and driving regeneration. This demonstrates the broad applicability and wide time window of IL-22's therapeutic potential, and also explains the need for IL-22 proteins with longer-lasting effects than hIL-22.

Therefore, according to a fifth aspect of the invention, a derivative of the first aspect or a pharmaceutical composition of the third aspect is provided for use in a method of treating metabolic, liver, lung, intestinal, kidney, or skin diseases, symptoms, or ailments. Any of the various derivatives of IL-22 described or contemplated herein is explicitly included in this aspect of the invention.

Metabolic diseases, conditions, or disorders can include obesity, type 1 diabetes, type 2 diabetes, hyperlipidemia, hyperglycemia, or hyperinsulinemia.

Liver diseases, conditions, or disorders can include NAFLD, NASH, cirrhosis, alcoholic hepatitis, acute liver failure, chronic liver failure, ACLF, acetaminophen-induced hepatotoxicity, acute liver injury, sclerosing cholangitis, biliary cirrhosis, or pathological conditions resulting from surgery or transplantation.

Lung diseases, conditions, or disorders can include COPD, cystic fibrosis, bronchiectasis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, chemical injury, viral infection, bacterial infection, or fungal infection.

Intestinal diseases, conditions, or disorders can include IBD, ulcerative colitis, Crohn's disease, GvHD, chemical damage, viral infection, or bacterial infection.

Kidney disease, condition, or disorder can be acute or chronic.

Skin diseases, conditions, or disorders can be wounds, inflammatory diseases, or GvHD.

A method is also provided for treating a subject with a condition (e.g., one or more of the aforementioned diseases, symptoms, or disorders) that responds to IL-22 treatment with a derivative of IL-22 or a pharmaceutical composition comprising the derivative.

The IL-22 derivative has all the features specified in the first aspect of the invention. The pharmaceutical composition has all the features specified in the third aspect of the invention. The method of treating a subject suffering from a condition (e.g., one or more of the aforementioned diseases, symptoms, or disorders) that responds to IL-22 treatment has all the features specified in the fourth aspect of the invention.

There are no restrictions on which derivative or composition of IL-22, as described herein, should be administered to which patient. Rather, it is intended to administer any derivative and composition described herein to any patient as described herein.

All features described herein (including any appended claims, abstracts and drawings) and/or all steps of any method or process so disclosed may be combined with any of the foregoing aspects in any combination, except for combinations in which at least some of such features and/or steps are mutually exclusive.

To better understand the present invention and to demonstrate how embodiments of the invention can be implemented, reference will now be made to embodiments that are not intended to limit the invention in any way.

Example

Unless otherwise stated, the materials and methods used in the studies described in the examples are as follows.

derivative

Table 4 provides an overview of the derivatives and comparisons of IL-22 represented in the dataset.

IL-22 derivatives possess a variety of backbones, fatty acid types, and covalent linkage sites, thus representing the diversity of derivatives covered by this invention. The linker used in all cases is γGlu-OEG-OEG-C ₂ DA-Ac. In all cases, the linker is attached to residue 1C, except for derivatives 2 (-7C), 4 (-7C), 8 (6C), and 9 (33C). While derivative 7 exemplifies covalent linkage at 1C, it lacks the GPG N-terminal peptide present in all other derivatives of fatty acids with covalent linkage at 1C.

The comparisons are hIL-22, hFc-hIL-22 (a recombinant fusion protein), and hIL-22 variants (i.e. hIL-22 with only one or more skeletal variations).

Table 4: Overview of key derivatives and comparatives represented in the dataset

The quality control analysis of the derivatives generated in the examples was carried out as follows.

The intact protein mass in the deglycosylated sample was determined by adding 20 μl of 1 mg/mL sample to 2 μl of N-glycosidase F at room temperature for 48 h. The sample was then diluted to 0.2 mg/mL with PBS at pH 7.4 and analyzed using a Synapt G2 and a Waters MassLynx 4.1 connected to a Waters Synapt G2. A 10-90 column Acquity UPLC protein BEH C4 1.7 μm 1×100 mm was used with the following mobile phases: A: 0.1% formic acid aqueous solution; and B: acetonitrile, 0.09% formic acid. The flow rate was 120 μl/min, UV 214 nm (20 pts/s), and the gradient is shown in Table 5.

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

Table 5: Gradients (%) and min) for quality control of IL-22 derivatives

The results are shown in Table 6.

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

Therefore, the quality control data confirms that the expected derivatives have indeed been produced.

The following are exemplary embodiments, intended only to illustrate the claimed invention. As those skilled in the art will know, the exact number of animals and the timeframe used in the study can vary.

Example 1 - Pharmacokinetic Study

method

Pharmacokinetic studies of selected derivatives were performed in mice (n=27), rats (n=4–8), and miniature pigs (n=2–5). IL-22 derivatives were tested in conjunction with hIL-22, hFc-hIL-22, and/or hIL-22 variants used as comparisons.

(i) Mice and rats

Thirty 8-week-old male C57Bl/6 mice and 5 male Sprague Dawley rats were obtained from Taconic Biosciences. Mice were housed in groups of 10. Animals were acclimatized for one week prior to the experiment. Body weight was measured before drug administration, which is important for pharmacokinetic calculations. Throughout the experiment, animals were kept awake and had access to food and water.

All derivatives and comparatives were prepared as 0.3 mg/ml solutions in PBS at 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.

The derivative and comparison compound were administered subcutaneously to animals. Blood samples were collected at specific time points after administration.

Sparse sampling was used in the mice; therefore, 27 mice were administered either an IL-22 derivative or a comparative, 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. The mice were euthanized by cervical dislocation after the last sample was taken.

Five rats were administered either a derivative or a comparative of IL-22, and three blood samples were collected 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. A total of 17 samples were collected from each rat during the study. The rats were euthanized with carbon dioxide after the last sample was collected.

Blood samples (100 μl) were collected from mice and rats via tongue blood collection and transferred to EDTA tubes (VetMed 200K3E, Sarstedt NR 09.1293.100). The blood was centrifuged at 8000 G, 4 °C for five minutes within 20 minutes of collection. Plasma samples (40 to 50 μl) were then transferred to semi-micronic tubes.

(ii) Miniature pigs

Nine-month-old female miniature pigs weighing approximately 15 kg were obtained from Ellegaard Minipigs A/S. An acclimatization period of approximately 18 days was allowed prior to surgery (catheter insertion), during which the pigs were grouped and trained for subcutaneous administration and catheter blood collection. The pigs were isolated for three to five days prior to surgery. Six days prior to administration, all pigs had two central venous catheters inserted (Cook Medical, C-TPNS-6.5-90-REDO, silicone, French size 6.5, 106 cm long TPN type), allowing for a postoperative recovery time of at least five days before the start of the study (administration).

All derivatives and comparative compounds were prepared as solutions in PBS at pH 7.4. The dosage used was 0.1 mg/kg (intravenous) or 0.2 mg/kg (subcutaneous).

Gently anesthetize miniature pigs with propofol during administration. Administer intravenous injection via a long central catheter. After administration, flush the catheter with 10 ml of sterile saline. Perform subcutaneous injection at a depth of 5 mm using a 25G needle. Hold the needle in the skin for 10 seconds after injection to prevent backflow.

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.

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.

(iii) Sample processing

Plasma levels of IL-22 derivatives or comparators were measured using an internally developed luminescent oxygen channel assay as described previously (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 on a Perkin Elmer Envision reader. Antibody conjugation to the beads, antibody biotinylation, and LOCI assay procedures were performed as described previously (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%.

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

Plasma concentration-time curves in miniature pigs were measured using non-compartmental analysis (NCA) in Phoenix WinNonlin Professional 6.4 (Pharsight Inc). Calculations were performed using single concentrations, weighted 1/(Y*Y), and linear logarithmic trapezoidal methods. Intravenous administration 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 frequent blood sampling on day 1 of the study.

The only parameter measured to assess pharmacokinetics in mice and rats was the circulating elimination half-life (T _{_1/2} ). In miniature pigs, other parameters measured were the maximum (peak) plasma concentration (C _{_max}) after drug administration, the time to reach C _{_max} (T _{_max} ), the area under the plasma drug concentration-time curve normalized to the drug dose (AUC; which reflects the actual exposure to the drug after administration of a given dose) (AUC/D), the mean residence time (MRT; i.e., the time the drug spends in the body after absorption and before elimination), the mean absorption time of the administered dose (MAT), and systemic utilization (i.e., bioavailability; F). MAT was calculated as the MRT after subcutaneous administration (MRT _{_SC} ) minus the MRT after intravenous administration (MRT _{_IV} ).

result

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 miniature pigs. ND = Undetermined. IV = Intravenous administration. SC = Subcutaneous administration.

Table 7: Pharmacokinetic data obtained in mice

As shown in Table 7, hIL-22 variants with only skeletal variations (Comparatives 1 and 3) exhibited short circulating half-lives regardless of the route of administration. Elongation with the Fc fusion (hFc-hIL-22) significantly increased the half-life. Covalent linkage of fatty acids (C18 diacids; derivatives 1 and 6) resulted in moderate circulating half-lives in mice. Subcutaneous administration of IL-22 derivatives resulted in longer circulation times in mice compared to intravenous administration.

Table 8: Pharmacokinetic data obtained in rats

As shown in Table 8, hIL-22 variants with only skeletal variations (Comparative 1) exhibit short circulating half-lives. Covalent linkage of fatty acids (derivatives 1, 3, and 6) leads to increased circulating half-lives in rats, regardless of the fatty acid used (C16 vs. C18 diacid) or the route of administration. Subcutaneous administration generally results in longer circulation times for IL-22 derivatives compared to intravenous administration.

Table 9: Pharmacokinetic data obtained in miniature pigs

As shown in Table 9, hIL-22 variants with only skeletal variations (Comparatives 1 and 2) have short circulating half-lives comparable to hIL-22. Comparatives Fc fusions (hFc-hIL-22) and all derivatives of IL-22 (Derivatives 1, 6, and 10) have significantly increased circulating half-lives. When administered intravenously, the derivatives of IL-22 have circulating half-lives exceeding 50 hours in miniature pigs, comparable to the comparative IL-22-Fc fusion.

Table 10: Pharmacokinetic data obtained in miniature pigs

As shown in Table 10, the derivative of IL-22 (derivative 6) demonstrated a faster MAT compared to the comparative Fc fusion (hFc-IL-22). MAT measures drug uptake more accurately than simply comparing _Tmax because it also takes into account differences in _Cmax ( _Tmax is affected by both dose and _Cmax ). This study used miniature pigs instead of mice or rats because they are similar to humans.

in conclusion

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

The proven half-life of the IL-22 derivative in miniature pigs is at least 40 hours when administered subcutaneously and more than 50 hours when administered intravenously, thus it is assumed to correspond to a once-weekly dosing curve with a peak-to-trough ratio of 2 in humans.

Therefore, data show that the derivatives of the present invention improve the circulating half-life of IL-22 and exhibit optimized pharmacokinetic and pharmacodynamic properties, thus providing new and improved treatments for a variety of indications, including metabolic, liver, lung, intestinal, kidney, eye, thymus, pancreas and skin diseases, conditions and disorders.

Example 2 - In vitro efficacy study

method

Two in vitro assays were used to study efficacy.

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

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

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

Derivatives of IL-22 were tested together with hIL-22 and hIL-22 variants with only skeletal variations as comparisons. The number of animals ‘n’ used to test each derivative or comparison ranged from 1 to 36.

Therefore, 50 μl of diluted derivative or comparison (diluted in basal medium) was added to each well, and the plate was incubated for four hours. Thus, the derivative and comparison were 2-fold diluted as they were diluted into the existing 50 μl of medium in the wells. Stimulation was terminated after four hours by adding 100 μl of Steadylite plus reagent (Perkin Elmer catalog number 6066759). The plate was sealed with TopSeal A, shaken at 450 rpm for 15 minutes, and read using Mithras or a similar system no later than 12 hours later.

Data analysis was performed using Graphpad Prism. The half-maximal effective concentration ( _EC50 ) of each derivative or comparative was evaluated as a measure of its potency. _EC50 was determined using the logarithm (for compounds) and the reaction-variable slope (4p). As a standard, the Hill slope was limited to 1.

The second in vitro power assay measured pSTAT3 in HepG2 cells, a human liver-derived cell line that endogenously expresses IL-22Ra and IL-10Rb.

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.

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.

IL-22-induced STAT3 phosphorylation was measured following the STAT3 (p-Tyr705) assay protocol (PerkinElmer catalog number TGRS3S(500-10K-50K)). For this purpose, 4 μl of lysate was transferred to a 384-well proxiplate for assay (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.

Data analysis was performed using Graphpad Prism. First, a nonlinear regression was performed in Prism using logarithmic (compound) versus reaction-variable slope (4p) analysis. The Hill slope was constrained to 1. Then, the Y=top from the control compound (His-tagged hIL-22 or hIL-22) was normalized in Prism. 0% was set to the minimum value in each dataset, and 100% was set to the Y=top of the aforementioned nonlinear regression (for the control). The nonlinear regression was repeated as described above, and the activity/weight percentage and _EC50 of the tested derivatives were read from the results at the top and below _EC50 , respectively.

result

Table 11 shows the EC ₅₀ of key derivatives and comparatives measured in the BHK cell reporter gene assay for IL-22 receptor-mediated STAT3 activation.

ID <![CDATA[EC₅₀(nM)]]> hIL-22 0.07 Comparative object 1 0.06 Comparative object 5 0.19 Comparative object 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

Table 11: _EC50 values of key derivatives and comparatives in BH cell assays

Because BHK cell assays contain a large amount of albumin, the measured _EC50 when testing derivatives reflects the effect of albumin binding.

Comparative compound 4, an IL-22 variant with only a skeletal variation, was shown to be equivalent to hIL-22. Derivative 3, having the same skeletal structure as comparative compound 4 but covalently linked to a medium-affinity albumin-binding agent (C16 diacid), exhibited a four-fold reduction in potency compared to hIL-22. Derivative 1, also having the same skeletal structure but covalently linked to a high-affinity albumin-binding agent (C18 diacid), showed only a seven-fold reduction in potency compared to hIL-22.

By comparing the results of derivatives 6 to 9, scans were performed on alkylation sites and skeletal variations in the 35Q and 64Q backgrounds (i.e., mutations at two of the three IL-22 glycosylation sites). The covalent linkage sites in these derivatives were selected based on analytical analysis of the IL-22 structure, which identified sites expected to be exposed on the surface and not involved in receptor binding. The results obtained with these derivatives indicate that Cys substitution and fatty acid covalent linkage are tolerable at several (selected) sites, which was unexpected for the inventors.

Table 12 shows the _EC50 of key derivatives and comparisons measured in the HepG2 cell assay of pSTAT3.

ID <![CDATA[EC₅₀(nM)]]> hIL-22 3.88 Comparative object 1 4.73 Comparative object 4 12.11 Derivative 1 10.13 Derivative 2 6.98 Derivative 6 14.86

Table 12: _EC50 values of key derivatives and comparatives in HepG2 cell assays

In HepG2 cell assays with endogenous receptor expression levels, minimal signal amplification, and no albumin, derivative 1 was 2.5-fold less potent than hIL-22 (similar to Comparative 4, an hIL-22 variant with the same backbone as derivative 1 but without fatty acids).

Table 13 summarizes the results of BHK and HepG2 cell assays to assess mutations in fatty acid covalent linkages and glycosylation sites during N-terminal extension. ND = Undetermined.

Table 13: _EC50 values of key derivatives and hIL-22 in BHK and HepG2 cell assays

Derivative 6 differs from Derivative 1 in that it has additional N35Q and N64Q substitutions (two of the three glycosylation sites are mutated), but it is equivalent (with a slight tendency for Derivative 6 to be slightly less potent).

Although derivatives 2 and 4 have N-terminal extensions of 15-mers with Cys residues (-7C) for fatty acid linkage, they surprisingly exhibit good tolerance.

in conclusion

The reduced potency observed in the tested derivatives of this invention via fatty acid covalent linkage was primarily driven by albumin binding, with minimal contribution from skeletal substitution. This is evidenced by the striking equivalence of Comparative Compound 4 and hIL-22. In contrast, and as previously mentioned, Genentech reported a 34-fold reduction in in vitro potency of its Fc fusion of IL-22.

In HepG2 cell assays with extremely low albumin levels, derivative 1 (an IL-22 derivative that showed a seven-fold decrease in potency in BHK assays (binding to albumin)) showed only a 2.5-fold decrease in potency compared to hIL-22.

The equivalence of derivatives 1 and 6 (Table 13) showed that the 35Q and 64Q mutations had unexpected tolerance without affecting efficacy.

Therefore, IL-22 derivatives maintain high potency in the presence of albumin and are nearly equivalent to hIL-22 in the absence of albumin. Cys substitution and fatty acid covalent linkage at several positions are tolerable.

Therefore, the data show that the derivatives of the present invention exhibit good bioavailability and efficacy, thus providing new and improved treatments for a variety of indications, including metabolic, liver, lung, intestinal, kidney and skin diseases, symptoms and disorders.

Example 3 - In vivo efficacy study in diabetes

This study aimed to investigate the effects of once-daily administration of the derivative of this invention for 8 to 16 days in a diabetic mouse model. The study was conducted in a therapeutic (not preventative) mode, meaning that the pathology of diabetes was established before administration began. Because the mouse model exhibited fatty liver (leptin receptor knockout), it could also be used as a metabolic model for liver disease.

method

Male C57BKSdb/db mice aged 7 to 8 weeks were obtained from the Charles River laboratory (Day -10) and acclimatized for at least one week before the start of the experiment. One week after arrival (Day -3), the mice were randomly assigned to groups of 10 and housed in captivity (or individually for food intake studies). Blood glucose and food intake were measured on Day -3 and each day from Day 1 to Day 16 of the study.

A derivative of IL-22 (Derivative 1) was tested together with an Fc fusion of IL-22 (hFc-hIL-22) as a comparative and a mediator used only as a negative control. Each agent was administered subcutaneously once daily at doses of 0.1, 0.25, 0.5, or 1.0 mg/kg on days 1 through 16 in diabetic db/db mice (n = 6 to 10 in each group). Food intake was reduced following administration of the derivative/comparative/control.

Blood glucose was measured daily throughout the study. At the end of the study, ocular blood samples were collected from anesthetized mice. 500 μl of blood was collected into an EDTA tube. The sample was kept on ice and centrifuged at 6000 G for 5 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.

In addition to measuring the concentrations of derivatives or comparisons, plasma levels of target-related biomarkers (hepatic acute-phase protein, haptoglobin, serum amyloid P fraction (SAP), and gut-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 using a commercial ELISA assay (ALPCO) that recognizes mouse and rat PYY according to the manufacturer's instructions. SAP was measured using a commercial ELISA assay (R&D Systems) that recognizes mouse Pentraxin2/SAP according to the manufacturer's instructions.

result

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

As can be seen from Figure 5, compared with the mediator control, hIL-22 and the hIL-22 variant with only skeletal variation (comparative 3) failed to lower blood glucose during the study.

As shown in Figure 6A, although hFc-hIL-22 had a higher target involvement, reflecting a higher steady-state exposure level in this particular study, both derivative 1 and hFc-hIL-22 reduced blood glucose to normal levels in a comparable manner during the final days of the study, with derivative 1 showing slightly higher efficacy. Reduced food intake was observed in the treated animals compared to the mediator control (see Figure 6B). Therefore, the tested derivatives normalized blood glucose in the db/db model in a similar manner to hFc-hIL-22; as mentioned above, this effect was not observed using hIL-22 or comparator 3.

The levels of the target-related biomarkers haptoglobin, SAP, and PYY, measured at the end of the study, are shown in Figures 7A through 7C. As can be seen from the figures, all three target-related biomarkers were upregulated by the tested derivative and hFc-hIL-22, with hFc-hIL-22 showing particularly greater upregulation than derivative 1.

Figure 8 shows dose-response data for derivative 6 (another derivative of the invention, identical to derivative 1 but with additional substitutions at two glycosylation sites). All three doses tested (0.1, 0.25, and 0.5 mg/kg) were effective in lowering blood glucose over time, and this was particularly evident with increasing concentration.

in conclusion

Both the tested derivative and hFc-hIL-22 normalized blood glucose in the db/db model, demonstrating in vivo therapeutic efficacy. Importantly, this effect was not observed during hIL-22 administration, suggesting that chronic exposure obtained with the long-acting derivative and Fc fusion is necessary for therapeutic efficacy. Although the mechanism of action for antidiabetic effects is not fully elucidated, the hepatic effects of IL-22 (gluconeogenesis and lipogenesis) are considered to be the major contributor.

Treatment with the derivatives of this invention also showed a reduction in food intake, thus demonstrating its efficacy as a treatment for obesity.

Target-related biomarkers were also observed to be upregulated by derivatives and hFc-hIL-22. Certain biomarkers measured in db/db mice are known to be transduced into humans.

It is noteworthy that the circulating half-life (T _{_1/2} ) of subcutaneously administered hFc-hIL-22 was longer than that of derivative 1, particularly in mice (T _{_1/2} were 20 and 8 hours, respectively; see Table 7). Therefore, hFc-hIL-22 exposure was higher at steady state. Observations further confirmed this, with higher levels of target-related biomarkers (haptoglobin, SAP, and PYY) in the hFc-hIL-22 group compared to the derivative 1 group (Figure 7), indicating higher target involvement itself in the experiments shown. Therefore, despite higher exposure and target involvement, the Fc fusion (hFc-hIL-22) showed inferior efficacy compared to the derivative of this invention (derivative 1) during the last three days of the 16-day dosing study.

Therefore, the data show that the derivatives of this invention exhibit good therapeutic efficacy in mouse models of diabetes and liver disease. Since specific biomarkers measured in db/db mice are known to translate into human biomarkers, it is reasonable to predict that this therapeutic efficacy will also translate.

Example 4 - In vivo efficacy study on liver injury (i)

This study aimed to investigate the effects of administering the derivatives of this invention in a mouse model of liver injury. The study was conducted in a preventative mode, meaning that liver injury was only induced after administration began.

method

Ten-week-old C57Bl/6Rj mice were obtained and acclimatized for one week prior to the start of the study. Liver injury was induced by a single intraperitoneal dose of APAP (300 mg/kg, 20 ml/kg). Test derivatives of IL-22 (derivatives 1 and 6) were subcutaneously administered at a dose of 1.5 mg/kg along with a mediator control 2 hours before APAP administration (n = 5 to 10). The study was terminated 24 hours after APAP administration. Peripheral blood was collected to measure plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

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

The liver was fixed in formalin and embedded in paraffin for histological analysis.

Proliferation was measured by Ki67 immunohistochemical (IHC) staining. IHC-positive staining was quantified by image analysis using VIS software (Visiopharm, Denmark).

Apoptosis was measured in a terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL) assay. In short, slides with paraffin-embedded sections were deparaffinized in xylene and rehydrated in a series of fractionated ethanol solutions. The slides were pretreated with proteinase K, and endogenous peroxidase activity was blocked with hydrogen peroxide. A TUNEL mixture (In Situ Cell Death Detection Kit, POD, Roche) was added to the slides, followed by amplification with horseradish peroxidase (HRP) and visualization with diaminobenzidine (DAB) (Chromogen). Finally, the slides were counterstained in hematoxylin and covered with coverslips.

result

Plasma ALT and AST levels at the end of the study are shown in Figures 9A and 9B, respectively. Compared to the mediator/APAP control, mice treated with derivatives 1 or 6 prior to liver injury showed significantly reduced levels of ALT and AST.

At the end of the study, the numbers of TUNEL and ki67 positive cells are shown in Figures 10A and 10B, respectively. The number of TUNEL positive cells was (significantly) reduced in mice treated with derivatives 1 or 6 before liver injury compared to the vector/APAP control. The number of ki67 positive cells was comparable in the APAP-treated groups.

in conclusion

ALT and AST are liver enzymes used as indicators of liver injury. Therefore, derivatives 1 and 6 were shown to protect the liver from APAP-induced damage.

TUNEL assays showed that derivatives 1 and 6 prevented liver injury-induced apoptosis compared to the mediator/APAP control. However, cell proliferation was not affected by these IL-22 derivatives. Since proliferation is physiologically upregulated as a response to injury (as seen in the control), the results indicate a proliferative effect of derivatives 1 and 6, as proliferation was not reduced after injury reduction (the proliferation-to-injury ratio increased).

Therefore, the data show that the derivatives of this invention exhibit good efficacy in protecting against liver damage in mouse models. It is known that specific biomarkers measured in mice can be translated into humans, so it is reasonable to predict that the observed protective effects will also be translated.

Example 5 - In vivo efficacy study on lung injury

This study aimed to investigate the effects of administering the derivatives of this invention in a rat model of lung injury. The study was conducted in a preventative and therapeutic mode, meaning that administration was initiated before inducing lung injury and then continued.

method

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

From day -1 to day 3, animals in groups 3, 4, and 5 were administered derivative 6 once daily (by subcutaneous injection) at doses of 0.5, 1.5, or 4.5 mg/kg, respectively. From day -1 to day 3, animals in group 6 were administered prednisolone once daily (by oral tube feeding) at doses of 10 mg/kg.

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

On day 4 (terminal euthanasia), all animals were submitted for necropsy. Right lungs from all animals were collected for histopathological examination and fixed with 10% neutral buffered formalin (NBF), then immersed in NBF for fixation. Three parallel longitudinal sections were trimmed from the right caudal lobe and fixed in box 01. The right parietal, middle, and accessory lobes were also longitudinally cut and fixed in box 02.

Two slides were made from each box; one slide was stained with hematoxylin and eosin (H&E), while the other slide was stained with hematoxylin and picrosirius red (H&PSR).

A random number generator was then used to assign a random number to each slide. The identification key was recorded in a Microsoft Excel spreadsheet, and a copy was provided to the research pathologist after the slides were evaluated. Thus, the six slides for each lung were read blindly.

The veterinary pathologist then scored the severity of inflammation for each section on each H&E-stained slide (where 0 = absent, 1 = very minor, 2 = mild, 3 = moderate, and 4 = severe). Mean and median scores 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). Mean and median scores were calculated for each group and subjected to nonparametric ANOVA and Kruskal-Wallis posttest analyses.

result

Table 14 presents a summary of the microscopic examination results, revealing the mean and median scores for inflammation and fibrosis in each group.

Table 14: Summary of Microscopic Examination Results in a Rat Model of Lung Injury

As demonstrated by comparing groups 1 and 2 in Table 14, bleomycin induced lung inflammation and fibrosis in the rat model. In groups 3 through 5, i.e., rats treated with the derivative of the present invention (derivative 6), the mean and median scores were lower. These lower scores were comparable to those observed in rats treated with prednisolone (group 6).

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

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

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

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

in conclusion

Microscopic studies have shown that the derivatives of this invention can prevent and/or reduce bleomycin-induced lung inflammation and fibrosis in a rat model. The effects observed on inflammation are comparable to those observed with prednisolone (a corticosteroid known for treating lung inflammation). However, the derivatives of this invention have a unique effect on fibrosis that prednisolone does not possess.

Example 6 - In vivo efficacy study for colitis

This study aimed to investigate the effects of administering the derivatives of this invention to a mouse model of colitis. The study was conducted in both preventative and therapeutic modes, meaning that administration was initiated on the same day as inducing colitis and continued thereafter.

method

Female C57Bl/6JRj mice were randomly assigned to five groups (n=8 per group) based on body weight. Disseminated intraperitoneal saline (DSS) was used to induce colitis in four of the five groups. These mice received DSS in their drinking water for seven days, from day 0 to day 6 of the study. In group 5, the animals received water without DSS and served as healthy controls. Starting from day 0 of the study, DSS mice were treated once daily for 10 days with either the mediator, a test derivative of IL-22 (derivative 6; administered intraperitoneally at 0.35 mg/kg or 1 mg/kg), or a comparison IL-22-Fc fusion (hFc-hIL-22; administered intraperitoneally at 0.5 mg/kg). Body weight, food, and water intake were monitored daily.

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

At the end of the procedure, the intestine is removed for stereoscopic analysis. Therefore, the intestine is rinsed with ice-cold saline and its contents are gently removed before sampling.

The intestine was infiltrated overnight in formalin (Tissue-Tek VIP) and then embedded in paraffin blocks. Using the Systematic Uniform Random Sampling (SURS) principle, samples were taken from proximal to distal ends of the formalin-fixed intestine, yielding a total of four sections, which were placed in multi-cassettes. All tissue sections were placed in this manner to allow for identification of individual sections at later stages. The paraffin blocks were trimmed, and a 5 μm top section was cut and fixed onto a Superfrost+ objective. For the large intestine, another section was cut 500 μm from the top portion, thus obtaining a total of eight colon sections from each animal.

Colonic inflammatory volume was measured stereotactically (i.e., using a three-dimensional interpretation of a two-dimensional cross-section of the colon). Stereoscopic volume estimation was performed on scanned H&E-stained slides using the newCAST system (Visiopharm). Total intestinal volume, mucosal volume, submucosa and muscularis propria volumes, and inflamed tissue volumes were estimated using point counting with an appropriately sized grid system, where all points hitting the structures of interest were counted. The number of points hitting the structures of interest was converted to volume according to the following mathematical relationship:

Vol _ref ＝∑p·A(p)·t

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 the parts. The average inflammatory volume for each group is calculated and statistical analysis is performed.

At the end of the procedure, colon morphology was also assessed by examining H&E stained slides.

result

The volume of colonic inflammation is shown in Figure 12. Mice treated with either dose of Derivative 6 showed prevention of inflammation compared to the mediator control (which also contained DSS). Notably, in the groups treated with Derivative 6, inflammation remained at normal levels, as evidenced by the fact that the volume of colonic inflammation in the treated groups was the same as that in the healthy control group (mediator without DSS). The same was true for the groups treated with hFc-hIL-22.

Representative H&E staining images of the colonic morphology at termination are shown in Figure 13. Mucosal epithelial damage (marked with black arrows) was observed in animals treated with the medium after DSS treatment, but not in animals treated with any dose of derivative 6 or hFc-hIL-22. This demonstrates a protective effect on the epithelial tissue.

Plasma Reg3g levels are shown in Figure 14. DSS treatment induced an increase in basal Reg3g levels (compared to the mediator without DSS). No further increase was detected in the low-dose (0.35 mg/kg) derivative 6 group, but further increases were 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 suggest higher target engagement despite the lower dose, which may be related to the longer half-life of hFc-hIL-22 in mice (the T1/2 of hFc-hIL-22 is 30 hours, while that of derivative 6 is 9.1 hours).

in conclusion

Therefore, the data show that the derivatives of this invention have demonstrated good efficacy in protecting against colitis and mucosal epithelial injury in mouse models. This indicates that a new and improved treatment for intestinal diseases, conditions, and disorders has been discovered. In particular, these findings demonstrate the potential for treating diseases characterized by mucosal epithelial damage, such as inflammatory bowel disease.

Example 7 - In vivo efficacy study on liver injury (ii)

This study aimed to investigate the effects of administering the derivative of the present invention to a second mouse model of liver injury (the first of which is described in Example 4 above). The study was conducted in a preventative mode, meaning that liver injury was only induced after administration was initiated.

method

Male C57Bl6/6j mice were randomly divided into five groups (n=8 per group). In contrast to ConA treatment, at -26 hours and -2 hours, two of the five groups received the test derivative of IL-22 (derivative 1) intraperitoneally at 1 mg/kg. The other two groups received the mediator only at these time points. ConA was administered to all four groups via intravenous bolus at a dose of 15 mg/kg over 30 seconds to induce liver injury. As a healthy control, the fifth group did not receive ConA (and received the mediator only as described above).

Eight or 24 hours after ConA injection, mice were anesthetized with isoflurane, and the largest possible volume of blood was collected via cardiac puncture (using polypropylene serum gel tubes containing a clot activator). Untreated mice (Group 5) were sacrificed at 8 hours. The blood in each tube was mixed with the clot activator by inverting the tubes several times. The tubes were kept at room temperature for 15 minutes, then centrifuged at 2000g for 10 minutes at 4°C. ALT and AST in the serum samples were measured using an automated system (Konelab 20) according to the manufacturer's instructions.

result

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

in conclusion

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

Example 8 - In vivo efficacy study on obesity and NASH

This study aimed to investigate the effects of administering the derivatives of this invention to obese and NASH mouse models. The study was conducted in a treatment (non-preventive) mode, meaning that obesity and NASH pathology were developed prior to the initiation of administration.

method

The diet-induced obesity mouse model was based on male C57BL/6JRj mice fed a high-fat diet for at least 30 weeks prior to the experiment. This diet was rich in fat (40%), fructose (22%), and cholesterol (2%) (Research DietsD09100310). This led to obesity, NAFLD, and ultimately NASH.

Animals were isolated for six days prior to the first dose of the test derivative (or others), and their weight was monitored daily throughout the experiment. Mice were divided into six groups (n = 12 per group). Administration began on day 0 of the study (indicated by the dashed line in Figure 16) and was administered subcutaneously once daily at the following doses.

Semaglutide, a long-acting GLP-1 receptor agonist, was used as a positive control in the first group and in combination with a test derivative of IL-22 (Derivative 6) in the second group. Semaglutide was titrated stepwise 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 groups, semaglutide was started on Day 0 after the plateau in weight loss observed in the semaglutide-treated groups, and Derivative 6 was started on Day 12 (indicated by dashed lines in Figure 16).

In the third "high-dose" group, the dose of derivative 6 was titrated stepwise 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. On day 14, the dose was switched from 0.25 mg/kg to 0.1 mg/kg (shown as 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 titration.

In the fifth group, the dosage of the IL-22-Fc fusion compound (hFc-hIL-22), used as a comparison, was titrated stepwise 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 correspondingly higher target engagement compared to derivative 6 as seen in Example 6 (see Figure 14), the dosage of hFc-hIL-22 was selected to match the target engagement of the 0.25 mg/kg derivative 6 group.

The sixth group of drug delivery vehicles served as a negative control only.

Plasma triglyceride (TG) levels were measured at baseline (day -2), week 2 (day 14), and week 4 (day 28) after the start of dosing. Specifically, tail blood samples were collected for analysis by injecting a volume equal to or less than 200 μl of tail blood into an open Microvette (100 μl or 200 μl) tube treated with an appropriate anticoagulant. The blood was incubated at 4°C until centrifuged at 3000g for 10 minutes. The plasma supernatant was transferred to a new tube and immediately frozen on dry ice and stored at -80°C. TG levels were measured on a C501 automated analyzer using a commercial kit (Roche Diagnostics) according to the manufacturer's instructions.

result

The body weight during the experiment is shown in Figure 16.

This study demonstrates the dose-dependent efficacy of derivative 6 in reducing body weight in an obese mouse model. Furthermore, it exhibits additive effects with the GLP-1 receptor agonist (semaglutide), which is being investigated in late-stage clinical trials for obesity treatment. Data indicate that derivative 6 is superior to 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 administered at half the dose of derivative 6. Therefore, the 0.1 mg/kg dose of hFc-hIL-22 used in this study was chosen for similar target engagement as the 0.25 mg/kg derivative 6 group.

Sensitivity to weight loss induced by derivative 6 was observed in diet-induced obese mice, but not in lean mice. For example, in a 10-day study of DSS-induced colitis administered once daily (Example 6), both the DSS/carrier group and the DSS/derivative 6 (0.35 mg/kg) group had a body weight of 19.0 g at the start of the study. At the end of the study, the body weight in the DSS/carrier group was 17.6 g, while the body weight in the DSS/derivative 6 (0.35 mg/kg) group was 17.4 g, with no difference between the two groups (p = 0.82 in the unpaired Student's t-test). In contrast, on day 10 of this study, the body weights of mice in the carrier group and the derivative 6 (0.25 mg/kg) group were 43.5 g and 35.2 g, respectively. Therefore, a significant weight loss was observed in the derivative 6 group compared to the carrier group (p < 0.0001 in the unpaired Student's t-test on day 10). At the start of the study, the body weight in the mediator group was similar to that in derivative group 6 (44.3 g and 44.1 g, respectively).

Plasma TG levels measured at baseline (day -2), week 2 (day 14), and week 4 (day 28) after the start of administration are shown in Table 15.

Table 15: Plasma TG levels (nmol/L) in obese and NASH mouse models

Δ refers to the change in TG level (nmol/L) at a specified treatment and time point relative to the baseline. As can be seen from Table 15, the level change in the medium group is positive (increase), but the level changes in all other groups are negative (decrease).

Derivative 6 was more effective than semaglutide in lowering TG levels, even at low doses, resulting in less weight loss than semaglutide (e.g., ΔTG level (nmol/L) at week 4 for semaglutide was -0.14 ± 0.066, for derivative 6 (0.05 mg/kg) it was -0.30 ± 0.034, and for derivative 6 (0.25/0.1 mg/kg) it was -0.35 ± 0.052). These results indicate that derivative 6 is highly effective in lowering TG, with its effect partially independent of weight loss. Furthermore, the effects of derivative 6 are completely additive with those of semaglutide. At week 4, TG levels (nmol/L) calculated as ΔTG decreased: -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). Although TG levels in the mediator groups increased during the study, they decreased compared to baseline (ΔTG).

in conclusion

Studies have shown that the derivative of this invention (Derivative 6) can induce weight loss in obese mice in a dose-dependent manner, at least to levels comparable to those observed with 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 with hFc-IL-h22 at doses selected to provide similar levels of target involvement. No weight loss induced by Derivative 6 was observed in DSS-treated lean mice as in diet-induced obese mice, indicating that obese mice are more sensitive to Derivative 6-induced weight loss. Since the weight loss observed in diet-induced obese mice is the same as the readout used in humans, it is reasonable to predict that the observed weight loss will also be converted.

Derivative 6 also demonstrated high efficacy in lowering TG levels. Derivative 6 showed greater efficacy than semaglutide at both tested doses, and a complete additive effect of the combined administration was observed. Given that derivative 6 at a dose of 0.05 mg/kg has greater efficacy than semaglutide, it can be concluded that the TG-lowering effect of derivative 6 is at least partially independent of weight loss, despite less weight reduction. 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 as that used in humans, it is reasonable to predict that the observed effect will also translate. Therefore, the results indicate that a novel treatment for conditions and disorders characterized by high TG levels has been discovered.

Although certain features of the invention have been described and illustrated herein, many modifications and equivalents will occur to those skilled in the art. Therefore, it should be understood that the claims are intended to cover all modifications and equivalents falling within the true spirit of the invention.

Claims (8)

1. A derivative of IL-22 having the following structure: 2. A derivative of IL-22 having the following structure: 3. A derivative of IL-22 having the following structure: 4. The derivative of any one of claims 1 to 3, or a pharmaceutical composition comprising the derivative of any one of claims 1 to 3, for use in a therapeutic manner. 5. A derivative of any one of claims 1 to 3 or a pharmaceutical composition comprising a derivative of any one of claims 1 to 3, for use in the treatment of metabolic, liver, lung, intestinal, kidney, or skin diseases, symptoms, or ailments. 6. The derivative or pharmaceutical composition for application as claimed in claim 5, wherein the application comprises administering a weekly dose of the derivative to a patient in need of it at a dose between 0.001 μg/kg body weight and 10 mg/kg body weight. 7. The derivative or pharmaceutical composition for application as described in claim 5, wherein: (i) The metabolic disease, condition or disorder is obesity, type 1 diabetes, type 2 diabetes, hyperlipidemia, hyperglycemia or hyperinsulinemia; (ii) The liver disease, condition or disorder mentioned is 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), acetaminophen-induced hepatotoxicity, acute liver injury, sclerosing cholangitis, biliary cirrhosis or pathological disorder caused by surgery or transplantation. (iii) The lung disease, condition or disorder is chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, chemical injury, viral infection, bacterial infection or fungal infection; (iv) The intestinal disease, condition or disorder mentioned is inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, graft-versus-host disease (GvHD), chemical injury, viral infection or bacterial infection; (v) The kidney disease, condition, or disorder described is acute or chronic kidney disease; or (vi) The skin disease, condition or ailment referred to is a wound, inflammatory disease or GvHD. 8. The derivative or pharmaceutical composition for application as described in claim 6, wherein: (i) The metabolic disease, condition or disorder is obesity, type 1 diabetes, type 2 diabetes, hyperlipidemia, hyperglycemia or hyperinsulinemia; (ii) The liver disease, condition or disorder mentioned is 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), acetaminophen-induced hepatotoxicity, acute liver injury, sclerosing cholangitis, biliary cirrhosis or pathological disorder caused by surgery or transplantation. (iii) The lung disease, condition or disorder is chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, chemical injury, viral infection, bacterial infection or fungal infection; (iv) The intestinal disease, condition or disorder mentioned is inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, graft-versus-host disease (GvHD), chemical injury, viral infection or bacterial infection; (v) The kidney disease, condition, or disorder described is acute or chronic kidney disease; or (vi) The skin disease, condition or ailment referred to is a wound, inflammatory disease or GvHD.