Dosage regimens for interleukin-22 derivatives

Abstract

The present invention relates to derivatives of interleukin-22 (IL-22), particularly derivatives comprising a fatty acid covalently attached to the IL-22 protein, and novel dosing regimens for treating metabolic, intestinal, and hepatic diseases, disorders, and conditions.

Description

[Technical Field]

[0001] Electronic Sequence Listing Reference The contents of the electronic sequence listing (CKTI-007 / F01EP_SeqList_ST26.xml, size: 46,749 bytes and created on December 19, 2022) are incorporated herein by reference in their entirety.

[0002] The present invention relates to derivatives of Interleukin-22 (IL-22), particularly derivatives comprising a fatty acid covalently attached to the IL-22 protein, and novel dosing regimens for treating metabolic, intestinal, and hepatic diseases, disorders, and conditions. [Background technology]

[0003] In the human body, IL-22 is secreted in response to cues reflecting pathogen infection and immune activation. The effects of IL-22 are the result of the orchestrated involvement of several activities / pathways. IL-22 acts on epithelial barrier tissues and organs upon injury to protect cells and maintain barrier function. It also accelerates repair, prevents fibrosis, and controls inflammation. IL-22 has been reported to be able to treat a range of medical conditions, including pancreatitis, renal failure, skin wounds, and conditions often observed in diabetic or overweight mammals, such as hyperglycemia, hyperlipidemia, and hyperinsulinemia.

[0004] However, IL-22 is generally rapidly cleared from the body by the kidney, limiting its use in clinical practice. This is a common feature of cytokines, and cytokine drug development candidates with extended half-lives have reached the drug development stage, for example, for oncology and immunotherapy treatments. Generally, these half-life-extended cytokines use Fc fusion solutions or PEGylation. Therefore, known methods for extending the half-life of circulating IL-22 attempt to artificially increase the size of IL-22 to greater than 70 kDa to avoid renal clearance. Genentech and Generon Shanghai both have long-acting IL-22-Fc fusions in clinical development. Modifying IL-22 with polyethylene glycol (PEGylation) is another known means to avoid renal clearance. However, these existing solutions are not without drawbacks, including immunogenicity, reduced and heterogeneous activity due to PEGylation, and poor distribution, receptor engagement kinetics, and potency of Fc fusions.

[0005] A new and improved class of IL-22 derivatives has been discovered and is described in WO 2019 / 101888, WO 2022 / 238503, and WO 2022 / 238510. These biocompatible derivatives contain fatty acids covalently attached to the IL-22 protein. They enhance circulating half-life and exhibit optimized pharmacokinetic (PK) and pharmacodynamic (PD) properties compared to the native molecule. They maintain the potency and other properties of the native molecule and avoid the toxicity, immunogenicity, and other adverse reactions exhibited by alternative derivatives of IL-22, such as the PEGylated derivatives and Fc fusions mentioned above.

[0006] Furthermore, these derivatives have been shown to be effective in the treatment and / or prevention of a range of diseases, disorders and conditions in animal models, including diabetes, liver injury, lung injury, colitis, obesity and non-alcoholic steatohepatitis (NASH).

[0007] Nevertheless, there remains a need for therapeutically effective dosing regimens in humans. It was therefore an object of the present invention to find dosing regimens that minimize adverse side effects without substantially compromising the beneficial effects of treatment, particularly in the treatment of metabolic, intestinal and hepatic diseases, disorders or conditions. Summary of the Invention

[0008] In a first aspect, there is provided a derivative of IL-22 comprising a fatty acid covalently attached to the IL-22 protein for use in a method of treating a metabolic, intestinal and / or hepatic disease, disorder or condition, the method comprising subcutaneously administering the derivative of IL-22.

[0009] In a second aspect, there is provided a derivative of IL-22 comprising a fatty acid covalently attached to the IL-22 protein for use in a method of treating a metabolic, intestinal and / or hepatic disease, disorder or condition, the method comprising intravenously administering the derivative of IL-22. [Brief explanation of the drawings]

[0010] [Figure 1] (A) C18 diacid, (B) C16 diacid, and (C) C14 diacid are exemplified, each connected to a linker containing a Cys reactive unit. These combinations of fatty acids and linkers are used in derivatives identified herein as Derivatives 1-12. [Figure 2] The structure of the derivative identified herein as Derivative 1 is illustrated. [Figure 3] The structure of the derivative identified herein as Derivative 6 is illustrated. [Figure 4] The structure of the derivative identified herein as derivative 10 is illustrated. [Figure 5] The structure of the derivative identified herein as derivative 11 is illustrated. [Figure 6]The structure of the derivative identified herein as derivative 12 is illustrated. [Figure 7] 1 illustrates the reduction of colonic inflammation volume in a murine model of colitis after subcutaneous administration of derivative 1 (see Table 7 for mouse group identification). [Figure 8] Figure 1 illustrates the curve of an in vitro signal transducer and activator of transcription 3 (STAT3) assay for Derivative 1. The y-axis shows luminescence in relative light units (RLU), and the x-axis shows the concentration of Derivative 1. "Replicate 01" and "Replicate 02" are two independent data sets obtained by repeating the assay. [Figure 9] 1 illustrates the normalization of blood glucose in a murine db / db diabetes model after subcutaneous administration of Derivative 1 (see Table 11 for mouse group identification). [Figure 10] Illustrates weight reduction in a murine diet-induced obesity model after subcutaneous administration of derivative 6 (see Table 12 for mouse group identification). [Figure 11] 1 illustrates the reduction of (A) body weight and (B) fasting plasma insulin in a murine diet-induced obesity model after subcutaneous administration of Derivative 1 (see Table 12 for mouse group identification). [Figure 12] 1 illustrates the reduction of plasma alanine aminotransaminase (ALT) levels in a murine inflammatory acute liver failure model after subcutaneous administration of Derivative 1 (see Table 13 for mouse group identification). [Figure 13] 1 illustrates an increase in IL-22 target-associated biomarkers, (A) regenerating islet-derived protein 3a (REG3a) and (B) highly sensitive C-reactive protein (hsCRP), after a single subcutaneous administration of derivative 1 in humans. [Figure 14]FIG. 1 illustrates a dose-dependent increase in the target engagement marker REG3a after SC administration of Derivative 1 in a multiple ascending dose study in humans. [Figure 15] 1 illustrates a dose-dependent reduction in total cholesterol in blinded data from a multiple ascending dose study of Derivative 1 administered SC in humans. [Figure 16] 1 includes a table outlining the timescales and events of the multiple ascending doses (MAD) protocol of Example 11. DETAILED DESCRIPTION OF THE INVENTION

[0011] In the following, Greek letters are represented by their symbols rather than by their written names, e.g., α = alpha, β = beta, ε = epsilon, γ = gamma, and μ = mu. Amino acid residues can be identified by their full name, three-letter code, or one-letter code, all of which are fully equivalent.

[0012] In a first aspect, there is provided a derivative of IL-22 comprising a fatty acid covalently attached to the IL-22 protein for use in a method of treating a metabolic, intestinal and / or hepatic disease, disorder or condition, the method comprising subcutaneously administering the derivative of IL-22.

[0013] In a second aspect, there is provided a derivative of IL-22 comprising a fatty acid covalently attached to the IL-22 protein for use in a method of treating a metabolic, intestinal and / or hepatic disease, disorder or condition, the method comprising intravenously administering the derivative of IL-22.

[0014] I. IL-22 Derivatives The term "derivative of IL-22" as used herein refers to an IL-22 protein having a covalently attached fatty acid. This term encompasses both derivatives in which the fatty acid is directly covalently attached to the IL-22 protein and derivatives in which the covalent attachment is via a linker that can be devised from various subunits.

[0015] Covalent attachment of fatty acids is a proven technique for extending the half-life of peptides and proteins, a method of subtending fatty acids from peptides or proteins, as is well known from marketed products for type 1 and type 2 diabetes, such as the insulin preparations Levemir® (detemir) and Tresiba® (degludec), and the glucagon-like peptide-1 (GLP-1) derivatives Victoza® (liraglutide) and Ozempic® (semaglutide).

[0016] Fatty acid conjugation allows binding to albumin, thereby preventing renal excretion and providing some steric protection against proteolysis. Advantageously, it provides minimal modification to IL-22 compared to Fc fusion or PEGylation. In this regard, while Fc fusion and PEGylation aim to increase the size of IL-22 above the threshold for renal clearance, derivatives containing fatty acids covalently bound to the IL-22 protein retain a small size similar to that of the IL-22 protein. Thus, because fatty acid conjugation is a minimal modification, the resulting derivatives are expected to maintain native-like properties, including distribution, diffusion rate, and receptor association (binding, activation, and transport), minimizing the risk of immunogenicity.

[0017] As mentioned above, fatty acid conjugation has proven therapeutic efficacy in insulin and GLP-1 derivatives for diabetes. However, IL-22 is a very different protein with respect to its size, sequence, and biological properties. Therefore, it was counterintuitive to the inventors at the time that fatty acids could be covalently conjugated to IL-22 while maintaining therapeutic efficacy. It was particularly surprising that such minimal modifications to IL-22 could result in high potency (same as or close to that of hIL-22) combined with a very long circulating half-life.

[0018] The term "IL-22 protein," as used herein, may refer to a native IL-22 protein, such as hIL-22, or a variant thereof. A "variant" may be a protein having an amino acid sequence similar to that of the native protein, as further defined herein.

[0019] In nature, human IL-22 protein is synthesized with a 33-amino acid signal peptide for secretion. The mature human IL-22 protein (i.e., hIL-22) is 146 amino acids long and shares 80.8% sequence identity with murine IL-22 (the latter is 147 amino acids long). The amino acid sequence of hIL-22 is identified herein as SEQ ID NO: 1. Similar to other IL-10 family members, the IL-22 structure contains six α-helices (termed helices A through F).

[0020] Thus, derivatives for use in the present invention may have the native amino acid sequence of hIL-22. Alternatively, they may have one or more amino acid sequence mutations within the native sequence. They may additionally or alternatively contain one or more amino acid sequence mutations relative to the native sequence (i.e., other than the native sequence). Thus, in one embodiment, the derivative comprises a fatty acid covalently attached to hIL-22 or a variant thereof.

[0021] Expressions such as "within," "against," "corresponding to," and "equivalent to" are used herein to characterize sites of fatty acid alteration and / or covalent attachment in the IL-22 protein by reference to the sequence of the native protein, e.g., hIL-22. In SEQ ID NO: 1, the first amino acid residue of hIL-22 (alanine (Ala)) is assigned to position 1.

[0022] Thus, mutations within the sequence of hIL-22 are mutations to any of residues 1-146 of SEQ ID NO: 1. For example, a Glu substitution for the native Asp at residue 10 in hIL-22 is designated herein as "D10E." When a derivative also has a fatty acid covalently attached at position 10, it is referred to herein as attachment at residue "10E."

[0023] However, the mutations to the sequence of hIL-22 are outside of residues 1-146 of SEQ ID NO:1. For example, Derivative 2, as defined herein, comprises a 15-amino acid long N-terminal peptide. The residues of the N-terminal peptide are negatively numbered, starting from the residue bound to residue 1 of hIL-22; i.e., the first residue in the N-terminal peptide bound to residue 1 of hIL-22 is designated "-1." Thus, Derivative 2 has a fatty acid covalently attached to the seventh residue of the N-terminal peptide starting at position -1, which is Cys; therefore, the covalent attachment site of Derivative 2 is referred to herein as "-7C." However, it should be understood that the numbering used in the sequence listing of Derivative 2 begins at 1 in accordance with WIPO Standard ST.26; therefore, position 1 in the sequence listing of Derivative 2 is actually residue -7, as referred to herein.

[0024] To form a derivative for use in the present invention, one, two, three, four, five or more mutations may be made in the native sequence. In this regard, for example, 10, 15, 20, 25, 50, 75, more than 100, or even more than 125 mutations may be made. Any of residues 1 to 146 in the native sequence may be altered. Exemplary residues 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, 78 of hIL-22. , 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. Mutations at residues 1, 21, 35, 64, 95, 106, 113 and / or 114 are particularly advantageous.

[0025] The mutation in the native sequence is typically an amino acid substitution. As used herein, the term "substitution" can mean that one amino acid in a native protein is replaced with another amino acid. These may be conservative or non-conservative substitutions.Exemplary substitutions include A1C, A1G, A1H, P2C, P2H, I3C, I3H, I3V, S4H, S4N, S5H, S5T, H6C, H6R, C7G, R8G, R8K, L9S, D10E, D10S, K11C, K11G, K11V, S12C, N13C, N13G, F14S, Q15C, Q15E, Q16V, P17L, Y18F, I19Q, T20V, N21C, N21D, N21Q, R22S, F24H, M25E, M25L, L26S, A27L, E29P, A30Q, L32C, L32R, A33C , A33N, D34F, N35C, N35D, N35H, N35Q, N36Q, T37C, T37I, D38L, V39Q, R40W, L41Q, I42P, E44R, K45A, F47T, H48G, H48R, G49N, V50S, S51C, M52A , M52C, M52L, M52V, S53C, S53K, S53Y, E54D, E54F, R55Q, R55V, C56Q, L58K, M59I, Q61E, V62D, L63C, N64C, N64D, N64Q, N64W, F65G, L67Q, E69D , E69L, V70S, L71C, F72D, F72L, P73C, P73L, Q74T, R77I, F78Q, Q79E, M82Y, Q83G, E84R, V86A, F88N, A90P, A90T, R91C, R91K, R91Y, L92R, S93Y , N94C, N94Q, R95C, R95K, R95Q, L96E, S97K, T98C, T98N, T98S, C99V, H100S, E102S, G103D, D104Y, D105Y, L106C, L106E, L106Q, H107L, H107N , I108L, Q109Y, R110C, R110K, N111K, V112E, Q113C, Q113R, K114C, K114R, L115V, K116Y, D117E, T118G, V119A, K120H, K121R, L122A, G123V, G126Y, E127C, I128V, K129V, G132Y, E133Q, L134P, D135M, L137D, F138R, M139L, M139R, L141Q, N143S, A144E, C145E, I146R and / or I146V.Advantageously, the substitutions may be selected from the group consisting of A1C, A1G, A1H, N21C, N21D, N21Q, N35C, N35D, N35H, N35Q, N64C, N64D, N64Q, N64W, R95C, L106C, Q113C, Q113R, K114C and K114R. Surprisingly, the substitutions used in the present invention do not adversely affect IL-22 activity.

[0026] Specific combinations of substitutions include: (i) A1G, N21D, N35D, and N64D; (ii) A1G, I3V, S4N, S5T, H6R, R8K, D10E, K11V, T20V, H48R, M52A, S53K, E54D, R55Q, E69D, F72L, A90T, R91K, R95Q, T98S, E102S, L106Q, H107N, R110K, Q113R, K114R, D117E, and I146V; and (iii) A1G, I3V, S4N, S5T, H6R, R8K, D10E, K11V, T20V, H48R, M52A, S53K, E5 4D, 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) A1 G and N35Q;(xv) A1G and N64Q;(xvi) A1G, N21Q and N35Q;(xvii) A1G, N21Q and N64Q;(xviii) A1G, N21Q, N35Q and N64Q;(xix) A1G and K11C;(xx) A1G and N13C;(xxi) N35Q and N64Q;(xxii) A1C, N35Q and N64Q;(xxiii) H6C, N35Q and N64Q;(xxiv) I3C, N35Q and N64Q;(xxv) P2C, N35Q and N64Q;(xxvi) L32C, N35Q and N64Q;(xxvii ) N35Q, M52C and N64Q; (xxviii) N13C, N35Q and N64Q; (xxix) N21C, N35Q and N64Q; (xxx) N35Q, N64Q and N94C; (xxxi) N35Q, N64Q and P73C; (xxxii) N35Q, N64Q and Q113C; (xxxiii) N35Q, N64Q and R91C; (xxxiv) N35Q, N64Q and R95C; (xxxv) N35Q, N64Q and L106C; (xxxvi) N35Q, N64Q and R110C; (xxxvii) S12C, N35Q and N64Q;(xxxviii) N35Q, S51C and N64Q; (xxxix) N35Q, S53C and N64Q; (xxxx) N35Q, T37C and N64Q; (xxxxi) N35Q, N64Q and T98C; (xxxxii) Q15C, N35Q and N64Q; (xxxxiii) N35C and N64Q; (xxxxiv) H6C, N35Q and N64Q; (xxxxv) A33C, N35Q and N64Q; and (xxxxvi) A1H, P2H, I3H, S4H, S5H, C7G, R8G, L9S, D10S, K11G, N13G, F14S, Q15E, Q16V, P17L, 18F, Y19Q, N21Q, R22S, F24H, M25E, L26S, A2 7L, E29P, A30Q, L32R, A33N, D34F, N35H, T37I, D38L, V39Q, R40W, L41Q, I42P, E44R, K45A, F47T, H48G, G49N, V50S, M52V, S 53Y, 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, D Examples of substitutions include 105Y, L106E, H107L, I108L, Q109Y, R111K, V112E, L115V, K116Y, D117E, T118G, V119A, K120H, K121R, L122A, G123V, G126Y, E127C, I128V, K129V, G132Y, E133Q, L134P, D135M, L137D, F138R, M139R, L141Q, N143S, A144E, C145E, and I146R. Any and all combinations of substitutions are contemplated and form part of the present invention.

[0027] Derivatives for use in the first or second aspects may typically comprise an amino acid substitution whereby Cys is substituted relative to the native residue, optionally at any of the above specified positions such as 1, 2, 3, 6, 11, 12, 13, 15, 21, 32, 33, 35, 37, 51, 52, 53, 63, 64, 71, 73, 91, 94, 95, 98, 106, 110, 113, 114 and / or 127. Advantageously, the IL-22 protein comprised in a derivative for use in the first or second aspect comprises a Cys residue at position 1 of hIL-22. The A1C substitution in combination with substitutions at the two glycosylation sites at positions 35 and 64 is particularly advantageous as it results in faster uptake without adversely affecting potency or half-life (see derivatives 6 and 10 in Examples 1 and 2 of WO 2021 / 089875, which are incorporated herein by reference). Alternatively, the IL-22 protein comprised in the derivative for use in the first or second aspect comprises the substitution R95C (as in derivative 14) or L106C (as in derivative 13). Such substitutions in combination with substitutions at the two glycosylation sites at positions 35 and 64 (as in derivatives 11 and 12) are particularly advantageous as it results in faster uptake without adversely affecting potency or half-life (see derivatives 1 and 2 in Example 1 of WO 2022 / 238510, which are incorporated herein by reference). In one advantageous embodiment, the derivative for use in the first or second aspect comprises the substitutions N35Q, N64Q and R95C. In another advantageous embodiment, the derivative for use in the first or second aspect comprises the substitutions N35Q, N64Q and L106C. However, in other embodiments, the derivative for use in the first or second aspect may advantageously comprise R95C or L106C without any additional substitutions or mutations in hIL-22 (SEQ ID NO: 1).

[0028] Alternatively or additionally, the mutation within the native sequence may be an amino acid insertion. 5, 10, 15, 20, 25, 30, 35, 40, 45, or even up to 50 amino acids may be inserted into the native sequence. Trimers, pentamers, heptamers, octamers, nonamers, and 44-mers are particularly advantageous in this regard. Exemplary sequences are shown in Table 1. Insertions can be made anywhere in the native sequence, but are preferred in helix A (e.g., residue 30), loop CD (e.g., residue 75), helix D (e.g., residue 85), and / or helix F (e.g., residue 124).

[0029] [Table 1]

[0030] The 1, 2, 3, 4, 5 or more mutations in the native sequence may be independently selected from the group consisting of substitutions and insertions.

[0031] Mutations in the native sequence may also or alternatively include deletion of one or more amino acids within SEQ ID NO: 1. Thus, the peptide may include deletion of up to five amino acids. Deletions of no more than three or two amino acids are preferred. The deletions may, for example, be at distant (i.e., non-contiguous) positions within SEQ ID NO: 1. Mutations may also or alternatively be deletions of 2, 3, 4 or 5 consecutive amino acids within SEQ ID NO: 1, meaning that a stretch of up to five adjacent amino acids may be deleted.

[0032] Sequence variations to the amino acid sequence of hIL-22, when present, typically involve extensions, such as the addition of a peptide at the N-terminus. The peptide may consist of 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 comprised in the derivative for use in the first or second aspect contains an N-terminal GPG. In a particularly preferred example, the derivative for use in the first or second aspect contains both a Cys residue at position 1 of hIL-22 (SEQ ID NO: 1) and an N-terminal GPG. This has been found to produce derivatives with very good half-lives and potency (see derivatives 1, 3 and 5 in Examples 1 and 2 of WO 2021 / 089875, which are incorporated herein by reference. In one embodiment, the IL-22 protein comprised in the derivative for use in the first or second aspect does not comprise an N-terminal GPG.

[0033] [Table 2]

[0034] Sequence variations to the amino acid sequence of hIL-22, if present, can include the addition of a peptide at the C-terminus. The peptide can consist of 5, 10, 15, 20, 25, 30, 35, 40, 45, or even up to 50 amino acids. Exemplary C-terminal peptide sequences include those shown in Table 2 (for the N-terminal peptide). The heptamer is particularly advantageous in this regard, optionally having the amino acid sequence GSGSGSC (SEQ ID NO: 22).

[0035] Derivatives for use in the present invention may include both N- and C-terminal peptides in addition to the native or variant hIL-22 amino acid sequences as described herein. Any combination of the N- and C-terminal peptides described herein is contemplated and expressly included in the present invention.

[0036] It is understood that the present invention extends to any derivative of IL-22, comprising a fatty acid covalently attached 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, a variant has at least 20% or even at least 30% sequence identity with hIL-22. A variant may have "substantially the amino acid sequence" of hIL-22, which may mean a sequence having at least 40% sequence identity with the amino acid sequence of hIL-22. Thus, in one embodiment, a derivative for use in the first or second 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 are set forth in SEQ ID NOS: 23-28.

[0037] Those skilled in the art understand how to calculate the percentage identity between two amino acid sequences. An alignment of the two sequences is first prepared, followed by calculating the sequence identity value. The percentage identity of two sequences can vary depending on (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented by different programs), or structural alignment by 3D comparison, and (ii) the alignment method, such as local versus global alignment, the pair-score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, including the parameters used, such as the function form and constants.

[0038] After alignment, there are many different ways to calculate the percentage of identity between two sequences.For example, the number of identities can be divided by (i) the length of the shortest sequence, (ii) the length of the alignment, (iii) the average length of the sequences, (iv) the number of non-gap positions, or (iv) the number of equivalent positions excluding overhangs.It is also understood that the percentage of identity is strongly length-dependent.Therefore, the shorter the sequence pair, the higher the sequence identity that can be expected to occur by chance.

[0039] Thus, it is understood that accurate alignment of amino acid sequences is a complex process. The general multiple alignment program ClustalW is a preferred method for generating multiple alignments of proteins according to the present invention. Suitable parameters for ClustalW may be as follows: for protein alignments, Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and protein alignments, ENDGAP = -1 and GAPDIST = 4. Those skilled in the art will recognize that it may be necessary to vary these and other parameters for optimal sequence alignment.

[0040] Preferably, the percentage identity between two amino acid sequences is calculated as (N / T) * The percentage identity between two sequences may be calculated by aligning sequences such as 100, where N is the number of positions where the sequences share identical residues, and T is the total number of positions compared, including gaps but excluding overhangs. Thus, the most preferred method for calculating the percentage identity between two sequences is to (i) prepare a sequence alignment using, for example, the ClustalW program with a suitable set of parameters as described above, and (ii) calculate the values ​​of N and T using the following formula: sequence identity = (N / T) * 100, including inserting.

[0041] Alternative methods for identifying similar sequences are known to those of skill in the art.

[0042] Preferably, a derivative for use in the first or second aspect contains 200 or fewer amino acids. For example, the derivative contains fewer than 190, fewer than 180, fewer than 170, fewer than 160, or even fewer than 150 amino acids. Preferably, the derivative contains at least 146 amino acids, which is the number of amino acids in hIL-22. The derivative may contain at least 150 amino acids, at least 160 amino acids, at least 170 amino acids, or even at least 180 amino acids. Derivatives for use in the present invention may include proteins of any length within the above ranges, but they are typically between 146 and 180 amino acids in length.

[0043] Derivatives for use in the present invention include fatty acids covalently attached to the IL-22 protein, regardless of whether they have a native or variant amino acid sequence. The fatty acid is typically covalently attached to the IL-22 protein via a linker. The fatty acid and linker are preferably connected to each other via an amide bond, and the linker is covalently attached to the IL-22 protein. Thus, the fatty acid and linker can be present as side chains on the IL-22 protein. It was surprising to the inventors at the time that the covalently attached fatty acid did not adversely affect IL-22 activity. It was particularly surprising that fatty acid attachment was associated with additional benefits, such as increased half-life.

[0044] The fatty acid may be any suitable fatty acid. In particular, the fatty acid may be of formula I: HOOC-(CH2) x -CO- * wherein x is an integer ranging from 10 to 18, optionally from 12 to 18, from 14 to 16, or from 16 to 18; *indicates the point of attachment to the IL-22 protein or linker. The fatty acid may be a fatty diacid, such as a C12, C14, C16, C18 or C20 diacid. Advantageously, the fatty acid is a C16 or C18 diacid, most advantageously a C18 diacid.

[0045] For example, -(CH2) in Formula I x may be a straight chain alkylene where x is 10. For convenience, this fatty acid may be referred to as a C12 diacid, i.e., an aliphatic dicarboxylic acid having 12 carbon atoms. Alternatively, the -(CH2) in Formula I x may be a straight chain alkylene where x is 12. For convenience, this fatty acid may be referred to as a C14 diacid, i.e., an aliphatic dicarboxylic acid having 14 carbon atoms. Similarly, -(CH2) in Formula I x may be a straight chain alkylene where x is 14 (C16 diacid), 16 (C18 diacid) or 18 (C20 diacid). Suitably, the derivative for use in the first or second aspect comprises a C14, C16, C18 or C20 diacid, more suitably a C16 or C18 diacid, even more suitably a C18 diacid.

[0046] The diacids can form non-covalent associations with albumin, thereby facilitating the circulation of the derivatives in the bloodstream. Shorter diacids (e.g., C16 diacids) have lower albumin affinity and therefore shorter half-lives than longer diacids (e.g., C18 diacids). However, they are still long-acting derivatives with expected half-lives of more than 1 day in humans.

[0047] The fatty acid linkage also itself stabilizes the IL-22 protein against proteolysis, and the resulting half-life is typically similar to that of an IL-22-Fc fusion (i.e., greatly improved compared to hIL-22).

[0048] Derivatives for use in the first or second aspect may include specific combinations of fatty acids and IL-22 proteins. For example, a C14, C16, C18, or C20 diacid may be attached to an IL-22 protein comprising a Cys residue at position 1 of hIL-22 and / or an N-terminal GPG. In one example, a derivative for use in the first or second aspect includes a C18 diacid, and the IL-22 protein comprises both a Cys residue at position 1 of hIL-22 and an N-terminal GPG. As another example, a derivative for use in the first or second aspect includes a C18 diacid, and the IL-22 protein comprises a Cys residue substituted at positions 95 or 106 of hIL-22. Such derivatives may further comprise Gln residues substituted at positions 35 and 64 of hIL-22 (e.g., derivatives 11 and 12). In these examples, the IL-22 protein may additionally comprise an N- or C-terminal pentamer having the sequence AEPEE (SEQ ID NO: 9).

[0049] As described above, the fatty acid is preferably connected to a linker that is bound to the IL-22 protein. The linker may contain several linker elements, including one or more amino acids, such as one or more Glu and / or Lys residues. The linker may contain an oxyethyleneglycine unit or multiple linked oxyethyleneglycine units, optionally 2 to 5 such units, preferably 2 units. One or more OEG residues, a C2DA group, and / or an Ac group may alternatively or additionally be included. The linker may contain a Cys-reactive unit. As used herein, "Cys-reactive unit" may refer to a functional unit that can react with the sulfur atom of Cys to create a carbon-sulfur covalent bond. The Cys-reactive unit can have any of several forms, but preferably contains a carbon atom bound to a leaving group, which is displaced by the sulfur atom of Cys during carbon-sulfur bond formation. The leaving group may be a halogen, optionally a bromine atom. The bromide leaving group may be alpha to the Ac functionality, and advantageously, it is a bromo-Ac functionality. Alternatively, the leaving group may be a functionalized hydroxyl group in the form of a mesylate or tosylate, or a non-functionalized hydroxyl group. Furthermore, the leaving group may be a maleimide or other functional group. Exemplary linkers include γGlu-OEG-OEG-C2DA-Ac, γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc, and γGlu-OEG-OEG-εLys-αAc, although any suitable linker may be used. The linker γGlu-OEG-OEG-C2DA-Ac is shown in Figure 1A (with a Br leaving group and a C18 fatty acid attached) and Figure 1B (with a Br leaving group and a C16 fatty acid attached). The linker γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc is shown in FIG. 1C (with a Br leaving group and a C14 fatty acid attached).

[0050] Variants comprising a fatty diacid covalently bonded to a linker selected from γGlu-OEG-OEG-C2DA-Ac and γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc may be preferred in some embodiments. Variants comprising a C14, C16, C18 or C20 fatty diacid covalently bonded to a linker selected from γGlu-OEG-OEG-C2DA-Ac and γGlu-γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc may be preferred in some embodiments. Variants comprising a C14 fatty diacid covalently bonded to the linker γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc may be preferred in some embodiments. Variants comprising a C16, C18 or C20 fatty diacid covalently bonded to the linker γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc may be preferred in some embodiments.

[0051] The linker may be a Cys-reactive linker attached to a Cys residue in SEQ ID NO: 1. The linker may be a Cys-reactive linker attached to a Cys residue in an extension C-terminal or N-terminal to SEQ ID NO: 1.

[0052] The fatty acid or linker can be attached to any amino acid residue of the IL-22 protein. Exemplary in this regard are residues -7, -5, 1, 6, 33, 95, 106, 113, 114, and 153 in or relative to the hIL-22 amino acid sequence. The native residue is typically substituted with Cys or Lys to allow for attachment of the fatty acid or linker. Alternatively, the fatty acid or linker can be attached to the native Cys or Lys residue. Preferably, the fatty acid or linker is attached to a Cys residue substituted at position 1, 6, 33, 95, 106, 113, or 114 of hIL-22, or to a Cys residue at position -5, -7, or 153 relative to hIL-22. In particular, the fatty acid or linker can be attached to a Cys residue substituted at position 1 of hIL-22. Alternatively, the fatty acid or linker can be attached to the substituted Cys residue at position 95 or 106 of hIL-22.

[0053] The attachment of the fatty acid or linker to the IL-22 protein is a covalent bond. For example, a Cys-reactive fatty acid or linker can be used to attach the fatty acid or linker to a Cys residue of the IL-22 protein. The fatty acid or linker can be covalently attached to the sulfur atom of the Cys residue via a thioether bond. Alternatively, a Lys-reactive fatty acid or linker can be used to attach the fatty acid or linker to a Lys residue of the IL-22 protein. Alternatively, the fatty acid or linker can be covalently attached to the free amine (-NH2) group at the N-terminus of the IL-22 protein (regardless of the amino acid at position 1). Attachment can proceed similarly to a Cys attachment, but with a substoichiometric amount of a fatty acid or linker containing a suitable N-reactive species. The fatty acid or linker may be present in the form of an aldehyde (N-reactive species) and can be covalently attached to the free amine using conventionally known reductive amination.

[0054] Thus, a derivative for use in the first or second aspect preferably comprises a C14, C16, C18 or C20 diacid attached by a linker to a variant of hIL-22, the variant comprising an N-terminal GPG and a Cys residue at position 1 of hIL-22, the linker optionally being attached to the Cys residue. Alternatively, a derivative for use in the first or second aspect preferably comprises a C14, C16, C18 or C20 diacid attached by a linker to a variant of hIL-22, the variant comprising a Cys residue at position 95 or 106 of hIL-22, the linker optionally being attached to the Cys residue. Optionally, a Gln residue is substituted at positions 35 and / or 64.

[0055] Exemplary derivatives for use in the first or second aspects include the IL-22 proteins set forth in any of SEQ ID NOS: 23 to 32. Particularly advantageous derivatives are shown in Table 3 and illustrated in Figures 1 to 6. Derivatives 1 and 6 are exemplified herein.

[0056] [Table 3]

[0057] Figure 1A illustrates a C18 diacid connected to a linker containing a Cys-reactive unit. This is the fatty acid and linker (side chain) used in derivatives 1, 2, 6-9, and 11-14. Figure 1B illustrates a C16 diacid connected 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 connected to a linker containing a Cys-reactive unit. This is the fatty acid and linker (side chain) used in derivative 5.

[0058] Derivatives 1, 6 and 10 to 14 are shown in Figures 2 to 6, respectively.

[0059] The derivatives for use in the present invention may exist in different stereoisomeric forms, all of which the present invention relates to.

[0060] II. Preparation process of IL-22 derivatives Derivatives for use in the first or second aspect may be prepared by a process comprising covalently attaching a fatty acid to the IL-22 protein.

[0061] This process can be used to produce any of the different derivatives of IL-22 described or contemplated herein, but is particularly advantageous when a fatty acid is covalently attached to the mutant IL-22 protein. Thus, the IL-22 protein used in this process may be a substituted form of hIL-22, optionally substituted at positions 1, 21, 35, 64, 95, 106, 113, and / or 114. Exemplary substitutions include A1C, A1G, A1H, N21C, N21D, N21Q, N35C, N35D, N35H, N35Q, N64C, N64D, N64Q, N64W, R95C, L106C, Q113C, Q113R, K114C, and / or K114R. Preferably, the IL-22 protein is substituted at position 1 with a Cys residue. Alternatively, the IL-22 protein is substituted at positions 95 or 106 with a Cys residue, and optionally at positions 35 and / or 64 with a Gln residue.

[0062] The fatty acids may be obtained by any means known in the art, including recombinant means. Suitable fatty acids are commercially available or readily derived from available starting materials using standard chemical synthesis.

[0063] IL-22 protein can be obtained by any means known in the art, including recombinant means. The production of recombinant hIL-22 has been previously described and is well known in the art. Desired mutant IL-22 proteins can be produced by similar methods. A skilled researcher in the art can easily identify a suitable nucleic acid sequence encoding the desired mutant IL-22 protein. Thus, one skilled in the art can easily carry out this part of the present invention based on existing knowledge in the art. Preferably, IL-22 protein is produced in a mammalian system, such as Chinese hamster ovary (CHO) cells, using standard techniques. A polyhistidine tag (His tag) may be used to assist in affinity purification of the recombinant protein.

[0064] In this regard, IL-22 proteins for use in the present invention can be prepared using a cleavable His tag after expression, i.e., an N- or C-terminal addition of fewer than 10, preferably six, histidine residues that can be purified by affinity to a nickel column. The His tag is linked to the N- or C-terminus of the protein via a linker that can leave the free IL-22 protein upon digestion with a known protease. The cleavable His tag can have the amino acid sequence HHHHHHGGSSGSGSEVLFQ (SEQ ID NO: 33), and the protease-cleavable linker can be a tobacco etch virus (TEV) linker with the natural cleavage site consensus sequence ENLYFQ\S (SEQ ID NO: 34), where "\" indicates a cleaved peptide bond or a human rhinovirus-14 3C (HRV14-3C) protease-cleavable linker with the EVLFQ consensus cleavage site. Cleavage can be achieved by incubating approximately 10 μg of protease with 2.5 μg of protein and 10 mM 2-mercaptoethanol for 4 hours at room temperature.

[0065] A representative process for protein preparation is provided as follows. This process involves preparing plasmid DNA encoding the desired amino acid sequence of the IL-22 protein. This plasmid may be transiently transfected into a cell line, e.g., CHO-K1, which allows it to grow in the relevant medium, followed by increased growth through the addition of known enhancers. The secreted IL-22 protein may then be harvested via known methods of centrifugation and sterile filtration, after which the protein is purified on a nickel column. Following concentration and buffer exchange, the His tag is removed using HRV14-3C protease, followed by alkylation with fatty acids (described further below), and final purification and buffer exchange. Analysis of the final product using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion chromatography, or liquid chromatography with tandem mass spectrometry (LC-MS-MS), with or without deglycosylation, can be used to ensure the quality of the final product.

[0066] The fatty acid can be covalently attached to the IL-22 protein directly or using a linker described in the first or second aspects. The linker can be obtained by any means known in the art. Representative methods for preparing the fatty acid and linker, if used, are as follows (exemplified by the C16 diacid used in derivative 10, but any derivative can be made by using similar methods):

[0067] A solution of N-(benzyloxycarbonyloxy)succinimide (100 g, 401 mmol) in dichloromethane (500 mL) is added to a solution of ethylenediamine (189 mL, 2.81 mol) in dichloromethane (750 mL). After 30 minutes, the suspension is filtered, washed, and concentrated in vacuo. The residue is diluted with toluene (750 mL), washed, extracted with dichloromethane (4 × 200 mL), dried over anhydrous sodium sulfate, filtered, concentrated in vacuo, and diluted with hexane (200 mL). A 4 M solution of hydrogen chloride in ether (100 mL, 400 mmol) is added to the solution, and the resulting suspension is concentrated in vacuo and diluted with hexane (1 L). The precipitated solid is filtered, washed with hexane, and dried in vacuo to give (2-aminoethyl)carbamic acid benzyl ester hydrochloride as a white powder.

[0068] 2-Chlorotrityl resin 100-200 was loaded with {2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid (Fmoc-Ado-OH, 17.5 g, 45.4 mmol). The Fmoc group was removed by adding a solution of 0-6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TCTU, 24.2 g, 68.1 mmol) and N,N-diisopropylethylamine (21.4 ml, 123 mmol) in N,N-dimethylformamide (140 ml) to the resin, and the mixture was shaken for 1 hour. The resin was filtered and washed. The Fmoc group was removed by treatment with 20% piperidine as described above. The resin was washed as described above.

[0069] A solution of (S)-2-(fluoren-9-ylmethoxycarbonylamino)-pentanedioic acid 1-tert-butyl ester (Fmoc-Glu-OtBu, 29.0 g, 68.1 mmol), TCTU (24.2 g, 68.1 mmol), and N,N-diisopropylethylamine (21.4 ml, 123 mmol) in N,N-dimethylformamide (140 ml) is added to the resin, and the mixture is shaken for 1 hour. The resin is filtered and washed as before. The Fmoc group is removed by treatment with 20% piperidine as before. The resin is washed as before.

[0070] 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 N,N-dimethylformamide / dichloromethane mixture (4:1, 200 ml) is added to the resin. The resin is shaken for 1 hour, filtered, and washed with N,N-dimethylformamide (3 x 250 ml), dichloromethane (2 x 250 ml), methanol (2 x 250 ml), and dichloromethane (6 x 250 ml). The product is cleaved from the resin by treatment with 2,2,2-trifluoroethanol (250 ml) for 18 hours. The resin is filtered off and washed with dichloromethane (2 x 250 ml), a 2-propanol / dichloromethane mixture (1:1, 2 x 250 ml), 2-propanol (250 ml) and dichloromethane (3 x 250 ml).

[0071] The solutions are combined, the solvent is evaporated, and the crude product is purified by flash column chromatography. Pure (S)-22-(tert-butoxycarbonyl)-41,41-dimethyl-10,19,24,39-tetraoxo-3,6,12,15,40-pentaoxa-9,18,23-triazadtetracontanoic acid is obtained as a pale yellow viscous oil after drying in vacuo.

[0072] Subsequently, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU, 11.4 g, 30.1 mmol) and triethylamine (8.77 ml, 62.9 mmol) are added to a solution of (S)-22-(tert-butoxycarbonyl)-41,41-dimethyl-10,19,24,39-tetraoxo-3,6,12,15,40-pentaoxa-9,18,23-triazadotetracontanoic acid (22.4 g, 27.4 mmol) in dry dichloromethane (110 ml). Triethylamine (72 ml, 41.0 mmol) is added to a suspension of (2-amino-ethyl)-carbamic acid benzyl ester hydrochloride (6.94 g, 30.1 mmol) in dry dichloromethane (165 ml), and the resulting mixture is added to the above solution. The mixture is stirred at room temperature overnight and then evaporated to dryness. The residue is redissolved, washed, dried over anhydrous sodium sulfate, and evaporated by column chromatography (silica gel 60, 0.040 mm 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]-pentadecanoic acid tert-butyl ester as a pale yellow viscous oil.

[0073] Palladium on carbon (10%, 1.27 g, 1.20 mmol) is added to a solution of the above compound (23.8 g, 24.0 mmol) in methanol (350 ml), and the resulting mixture is hydrogenated at atmospheric pressure for 4 hours. The catalyst is filtered off, and the filtrate is evaporated to dryness. The residue is evaporated several times from dichloromethane to remove residual methanol, and dried in vacuo to give tert-butyl (S)-1-amino-25-tert-butoxycarbonyl)-4,13,22,27-tetraoxo-6,9,15,18-tetraoxa-3,12,21,26-tetraazadotetracontan-42-oate as a viscous, colorless oil.

[0074] N,N-Diisopropylethylamine (4.98 ml, 28.6 mmol) is added to a solution of the above amine (20.5 g, 23.8 mmol) in dry dichloromethane (290 ml) under argon at −30° C. Bromoacetyl bromide (2.48 ml, 28.6 mmol) is added dropwise, and the resulting solution is stirred at −30° C. for a further 3 h. The cooling bath is removed, the mixture is stirred at room temperature for 1 h, and the solvent is removed in vacuo. The residue is redissolved in ethyl acetate (450 ml) and washed with a 5% aqueous solution of citric acid (300 ml). The phases are separated within 1 h. The organic layer is left to separate overnight to give three phases. The clear aqueous layer is removed, and the remaining two phases are shaken with a saturated aqueous solution of potassium bromide (100 ml). The phases are left to separate overnight, the aqueous phase is removed, and the organic phase is dried over anhydrous sodium sulfate. The solvent is removed in vacuo and the residue is purified by flash column chromatography: dichloromethane / methanol 95:5) to give tert butyl (S)-1-bromo-28-tert-butoxycarbonyl)-2,7,16,25,30-pentaoxo-9,12,18,21-tetraoxa-3,6,15,24,29-pentaazapentatetracontan-45-oate as a colorless solid.

[0075] The above compound (19.5 g, 19.8 mmol) is dissolved in trifluoroacetic acid (120 ml), and the resulting solution is stirred at room temperature for 1.5 hours. The trifluoroacetic acid is removed in vacuo, and the residue is evaporated from dichloromethane (6 x 200 ml). Diethyl ether (200 ml) is added to the oily residue, and the mixture is stirred overnight to give a suspension. The solid product is filtered, washed with diethyl ether and hexane, and dried in vacuo to give the desired product, 15-{(S)-1-carboxy-3-[2-(2-{[2-(2-{[2-(2-bromoacetylamino)ethylcarbamoyl]methoxy}ethoxyethylcarbamoyl]methoxy}ethoxyethylcarbamoyl]propylcarbamoyl}pentadecanoic acid, as a white powder.

[0076] Covalent attachment of a fatty acid or linker to an IL-22 protein can be carried out using standard procedures in the art. Thus, when used, a linker allows for covalent attachment of the IL-22 protein to the fatty acid. As a non-limiting example, a thioether bond can be formed by reacting a Cys-reactive fatty acid or linker with the sulfur atom of a Cys residue in an IL-22 protein. Suitable conditions for the covalent attachment step can be exemplified as follows: Tris in water is added to IL-22 protein (70 mg) in Tris and NaCl buffer (1.35 mg / ml) and adjusted to pH 8. Bis(p-sulfonatophenyl)-phenylphosphine dihydrate dipotassium (BSPP) salt (12 mg) dissolved in water is added and gently stirred at room temperature for 4 hours. 15-{(S)-1-carboxy-3-[2-(2-{[2-(2-{[2-(2-bromoacetylamino)ethylcarbamoyl]ethoxy}ethoxy)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]propylcarbamoyl}pentadecanoic acid (19 mg, 0.022 mmol) in ethanol (0.5 mL) is added and the mixture is gently stirred overnight. MiliQ water (150 mL) is added to reduce the conductivity to 2.5 mS / cm. The mixture is then purified using anion exchange on a MonoQ 10 / 100 GL column using binding buffer (20 mM Tris, pH 8.0), elution buffer (20 mM Tris, 500 mM NaCl, pH 8.0), a flow rate of 6 mL, and a gradient of 0% to 80% elution buffer over 60 column volumes.

[0077] Derivatives for use in the present invention may be purified using any suitable procedure known in the art, such as chromatography, electrophoresis, differential solubility, or extraction.

[0078] III. Therapeutic efficacy of derivatives of IL-22 As mentioned above, it was surprising to the inventors at the time to find that fatty acids could be covalently attached to the IL-22 protein while maintaining biological activity. It was particularly surprising that such minimal modifications to IL-22 could result in high potency (close to that of hIL-22) combined with a very long circulating half-life. This particular combination of properties may be highly desirable.

[0079] The efficacy of the derivatives can be determined in an in vitro assay using whole cells expressing the human IL-22 receptor. For example, the response of the human IL-22 receptor can be measured using baby hamster kidney (BHK) cells overexpressing IL-22R1 (also called IL-22 receptor α or IL22RA), IL-10R2 (also called IL-10 receptor β or IL10RB), and a phospho-STAT3 (pSTAT3)-responsive reporter gene, as demonstrated in Example 5. 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 STAT3-inducible promoter and a luciferase reporter gene or by assaying pSTAT3. In vivo efficacy can be determined by animal models or clinical trials, as is known in the art. By way of illustration, Example 10 describes a clinical trial that demonstrated surprisingly high efficacy for Derivative 1 using IL-22 target-associated biomarkers.

[0080] Half maximal effective concentration (EC 50 The EC value is often used as a measure of a drug's potency. It represents the concentration of a drug required to produce 50% of its maximum effect, and is therefore called the EC 50The lower the value, the better the potency. Derivatives for use in the present invention suitably have a potency (EC 2 H 2 O ) of less than 1.5 nM, less than 1.25 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM or even less than 0.1 nM, as measured using IL-22 receptor-mediated STAT3 activation in cells. 50 Advantageously, the potency is less than 1 nM. Derivatives for use in the present invention suitably have a potency (EC 50 value).

[0081] 99% effective concentration (EC 99 ) values ​​can also be used as a measure of drug efficacy. Again, the EC 99 The lower the value, the better the potency. Derivatives for use in the present invention suitably have an EC value of less than 2000 ng / mL, less than 1800 ng / mL, less than 1600 ng / mL, less than 1400 ng / mL, less than 1200 ng / mL, less than 1000 ng / mL or even less than 900 ng / mL, as measured using IL-22 receptor-mediated STAT3 activation in cells. 99 value) (as shown in Example 5).

[0082] Advantageously, the potency of IL-22 derivatives can be higher than that of IL-22-Fc fusions. For example, Genentech reported a 34-fold decrease in the in vitro potency of its IL-22-Fc fusion UTTR1147A compared to hIL-22 (Stefanich et al., Biochem Pharmacol, 2018, 152:224-235). In contrast, covalent attachment of a fatty acid to hIL-22 has been shown to result in only a 7-fold decrease in potency (see Derivatives 1 and 2 in Example 1 of WO 2022 / 238510 and Derivative 1 in the Examples of WO 2021 / 089875, both of which are incorporated herein by reference). While both IL-22-Fc fusions and derivatives for use in the present invention may, at least in some circumstances, be comparable in terms of their improved half-life and biological function relative to hIL-22, the derivatives for use in the present invention may have the added advantage of minimal loss of potency. Illustratively, Example 10 demonstrated the surprisingly high potency of Derivative 1 in clinical trials.

[0083] The circulating elimination half-life (t 1 / 2 ) can be determined in vivo in a suitable animal model, such as a mouse, rat, or minipig, by subcutaneously or intravenously administering the derivative. A suitable method is described in Example 1 of WO 2021 / 089875, incorporated herein by reference. As non-limiting examples, a derivative for use in the first or second aspect may have a circulating half-life of at least 1 hour, at least 3 hours, at least 5 hours, or even at least 8 hours after subcutaneous or intravenous administration to a mouse. The derivative may have a circulating half-life of at least 3 hours, at least 5 hours, at least 8 hours, at least 10 hours, or even at least 13 hours after subcutaneous or intravenous administration to a rat. The derivative may have a circulating half-life of at least 25 hours, at least 40 hours, at least 70 hours, or even at least 100 hours after subcutaneous or intravenous administration to a minipig.

[0084] The inventors have also previously found that derivatives for use in the present invention are rapidly absorbed in vivo. Advantageously, absorption of the derivatives after subcutaneous administration can occur faster than that of IL-22-Fc fusions. The mean absorption time is an accurate parameter for measuring uptake because it is independent of the dose and maximum plasma concentration after drug administration. It can be calculated based on the mean residence time, i.e., the time the drug spends in the body before complete absorption and excretion. The derivatives for use in the present invention preferably have a mean absorption time in pigs of less than 100 hours, less than 90 hours, less than 80 hours, less than 70 hours, or even less than 60 hours (e.g., as determined as described in Example 1 of WO 2021 / 089875, incorporated herein by reference).

[0085] Derivatives for use in the present invention also have good biophysical properties, such as high physical stability and / or solubility, which can be measured using standard methods in the art.

[0086] IV. Pharmaceutical Compositions Comprising Derivatives of IL-22 Thus, a pharmaceutical composition can be prepared comprising a derivative described or contemplated herein and a pharmaceutically acceptable vehicle.

[0087] The pharmaceutical composition may comprise any of the different derivatives of IL-22 described or contemplated herein. Suitably, the pharmaceutical composition comprises one of the derivatives of IL-22 identified herein as Derivatives 1 to 14. Advantageously, the pharmaceutical composition comprises one of the derivatives of IL-22 identified herein as Derivatives 1, 6, 11 or 12.

[0088] The derivatives described or contemplated herein, or pharmaceutical compositions comprising same, suitably exhibit an increased circulating elimination half-life compared to hIL-22, advantageously by at least 50%, at least 75%, at least 100% or more compared to hIL-22.

[0089] Pharmaceutical compositions can be prepared by combining a therapeutically effective amount of a derivative described or contemplated herein with a pharmaceutically acceptable vehicle. Formulation of pharmaceutically active ingredients with various excipients is well known in the art.

[0090] A "therapeutically effective amount" of a derivative described or contemplated herein is any amount that, when administered to a subject, is the amount of derivative needed to treat a disease, disorder, or condition or to produce a desired effect.

[0091] For example, the therapeutically effective amount of the derivative used can be about 0.001 mg to about 1000 mg, preferably about 0.01 mg to about 500 mg. The amount of the derivative is preferably about 0.1 mg to about 100 mg, and most preferably about 0.5 mg to about 50 mg. For reference, the dose of the derivative used in mice in Example 3 described herein was 0.0125 mg / kg to 0.5 mg / kg (subcutaneous administration), with 0.05 mg / kg being observed as the initial therapeutic dose. In Examples 6 to 8 described herein, doses ranging from 0.0125 mg / kg to 0.6 mg / kg (subcutaneous administration) were used, with 0.0125 mg / kg being observed as the initial therapeutic dose (Example 7).

[0092] A "pharmaceutically acceptable vehicle," as referred to herein, is any known compound or combination of known compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0093] The pharmaceutically acceptable vehicle is preferably liquid, and optionally the pharmaceutical composition is in the form of a liquid. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The derivatives for use in the present invention can be dissolved or suspended in a pharmaceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture of both, or a pharmaceutically acceptable oil or fat. The liquid vehicle may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickeners, colorants, viscosity adjusters, stabilizers, or osmolality adjusters. Suitable examples of liquid vehicles for parenteral administration include water (partially containing additives such as those described above, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.

[0094] Thus, the process for preparing the pharmaceutical composition may involve routine steps that are standard in the art.

[0095] V. Treatment method According to a first aspect of the present invention there is provided a derivative of IL-22 comprising a fatty acid covalently attached to the IL-22 protein for use in a method of treating a metabolic, intestinal and / or hepatic disease, disorder or condition, the method comprising subcutaneously administering the derivative of IL-22.

[0096] According to a second aspect of the present invention there is provided a derivative of IL-22 comprising a fatty acid covalently attached to the IL-22 protein for use in a method of treating a metabolic, intestinal and / or hepatic disease, disorder or condition, the method comprising intravenously administering the derivative of IL-22.

[0097] In either aspect, the derivative of IL-22 may be in the form of a pharmaceutical composition as described herein. Also provided are methods for treating a subject having a metabolic, intestinal, and / or hepatic disease, disorder, or condition, comprising subcutaneous or intravenous administration of such a derivative or a pharmaceutical composition comprising same. Any of the different derivatives of IL-22 described or contemplated herein are expressly included in these aspects of the invention.

[0098] Terms such as "treating" and "therapy" as used herein expressly include the treatment, amelioration, or prevention of a disease, disorder, or condition.

[0099] The IL-22 derivative or a pharmaceutical composition comprising it can be administered directly to the subject to be treated. It is preferably administered subcutaneously or intravenously by injection or infusion. Advantageously, it is administered subcutaneously, preferably by injection. Intravenous administration is preferably by infusion. Thus, the derivative has a clear advantage over Fc fusions in flexibility of administration due to its smaller size and higher potency. It will be understood that administration of the derivative or a pharmaceutical composition comprising it to the subject to be treated results in a prolonged circulation time compared to hIL-22, which aids in the treatment of the disease, disorder or condition. As noted above, "treating" also includes ameliorating and preventing the disease, disorder or condition.

[0100] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized by, for example, subcutaneous or intravenous injection or infusion. The derivatives can also be prepared as sterile solid compositions that can be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile medium.

[0101] The derivatives or pharmaceutical compositions described or contemplated herein can be administered to any subject in need thereof. As used herein, a "subject" may be a vertebrate, mammal, or livestock. Thus, the derivatives and compositions used in the present invention can be used to treat any mammal, for example, livestock (e.g., horses), pets, or in other veterinary applications. Most preferably, the subject is a human. The derivatives and compositions need not only be administered to those who already show signs of a disease, disorder, or condition. Rather, they can be administered to apparently healthy subjects as a purely preventative measure against the possibility of such a disease, disorder, or condition in the future.

[0102] It will be understood that the IL-22 derivatives and compositions described or contemplated herein can be used in monotherapy (i.e., use of the derivative or composition alone) to treat a disease, disorder, or condition. Alternatively, such derivatives and compositions can be used as an adjunct to, or in combination with, known therapies for treating a disease, disorder, or condition.

[0103] It is understood that the amount of IL-22 derivative required will be determined by its biological activity, half-life, and bioavailability, which in turn will depend on the mode of administration, the physiochemical properties of the derivative and composition, and whether it is used as monotherapy or in combination therapy. The frequency of administration will also be affected by the half-life of the derivative in the subject being treated. The optimal dosage to be administered can be determined by one skilled in the art and will vary depending on the particular derivative used, the strength of the pharmaceutical composition, the mode of administration, and the progression of the disease, disorder, or condition. Additional factors depending on the particular subject being treated, including the subject's age, weight, sex, diet, and time of administration, will result in the need to adjust the dosage.

[0104] Generally, a daily dose of 0.1 μg / kg to 60 μg / kg of body weight of a derivative of IL-22 described or contemplated herein can be used to treat a disease, disorder, or condition, depending on which derivative or composition is used. More preferably, the daily dose is 0.1 μg / kg to 40 μg / kg of body weight, more preferably 0.1 μg / kg to 30 μg / kg of body weight, and most preferably about 0.1 μg / kg to 15 μg / kg of body weight.

[0105] In a particularly preferred embodiment of the first aspect, when the derivative is for subcutaneous administration, the weekly dose is approximately 1 μg / kg to 500 μg / kg body weight, more preferably approximately 1 μg / kg to 400 μg / kg body weight, and most preferably approximately 1 μg / kg to 15 μg / kg body weight.Further preferred ranges for weekly doses are approximately 1 μg / kg to 300 μg / kg body weight, 1 μg / kg to 200 μg / kg body weight, 1 μg / kg to 150 μg / kg body weight, 1 μg / kg to 80 μg / kg body weight, 1 μg / kg to 50 μg / kg body weight, 1 μg / kg to 30 μg / kg body weight, 1 μg / kg to 10 μg / kg body weight, 1 μg / kg to 7.5 μg / kg body weight, 1 μg / kg to 5 μg / kg body weight, 1 μg / kg to 2.5 μg / kg body weight, 1 μg / kg to 1.25 μg / kg body weight, 1.25 μg / kg to 7.5 μg / kg body weight, 1.25 μg / kg to 1.2 ... g / kg~5μg / kg body weight, 1.25μg / kg~2.5μg / kg body weight, 2.5μg / kg~15μg / kg body weight, 2.5μg / kg~10μg / kg body weight, 2.5μg / kg~7.5μg / kg body weight, 2.5μg / kg~5μg / kg body weight, 3μg / kg~1 00μg / kg body weight, 3μg / kg~80μg / kg body weight, 3μg / kg~50μg / kg body weight, 3μg / kg~30μg / kg body weight, 3μg / kg~15μg / kg body weight, 3μg / kg~10μg / kg body weight, 3μg / kg~5μg / kg body weight, 5μg / kg~80 μg / kg body weight, 5μg / kg~50μg / kg body weight, 5μg / kg~30μg / kg body weight, 5μg / kg~15μg / kg body weight, 5μg / kg~10μg / kg body weight, 5μg / kg~7.5μg / kg body weight, 10μg / kg~150μg / kg body weight, 10μg / k g~100μg / kg body weight, 10μg / kg~80μg / kg body weight, 10μg / kg~50μg / kg body weight, 10μg / kg~15μg / kg body weight, 20μg / kg~150μg / kg body weight, 20μg / kg~100μg / kg body weight, 20μg / kg~80μg / kg body weight, 25 μg / kg to 300 μg / kg body weight, 25 μg / kg to 200 μg / kg body weight, 25 μg / kg to 150 μg / kg body weight, 30 μg / kg to 150 μg / kg body weight, 30 μg / kg to 100 μg / kg body weight, 50 μg / kg to 350 μg / kg body weight, 50 μg / kg to 250 μg / kg body weight, 50 μg / kg to 150 μg / kg body weight, 100 μg / kg to 400 μg / kg body weight, 100 μg / kg to 250 μg / kg body weight, 200 μg / kg to 400 μg / kg body weight, and 200 μg / kg to 300 μg / kg body weight. Any combination of these range endpoints is envisioned and forms part of the present invention.Preferably, the weekly dose is 1 μg / kg to 10 μg / kg body weight, more preferably 1 μg / kg to 5 μg / kg body weight, more preferably approximately 1.25 μg / kg to 3 μg / kg body weight, more preferably approximately 2 μg / kg to 3 μg / kg body weight. Preferably, the weekly dose is approximately 2 μg / kg body weight, 2.5 μg / kg body weight or 3.0 μg / kg body weight, more preferably approximately 2.5 μg / kg body weight. Advantageously, the weekly dose is 10 μg / kg body weight or less.

[0106] In a particularly preferred embodiment of the second aspect, when the derivative is for intravenous administration, the weekly dose is approximately 1 μg / kg to 400 μg / kg body weight, more preferably approximately 1 μg / kg to 300 μg / kg body weight, and most preferably approximately 1 μg / kg to 15 μg / kg body weight.Further preferred ranges for weekly doses are approximately 1 μg / kg to 200 μg / kg body weight, 1 μg / kg to 100 μg / kg body weight, 1 μg / kg to 80 μg / kg body weight, 1 μg / kg to 50 μg / kg body weight, 1 μg / kg to 30 μg / kg body weight, 1 μg / kg to 10 μg / kg body weight, 1 μg / kg to 7.5 μg / kg body weight, 1 μg / kg to 5 μg / kg body weight, 1 μg / kg to 2.5 μg / kg body weight, 1 μg / kg to 1.25 μg / kg body weight, 1.25 μg / kg to 7.5 μg / kg body weight, 1.25 μg / kg to 5 μg / kg body weight, 1 μg / kg to 1. ... .25μg / kg~2.5μg / kg body weight, 2.5μg / kg~15μg / kg body weight, 2.5μg / kg~10μg / kg body weight, 2.5μg / kg~7.5μg / kg body weight, 2.5μg / kg~5μg / kg body weight, 3μg / kg~100μg / kg body weight, 3μg / kg~80μg / kg body weight, 3μg / kg~50μg / kg body weight, 3μg / kg~30μg / kg body weight, 3μg / kg~15μg / kg body weight, 3μg / kg~10μg / kg body weight, 3μg / kg~5μg / kg body weight, 5μg / kg~80μg / kg body weight, 5μ g / kg~50μg / kg body weight, 5μg / kg~30μg / kg body weight, 5μg / kg~15μg / kg body weight, 5μg / kg~10μg / kg body weight, 5μg / kg~7.5μg / kg body weight, 10μg / kg~150μg / kg body weight, 10μg / kg~100μg / kg body weight, 10μg / kg~80μg / kg body weight, 10μg / kg~50μg / kg body weight, 10μg / kg~15μg / kg body weight, 20μg / kg~150μg / kg body weight, 20μg / kg~100μg / kg body weight, 20μg / kg~80μg / kg body weight, 25 μg / kg to 300 μg / kg body weight, 25 μg / kg to 200 μg / kg body weight, 25 μg / kg to 100 μg / kg body weight, 30 μg / kg to 150 μg / kg body weight, 30 μg / kg to 100 μg / kg body weight, 50 μg / kg to 350 μg / kg body weight, 50 μg / kg to 250 μg / kg body weight, 50 μg / kg to 150 μg / kg body weight, 100 μg / kg to 300 μg / kg body weight, 100 μg / kg to 250 μg / kg body weight, 150 μg / kg to 300 μg / kg body weight and 150 μg / kg to 250 μg / kg body weight. Any combination of these range endpoints is also envisioned and forms part of the present invention.Preferably, the weekly dose is 1 μg / kg to 10 μg / kg body weight, more preferably 1 μg / kg to 5 μg / kg body weight, more preferably approximately 1.25 μg / kg to 3 μg / kg body weight, more preferably approximately 2 μg / kg to 3 μg / kg body weight. Preferably, the weekly dose is approximately 2 μg / kg body weight, 2.5 μg / kg body weight or 3.0 μg / kg body weight, more preferably approximately 2.5 μg / kg body weight. Advantageously, the weekly dose is 10 μg / kg body weight or less.

[0107] The IL-22 derivative or composition may be administered before, during, or after the onset of a disease, disorder, or condition. A weekly dose may be given as a single administration (e.g., a weekly injection). Suitable dose ranges for a single weekly dose are the same as those set forth above, e.g., a single dose may be approximately 1 μg / kg to 400 μg / kg, 1 μg / kg to 300 μg / kg, 1 μg / kg to 200 μg / kg, 1 μg / kg to 80 μg / kg, 1 μg / kg to 50 μg / kg, 1 μg / kg to 30 μg / kg, 1 μg / kg to 15 μg / kg, 1 μg / kg to 10 μg / kg, 1 μg / kg to 7.5 μg / kg, 1 μg / kg to 5 μg / kg, 1 μg / kg to 2 μg / kg, or 1 μg / kg to 2 μg / kg. 5μg / kg, 1μg / kg~1.25μg / kg, 1.25μg / kg~7.5μg / kg, 1.25μg / kg~5μg / kg, 1.25μg / kg~2.5μg / kg, 2.5μg / kg~15μg / kg, 2.5μg / k g~10μg / kg, 2.5μg / kg~7.5μg / kg, 2.5μg / kg~5μg / kg, 3μg / kg~100μg / kg, 3μg / kg~80μg / kg, 3μg / kg~50μg / kg, 3μg / kg~30μg / k g, 3μg / kg~15μg / kg, 3μg / kg~10μg / kg, 3μg / kg~5μg / kg, 5μg / kg~80μg / kg, 5μg / kg~50μg / kg, 5μg / kg~30μg / kg, 5μg / kg~15μg / kg, 5μg / kg~10μg / kg, 5μg / kg~7.5μg / kg, 10μg / kg~150μg / kg, 10μg / kg~100μg / kg, 10μg / kg~80μg / kg, 10μg / kg~50μg / kg, 10μ g / kg~15μg / kg, 20μg / kg~150μg / kg, 20μg / kg~100μg / kg, 20μg / kg~80μg / kg, 25μg / kg~300μg / kg, 25μg / kg~200μg / kg, 30μg / k g~150μg / kg, 30μg / kg~100μg / kg, 50μg / kg~350μg / kg, 50μg / kg~250μg / kg, 50μg / kg~150μg / kg, and 100μg / kg~250μg / kg.Advantageously, a single weekly dose of approximately 1 μg / kg to 15 μg / kg body weight (preferably approximately 1 μg / kg to 10 μg / kg body weight, 1.25 μg / kg to 7.5 μg / kg body weight, or 1 μg / kg to 5 μg / kg body weight, for example, approximately 1.25 μg / kg body weight, 2.5 μg / kg body weight, 5 μg / kg or 7.5 μg / kg body weight) is given subcutaneously or intravenously (preferably subcutaneously). Preferably, a single weekly dose of 1 μg / kg to 10 μg / kg body weight, more preferably 1 μg / kg to 5 μg / kg body weight, more preferably approximately 1.25 μg / kg to 3 μg / kg body weight, more preferably approximately 2 μg / kg to 3 μg / kg body weight, most preferably approximately 2.5 μg / kg body weight is given subcutaneously or intravenously (preferably subcutaneously). Alternatively, the derivative or composition may require administration more than once a week, with the single doses above being adjusted accordingly.

[0108] The present specification contemplates various dosing regimens, including dosing at different time intervals (e.g., daily, weekly, biweekly, monthly, etc.), and also contemplates various dosing ranges and amounts. Generally, dosages are discussed in terms of the amount of IL-22 derivative relative to the mass / body weight of the recipient of IL-22. Recipients of IL-22 generally have a weight range of 65 kg (or less) to 160 kg (or more), including weights such as 70 kg, 100 kg, and 130 kg. Body weight can be, for example, approximately 65 kg, 70 kg, 75 kg, 80 kg, 85 kg, 90 kg, 95 kg, 100 kg, 105 kg, 110 kg, 115 kg, 120 kg, 125 kg, 130 kg, 135 kg, 140 kg, 145 kg, 150 kg, 155 kg, or 160 kg.

[0109] Compositions containing a specific amount of IL-22 derivative (dose per individual), such as 0.2 mg or 0.3 mg, may be available. Such compositions can be used for patients whose weight and / or BMI are within a specific range, or above or below a specific value. For example, in the indication of obesity, treatment is typically tailored to a specific BMI, with the lower limit of the range specified (e.g., a BMI of 27 or 30) and no upper limit specified. This is then used to treat any patient who is eligible for such treatment (e.g., based on BMI or blood glucose / Hb1Ac levels), regardless of their specific weight.

[0110] The relative amount of IL-22 / body weight of the recipient will, of course, vary depending on the amount of IL-22 derivative in the dose and the body weight of the recipient.

[0111] Thus, if the amount of IL-22 derivative is 0.2 mg administered weekly, this corresponds to about 2.9 μg / kg in a patient weighing 70 kg, 2.0 μg / kg in a patient weighing 100 kg, about 1.5 μg / kg in a patient weighing 130 kg, etc. Similarly, if the amount of IL-22 derivative is 0.3 mg administered weekly, this corresponds to about 4.3 μg / kg in a patient weighing 70 kg, 3.0 μg / kg in a patient weighing 100 kg, about 2.3 μg / kg in a patient weighing 130 kg, etc.

[0112] Dosages contemplated herein include dosage ranges and values ​​for the IL-22 derivatives disclosed herein, as well as dosages expressed in relative amounts (e.g., in μg / kg) and absolute amounts based on the body weight value of the recipient of the IL-22 derivatives disclosed herein.

[0113] The following table shows dose values ​​and ranges expressed in μg / kg, as well as exemplary doses (expressed in mg) based on body weight in kg. Thus, a patient weighing 70 kg receiving a dose of 1 μg / kg would receive an amount of 0.07 mg of IL-22 derivative. An obese patient weighing, for example, 100 kg receiving the same dose of 1 μg / kg would receive an amount of 0.1 mg of IL-22 derivative. Preferably, the amount of IL-22 derivative in a dose is approximately 0.05 mg to 2.0 mg, more preferably approximately 0.07 mg to 1.75 mg, more preferably approximately 0.1 mg to 1.5 mg, more preferably approximately 0.1 mg to 1.0 mg, more preferably approximately 0.1 mg to 0.5 mg, and most preferably approximately 0.1 mg to 0.4 mg.

[0114] [Table 4]

[0115] Alternatively, doses may be given daily, biweekly (once every two weeks) or once a month.

[0116] Repeated dosing can range from 1 μg / kg body weight to 7.5 μg / kg body weight, for example, approximately 1.25, 2.5, 5.0, or 7.5 μg / kg body weight. Preferably, a single biweekly (every two weeks) dose of 1 μg / kg body weight to 15 μg / kg body weight, more preferably 1.25 μg / kg body weight to 7.5 μg / kg body weight, more preferably approximately 3 μg / kg body weight to 7.5 μg / kg body weight, and most preferably approximately 5 μg / kg body weight is given subcutaneously or intravenously (preferably subcutaneously). In some embodiments, a single biweekly (every two weeks) dose of 1 μg / kg body weight to 15 μg / kg body weight is envisioned. In some embodiments, a single biweekly (every two weeks) dose of 10 μg / kg to 15 μg / kg body weight, more preferably about 10 μg / kg to 14 μg / kg body weight, more preferably about 10 μg / kg to 13 μg / kg body weight, more preferably about 10 μg / kg to 12 μg / kg body weight is contemplated.

[0117] In some embodiments, a single biweekly (every two weeks) dose of 1 μg / kg body weight to 10 μg / kg body weight, more preferably 1 μg / kg to 5 μg / kg body weight, more preferably approximately 1.25 μg / kg to 3 μg / kg body weight, more preferably approximately 2 μg / kg to 3 μg / kg body weight is contemplated. In some embodiments, a single biweekly (every two weeks) dose of approximately 2 μg / kg body weight, 2.5 μg / kg body weight, or 3.0 μg / kg body weight, more preferably approximately 2.5 μg / kg body weight is contemplated. In some embodiments, a weekly dose of 10 μg / kg body weight or less is contemplated.

[0118] Advantageously, the biweekly dose is equal to or less than 15 μg / kg body weight.

[0119] Preferably, a single monthly dose of 1 μg / kg to 15 μg / kg body weight, more preferably 1.25 μg / kg to 10 μg / kg body weight, more preferably approximately 3 μg / kg to 7.5 μg / kg body weight, and most preferably approximately 5 μg / kg body weight is given subcutaneously or intravenously (preferably subcutaneously). Advantageously, the monthly dose is 15 μg / kg body weight or less. Known procedures, such as those conventionally used by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), can be used to formulate the specific formulations of the derivatives and compositions according to the invention, as well as the exact treatment regimen (e.g., the weekly dose and administration frequency of the drug).

[0120] It may be advantageous to use a loading dose where the first dose administered to a patient is higher than subsequent doses, with the intention of achieving steady state after the first dose. For example, in a once-weekly dosing regimen, the first dose in week 1 may be a higher loading dose than the doses administered in weeks 2, 3, and potentially thereafter (as exemplified in Example 10). The loading dose is suitably the weekly dose plus approximately 0.2 to 4 times the weekly dose, preferably approximately 0.4 to 2 times the weekly dose, more preferably approximately 0.6 to 2 times the weekly dose, even more preferably approximately 0.8 to 2 times the weekly dose, and most preferably approximately 1 to 2 times the weekly dose. Suitably, the loading dose is approximately 1 μg / kg to 15 μg / kg body weight (preferably approximately 1 μg / kg to 10 μg / kg, 1 μg / kg to 5 μg / kg, 1.25 μg / kg to 12.5 μg / kg, 1.25 μg / kg to 7.5 μg / kg, 2.5 μg / kg to 15 μg / kg, 2.5 μg / kg to 10 μg / kg or 2.5 μg / kg to 5 μg / kg body weight, for example, approximately 2.5 μg / kg, 5 μg / kg, 10 μg / kg or 15 μg / kg body weight). Advantageously, the loading dose is twice the weekly dose; for example, the loading dose may be 15 μg / kg body weight and the weekly dose may be 7.5 μg / kg body weight. Other suitable examples include a loading dose of 2.5 μg / kg body weight followed by a weekly dose of 1.25 μg / kg body weight, a loading dose of 5 μg / kg body weight followed by a weekly dose of 2.5 μg / kg body weight, and a loading dose of 10 μg / kg body weight followed by a weekly dose of 5 μg / kg body weight. In a particularly preferred embodiment, the loading dose is approximately 2.5 μg / kg body weight to 15 μg / kg body weight, and the repeated weekly doses are approximately 2.5 μg / kg body weight to 7.5 μg / kg body weight. Preferably, the loading dose is given in the first week, and weekly doses are given for the remainder of the treatment period. The loading dose is preferably given as a single administration (e.g., a single subcutaneous injection or a single intravenous infusion).

[0121] Example 10 provides evidence of the surprisingly high efficacy of Derivative 1 in humans. As described in Example 10, the escalating dose study was originally designed to investigate doses of 3 μg / kg body weight to 400 μg / kg body weight. It was never anticipated that doses below 3 μg / kg body weight could be therapeutically effective. Thus, the low doses of the derivatives described herein that can be administered for therapeutic purposes are both novel and surprisingly advantageous. In comparison, Genentech has disclosed much higher doses of IL-22-Fc fusion UTTR1147A, ranging from 30 μg / kg body weight to 90 μg / kg body weight (Wagner F., et al. Gut 2023;0:1-11).

[0122] Numerous studies have demonstrated the key effects of IL-22 in multiple epithelial injury models, particularly in the lung, liver, intestine, kidney, skin, pancreas, and thymus. Mechanistically, several pathways, such as anti-apoptosis, proliferation, innate immunity, anti-oxidative stress, anti-fibrosis, and stem / progenitor cell mobilization, have been well established to mediate IL-22 effects in studies by multiple investigators. Key mechanistic findings have been further confirmed in vitro using human cell lines or in human ex vivo models (e.g., primary human intestinal organoids). Thus, the strong role of IL-22 in preventing cell death, ensuring regeneration, and controlling inflammation in epithelial injury has been well documented.

[0123] Many studies have been performed by analyzing genetic models of injury (IL-22 knockout or transgenic overexpression). In these studies, a lack of IL-22 or overexpression of IL-22 is present at the time of injury. In other studies, IL-22 is neutralized with antibodies at the time of injury, and in some cases, IL-22 is neutralized beyond the acute injury phase (e.g., subacutely or well into the regeneration phase). Other studies approach treatment scenarios by looking at the effects of exogenously administered IL-22. When reviewing the available literature overall, it is important to note that different models, whether knockout, overexpression, pre- or post-injury IL-22 neutralization, or exogenous protein administration, paint the same picture of IL-22 protecting injured organs and driving regeneration. This demonstrates the broad applicability and broad time frame of potential IL-22 treatment and also explains why a longer-acting IL-22 protein than hIL-22 is needed.

[0124] However, according to the first and second aspects of the invention, the derivatives of IL-22 (or pharmaceutical compositions comprising same) are for use in methods of treating metabolic, intestinal and / or hepatic diseases, disorders or conditions. Any of the different derivatives of IL-22 described or contemplated herein are expressly included in these aspects of the invention. Any of the dose ranges described can be used for any metabolic, intestinal and / or hepatic disease, disorder or condition.

[0125] The term "metabolic disease, disorder, or condition" can refer to any disease, disorder, or condition that disrupts the body's ability to convert food into energy (i.e., metabolism). The metabolic disease, disorder, or condition can be obesity, type 1 diabetes, type 2 diabetes, hyperlipidemia, hyperglycemia, or hyperinsulinemia.

[0126] The intestinal disease, disorder or condition may be inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, graft-versus-host disease (GvHD), chemical injury, viral infection, bacterial infection or short bowel disease.

[0127] The liver disease, disorder, or condition can be non-alcoholic fatty liver disease (NAFLD), 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 a condition caused by surgery or transplantation.

[0128] Also provided are methods of treating a subject having a metabolic, intestinal and / or hepatic disease, disorder or condition, such as one or more of those described above, using a derivative of IL-22 described or contemplated herein or a pharmaceutical composition comprising same.

[0129] The IL-22 derivatives described or contemplated herein, or pharmaceutical compositions comprising same, may be useful for treating patients or patient groups with certain characteristics. As used herein, the term "patient" may refer to an otherwise healthy recipient of an IL-22 derivative. For example, a patient or patient group may not respond to GLP-1 receptor agonists to a level that achieves a desired outcome.

[0130] The patient or patient group may have a condition that would benefit from the use of a derivative of IL-22 described or contemplated herein, or a pharmaceutical composition comprising same. In particular, such a patient or patient group may have a co-morbidity such as cardiovascular disease (CVD) and / or low-grade inflammation (defined by elevated CRP and / or IL-6 levels).

[0131] The patient or patient group may have impaired glucose tolerance and / or metabolic syndrome (also called metabolic syndrome X or insulin resistance syndrome).

[0132] A patient or group of patients may have elevated cholesterol levels and / or may desire to lower their cholesterol levels. A patient or group of patients may have elevated cholesterol levels and / or may desire to lower their cholesterol levels in conjunction with having or having a family member with familial hypercholesterolemia (FH). In some countries, cholesterol is measured in milligrams (mg) per deciliter (dL) of blood, while in other countries, cholesterol is measured in millimoles per liter (mmol / L). Generally, total cholesterol is measured after 9 to 12 hours of fasting, although fasting is not required in some cases.

[0133] Elevated cholesterol levels can be measured and assessed by the level of total cholesterol, a measurement derived from high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides (TG) in a patient's blood. Particular patients or patient groups may have elevated cholesterol levels of greater than 200 mg / dL or 5.2 mmol / L, or greater than 240 mg / dL or 6.2 mmol / L total cholesterol.

[0134] Elevated cholesterol levels can be measured and assessed by low-density lipoprotein (LDL) cholesterol levels. Particular patients or patient groups can have elevated cholesterol of greater than 130 mg / dL or 3.4 mmol / L, greater than 160 mg / dL or 4.1 mmol / L, or greater than 190 mg / dL or 4.9 mmol / L.

[0135] Elevated cholesterol levels can be measured and assessed by the level of non-high density lipoprotein (non-HDL) cholesterol. Elevated cholesterol levels can be measured and assessed by subtracting the level of high density cholesterol (HDL) from the total cholesterol level. Certain patients or patient groups may have elevated cholesterol levels of non-high density lipoprotein (non-HDL) cholesterol greater than 130 mg / dL or 3.4 mmol / L.

[0136] A patient or group of patients may have elevated triglyceride (TG) levels and / or may desire to lower triglyceride (TG) levels. Elevated triglyceride (TG) levels can be measured and assessed by triglyceride (TG) levels. A particular patient or group of patients may have elevated triglyceride (TG) levels greater than 150 mg / dL or 1.7 mmol / L, greater than 200 mg / dL or 2.3 mmol / L, or greater than 500 mg / dL or 5.6 mmol / L.

[0137] A patient or patient group may wish to achieve, manage, or maintain weight loss. The IL-22 derivatives described or contemplated herein, or pharmaceutical compositions comprising same, may be useful for treating a patient or patient group to achieve and / or manage weight loss. This may be the case when the patient or patient group does not tolerate other medications, such as GLP-1 receptor agonists (e.g., semaglutide, tirzepatide). This may be due to undesirable effects, such as adverse GI effects, rapid weight loss, and / or excessive weight loss, caused by other medications, such as GLP-1 receptor agonists (e.g., semaglutide, tirzepatide). These considerations apply to otherwise healthy patients, and also to patients with conditions such as diabetes (particularly type 2 diabetes). The present invention contemplates the use of IL-22 derivatives for the treatment of diabetes (particularly type 2 diabetes), and also for the treatment of obesity. A patient can have type 2 diabetes and not be obese, or can have type 2 diabetes and be obese, or a patient can be obese and not have type 2 diabetes.

[0138] The IL-22 derivatives disclosed herein are useful, in relevant doses, for treating the indications discussed herein, whether as first- or second-line treatment, monotherapy or combination treatment.

[0139] There are no limitations as to which of the IL-22 derivatives or compositions described herein should be administered to which patients, rather it is contemplated that any of the derivatives and compositions described herein can be administered to any of the patients described herein.

[0140] Every feature described in this specification (including any accompanying claims, abstract and drawings), and / or every step of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0141] For a better understanding of the present invention and to illustrate how embodiments of the present invention may be practiced, reference will now be made to examples which are not intended to limit the invention in any way. [Example]

[0142] Derivative 1 (see Table 3 and Figure 2) is a novel, long-acting IL-22 analogue being developed for the long-term treatment of intestinal and liver diseases, disorders, or conditions. The intended clinical route of administration is either subcutaneous or intravenous. The dosing regimen in humans was devised based on the results of the following animal studies.

[0143] Example 1 - Subcutaneous Toxicokinetic (TK) Study in Minipigs the purpose This study aimed to establish the TK profile of derivative 1 when administered subcutaneously to Göttingen minipigs once a week for 6 weeks, followed by a 4-week recovery period for selected animals.

[0144] overview Minipigs were selected as a test model because they have demonstrated pharmacological response to treatment in PK / PD minipigs, single and four subcutaneous doses of Derivative 1 were well tolerated in the same study, and because they are well tolerated and suitable for this type of study. Subcutaneous injection was selected to correspond to one of the intended human administration routes. The dose was selected based on the results of a dose range-finding study in minipigs; 9.48 mg / kg was found to be intolerable over a 4-week period, while 1 mg / kg did not produce any adverse findings. Furthermore, one animal tolerated weekly dosing at 9.48 mg / kg for 2 weeks, and findings in other animals led to a reduction in the dose to 3 mg / kg for the remainder of the study, which was tolerated.

[0145] method (i)Animals Forty Göttingen specific pathogen-free (SPF) minipigs (20 males and 20 females) were obtained from Ellegard Göttingen Minipigs A / S, Denmark. The animals were 3-4 months old and weighed 7-9 kg upon arrival. The animals were housed in accordance with relevant guidelines (males housed individually and females housed in groups) and fed minipig diet (SMP(E)SQC, Special Diets Services, UK) twice daily at approximately 125 g per animal per meal. From day 1, treated animals (groups 2-4, see Table 4) received an additional 50 g of food in the morning. All animals had free access to domestic quality drinking water.

[0146] On the day of arrival, the minipigs were randomly assigned to four groups (Groups 1-4) using a randomization scheme, with each litter being equally distributed among the groups.

[0147] A pretreatment period of approximately 3 weeks (including a 5-day acclimation period) was allowed during which the animals were observed daily to exclude animals in poor condition.

[0148] Approximately one week before the start of the study, animals were assessed for body weight, and if group means were significantly unequal, animals were reassigned to achieve uniformity in mean group weights. Data available from pre-treatment observations, clinical signs, and laboratory investigations were also taken into consideration when reassigning animals.

[0149] Prior to the start of treatment, two injection sites, one on each side, were clipped and marked on the animal's neck area. Injection sites were marked on each corner. The tattoo area was approximately 2 x 5 cm, as illustrated below.

[0150] [ka]

[0151] Prior to tattooing, anesthesia was achieved by intramuscular injection (1.0 mL / 10 kg body weight) of a mixture of Zoletil 50® Vet (125 mg tiletamine and 125 mg zolazepam, Virbac, France), 20 mg xylazine / mL (6.25 mL), 100 mg ketamine / mL (1.25 mL), and 10 mg butorphanol / mL (2.5 mL) into the left hind limb. If supplementation was required, a dose of 1 / 3 of the original dose was usually given. However, individual needs were taken into consideration.

[0152] (ii) Preparation of dosage formulations Quality control analysis of Derivative 1 was performed using standard procedures to establish its identity, purity, and stability. Following characterization, Derivative 1 was supplied as a 9.25 mg / mL stock solution in phosphate buffered saline (PBS): 1.96 mM KH2PO4, 8.05 mM Na2HPO4, 140 mM NaCl, pH 7.4. Dose formulations were prepared by diluting the stock solution to the required concentration in sterile-filtered PBS. Analysis of test samples was performed by high-pressure liquid chromatography (HPLC) with ultraviolet (UV) detection. To be considered acceptable, the concentration of each formulation had to be within ±10% of the nominal value.

[0153] (iii) Treatment The groups, dose levels and numbers of animals (1-40) in the main study and recovery period were as follows:

[0154] [Table 5]

[0155] The first day of treatment was designated as day 1.

[0156] Weekly doses of Groups 1-3 were given by subcutaneous injection on days 1, 8, 15, 22, 29 and 36 to both main and recovery animals according to recent weight data.

[0157] Main and recovery animals in Group 4 were given weekly doses by subcutaneous injection according to the most recent body weight data on days 1, 15, 22, 29, and 36. There was a washout period at week 2 depending on clinical signs.

[0158] Treatments were administered at site 1 (left) on days 1, 15, and 29, and at site 2 (right) on days 8, 22, and 36.

[0159] Dose volume was 0.40 mL / kg. Dosing was performed using a butterfly needle to allow for slower injection rates as needed depending on the volume and size of the animal.

[0160] The weight of the dosage formulation for each group was recorded before and after dosing. After dosing, the amount of dosage formulation used for each group was compared to the expected daily amount.

[0161] For recovery animals, a 6-week treatment period was followed by a 4-week treatment-free period.

[0162] (iv) Blood sampling and observation Blood samples for TK analysis were collected from the jugular vein / bijugular trunk at specific time points during the study. Specifically, on the first day of dosing (Day 1), blood samples for TK were collected from all animals at the following time points: 1.5, 4, 8, 12, 24, 48, 72, and 168 hours (the latter immediately prior to dosing on Day 8). Additionally, on Day 36, blood samples were collected pre-treatment and 1.5, 4, 8, 12, 24, 48, 72, and 168 hours post-dosing.

[0163] Animals were monitored daily throughout the study for clinical observations, including injection site reactions and behavioral changes. Animals were weighed weekly at the same time of day and weight gain was calculated.

[0164] (v) Bioanalysis Minipig serum samples were analyzed to quantify derivative 1 using a fully validated assay (the "V-PLEX Human IL-22 Kit" commercially available from Meso Scale Discovery (MSD)). Briefly, the kit (MSD Multi-Array®) contained a 96-well Small Spot plate pre-coated with a capture antibody against human IL-22 on a well-defined single spot. After washing (three times with 150 μl / well of wash buffer), 50 μl / well of calibration standards, quality control samples, and unknown samples were added to each well. After incubation (2 hours, 600 rpm, room temperature), the plate was washed (as described above) and 25 μl / well of SULFO-TAG®-conjugated detection antibody was added. After incubation (as described above), the plate was washed again (as described above) and 150 μl / well of 2× Read Buffer T (MSD) was added to each well. Plates were analyzed using a QuickPlex SQ 120 reader (MSD) to generate electrochemiluminescent (ECL) signals. Signals from calibration standards, quality control samples, and unknown samples were converted to concentrations using a regression curve calculated based on the signals from the calibration samples. Calculations were performed using Workbench® software from MSD and / or Watson LIMS.

[0165] TK evaluation of analytical results was performed using the commercially available validated software Phoenix WinNonLin (version 8.3 or later). All critical operations and methods were performed and documented in accordance with current written local standard operating procedures unless otherwise stated. TK analysis was performed using the maximum concentration (C max ), C max Time to obtain (T max ), time of last measurable concentration (T last ), t 1 / 2, area under the concentration-time curve for the last measurable concentration (AUC last ), AUC of the dosing interval (AUC tau ), extrapolated AUC as a percentage of the total (AUC%extrap ), AUC extrapolated to 168 hours (AUC 0-168h ), AUC extrapolated to infinity (AUC inf ), and drug accumulation ratio (R ac The study consisted of assessment of standard parameters, including dose-corrected (linear) values ​​of exposure (AUC and C) when possible. max ) were also obtained. Sex-specific and pooled estimates were generated for each of the TK parameters. Other TK parameters were assessed as deemed appropriate.

[0166] Data were processed to obtain group means and standard deviations where appropriate. Statistical analysis was performed using Instem Provantis® (version 9.3.0.0).

[0167] result Table 5 shows the main TK parameters after weekly subcutaneous administration of Derivative 1 to minipigs.

[0168] [Table 6]

[0169] The results showed that the mean serum concentration of derivative 1 after subcutaneous administration reached its maximum concentration (T max Serum concentrations exceeded the lower limit of quantitation (BLOQ, 0.0488 ng / mL) at all dose levels and remained above the 168-hour (T last After absorption, derivative 1 was detectable for a mean elimination half-life (t 1 / 2 ) was discharged. C max The increase was dose-proportional in both males and females after a single subcutaneous dose when comparing the lowest and highest dose levels.

[0170] conclusion These data from a 6-week repeated dose study by subcutaneous administration demonstrate the exposure of minipigs to derivative 1. The observed long elimination half-life (t 1 / 2) is evidence that the compound is suitable for once-weekly dosing in humans.

[0171] Example 2 - Bioavailability study in minipigs the purpose This study aimed to determine the PK parameters and bioavailability of Derivative 1 by a single-dose subcutaneous to intravenous crossover study in Göttingen minipigs.

[0172] overview Minipigs were chosen as the test model for the same reasons provided in Example 1. Subcutaneous injection and intravenous infusion were chosen to be compatible with both intended human administration routes.

[0173] method The method used was identical to that described in Example 1. The only difference was an additional PK parameter measured for the intravenous route of administration, which was the apparent volume of distribution (V) at terminal phase. z ), drug clearance rate (CL) and bioavailability (i.e., the percentage of the administered dose that reached the systemic circulation; F).

[0174] result Tables 6A and 6B show the PK and bioavailability parameters of Derivative 1 after intravenous or subcutaneous administration to minipigs.

[0175] [Table 7]

[0176] [Table 8]

[0177] The results showed that derivative 1 was eliminated with a two-compartment decay after a single intravenous dose of 0.1 mg / kg, with a (mean) terminal elimination half-life (t 1 / 2 ) was 46.4 hours in males and 35.4 hours in females.

[0178] After a single subcutaneous dose to minipigs, the maximum concentration of derivative 1 was reached 4 hours after administration in all animals except animal number 3, where the maximum concentration was observed 8 hours after administration (T max Serum concentrations after a single subcutaneous dose of 0.5 mg / kg were 0.0168 hours post-dose (T last ) above the lower limit of quantitation (LLOQ). After absorption, derivative 1 had a terminal elimination half-life (t 1 / 2 ) was released.

[0179] After intravenous administration, the (mean) maximum concentration (C max ) ranged from 187 ng / mL to 204 ng / mL at 0.1 mg / kg and from 104 ng / mL to 164 ng / mL at 0.5 mg / kg after subcutaneous administration. The corresponding AUC INF 0.1 mg / kg intravenously for 3300 hours * ng / mL~3600h * ng / mL, and 0.5 mg / kg subcutaneously for 8280 h * ng / mL~11100h * ng / mL.

[0180] Bioavailability (F) after subcutaneous administration was moderate, averaging 62% in male minipigs and 52% in female minipigs.

[0181] conclusion These data show the bioavailability of derivative 1 in minipigs. The bioavailability after subcutaneous administration was moderate, averaging 57%, with no significant sex differences.

[0182] Example 3 - Therapeutic effect in a mouse model of colitis the purpose This study aimed to evaluate the effects of derivative 1 on intestinal parameters in a dextran sodium sulfate (DSS)-induced colitis mouse model.

[0183] method (i)Animals Sixty 10-week-old female C57Bl / 6JRj mice weighing approximately 20-25 g were obtained from Janvier (France) and acclimatized in group housing (5 mice per box) for one week prior to the start of the study. Animals were housed according to standard procedures, provided with regular chow and tap water, and observed daily for any signs of ill health. The day before the start of the study, mice were randomized according to body weight into six groups (n = 10 mice per group), and baseline measurements, including body weight, were taken.

[0184] (ii) Induction of colitis On day 1 of the study, acute enteritis (colitis) was induced by dissolving DSS in drinking water (2.5% (w / v)) and administering it to all mice except the control group. This was repeated once daily for 6 days, and normal drinking water was restored on days 7-11. Control mice received normal drinking water throughout the study period. Body weights, as well as food and water intakes, were measured daily on days 1-10 for all mice. Disease activity indexes were determined by scoring changes in weight loss and rectal bleeding daily on days 1-10.

[0185] (iii) Preparation of dosage formulations Derivative 1 was prepared as a 12 mg / mL stock solution in PBS at room temperature in a clean laboratory. At the time of testing, the stock solution was found to be 88.6% pure and therefore contained 10.6 mg / mL of derivative 1. The stock solution was diluted with sterile-filtered PBS to provide dosing solutions at concentrations of 0.01, 0.03, 0.08, and 0.25 mg / mL. These were stored at 4°C until needed. PBS was also used as a control vehicle and was prepared according to standard procedures. 2 mL / kg of derivative 1 or PBS was administered subcutaneously to mice every morning on days 1 through 10 of the study, as detailed in Table 7.

[0186] [Table 9]

[0187] (iv) Bioanalysis On day 11, all mice were sacrificed by cardiac puncture under isoflurane anesthesia.

[0188] The intestine was dissected, flushed with ice-cold saline, and the contents gently removed. Then, using the systematic random uniform sampling (SURS) principle, the colon was sampled into four slabs and placed in a multi-cassette. The tissue was infused overnight in formalin (Tissue-Tek VIP®, Sakura) and subsequently embedded in paraffin blocks. All tissue slabs were arranged so that individual slabs could be identified at a later stage. The paraffin blocks were trimmed, and 5 μm upper sections were cut and mounted on a SuperFrost® Plus objective glass (Thermo Fisher Scientific). Another section was cut at a distance of 500 μm to the upper section, thereby obtaining a total of eight colon sections from each animal.

[0189] Stereological volume estimation was performed on scanned hematoxylin and eosin-stained slides using the newCAST system (Visiopharm). Total colon volume, mucosal and submucosal volume, muscularis, and inflamed tissue volume were estimated by point counting using an appropriately sized grid system, counting all points hitting structures of interest. The number of points hitting structures of interest was converted to volume according to the following mathematical relationship: Vol ref =Σp×A(p)×t where A(p) is the area per point, p is the total number of points that hit the structure of interest, and t is the distance between intercepts.

[0190] result Figure 7 compares the colonic inflammation volume of mice in groups 2 to 5. Mice in group 2 (administered only the PBS vehicle) were characterized by DSS-induced inflammatory colons (3 mm 3(The mean volumes before and after treatment are shown.) A reduction in the volume of colonic inflammation was observed in groups 4 to 6 (administered at least 0.05 mg / kg of derivative 1). However, no reduction was observed in group 3 (administered at 0.0125 mg / kg of derivative 1).

[0191] conclusion A reduction in colonic inflammation volume was achieved in a murine model of colitis after subcutaneous administration of derivative 1. The first therapeutic effect was observed at a dose of 0.05 mg / kg.

[0192] Example 4 - Subcutaneous PK study in mice the purpose This study aimed to determine the PK profile of Derivative 1 when administered as a single dose by subcutaneous injection to C57BL / 6J mice.

[0193] method (i)Animals Forty-eight male, 10-week-old lean C57Bl / 6JRj mice were obtained from Janvier (France) and acclimatized in group housing (3 per box) for 2 weeks before the start of the study. Animals were housed according to standard procedures, provided with regular chow and tap water, and observed daily for any signs of ill health. Ten days before the start of the study, mice were randomized into four groups (n = 12 per group) according to body weight, and baseline measurements, including body weight, were taken.

[0194] The day before dosing, the planned injection site of each animal was shaved to better monitor for injection site reactions.

[0195] (ii) Preparation of dosage formulations Derivative 1 was prepared as a 10 mg / mL stock solution in PBS at room temperature in a clean laboratory. The stock solution was diluted with sterile-filtered PBS to provide dosing solutions at concentrations of 0.01, 0.05, 0.15, and 0.5 mg / mL. These were stored at 4°C until needed. A single 2 mL / kg dose of Derivative 1 was administered subcutaneously to each mouse in the neck on the morning of Day 0 of the study, as detailed in Table 8.

[0196] [Table 10]

[0197] (iii) Blood sampling and observation At 1, 2, 4, and 8 hours after dosing, 75 μl blood samples were collected from the tail vein of three mice in each group (all 12 mice in a group were rotated over four time points) into appropriately sized Microvette tubes, mixed by inversion five times, and allowed to clot at room temperature. The blood was centrifuged at 3000 g for 10 minutes at room temperature. Collected serum samples (at least 25 μl) were kept at -70°C in serum separator tubes until required for PK analysis. This serum sampling was repeated on days 1 through 4.

[0198] Body weights were measured on the mornings of days 0, 1, 3 and 4. Animals were observed daily for any clinical signs of ill health, including injection site reactions such as bruising, erythema, swelling and crusting.

[0199] (iv) Bioanalysis PK parameters were calculated based on the quantitative data using PKSolver. Data were analyzed by noncompartmental analysis (NCA).

[0200] Mouse serum samples were also analyzed to quantitatively determine Derivative 1 using the commercially available "V-PLEX Human IL-22 Kit" from MSD as described in Example 1.

[0201] Continuous data from a single time point were fitted to a one-factor linear regression model with treatment group as the distinct independent (predictor) variable, and treatment was compared to control using a Dunnett's test. Continuous data from repeated sample collections over time were fitted to a two-factor linear regression model with treatment group and time point as distinct independent (predictor) variables with an interaction. A Dunnett's test was again used to compare treatment to control for each time point. Data from distinct endpoints, such as histopathological score values, were partitioned into a 2 × 2 contingency table containing responders and non-responders in the control and treatment groups. Fisher's exact test assumed that row and column sums in the contingency table were fixed, sampling was random, and observations could be classified into only one cell. Reported p-values ​​from all pairwise comparisons were adjusted using a Bonferroni correction.

[0202] result Table 9 shows the PK parameters after a single subcutaneous administration of Derivative 1 to mice.

[0203] [Table 11]

[0204] As can be seen from Table 9, a single subcutaneous dose of 0.05 mg / kg of derivative 1 resulted in a C of 1.12 ng / mL in mice. max and 22.7h * AUC in ng / mL INF This was also the dose that produced the first therapeutic effect observed in the murine acute DSS model of Example 3.

[0205] conclusion These data from a single-dose mouse PK study demonstrate the exposure of mice to Derivative 1. Serum concentrations of Derivative 1 after a single subcutaneous administration to mice increased in a dose-dependent manner, with an elimination half-life (T 1 / 2 ) was an average of 8.2 hours.

[0206] Example 5 - In vitro STAT3 potency assay the purpose This study aimed to determine the in vitro potency of Derivative 1.

[0207] overview Derivative 1 can bind to a specific receptor overexpressed on the surface of BHK cells, activating the luciferase reporter, which can be detected using the One-Glo® Luciferase Assay Detection Kit (Promega, US) according to the manufacturer's instructions.

[0208] method In this assay, 100 μl / well of BHK cells overexpressing IL10RB and IL22RA (hereafter referred to as "BHK IL22 cells") was added to 2 × 10 5 The cells were seeded into a 96-well cell culture plate at a viable cell density of 1000 cells / mL and incubated under a cover at 37°C and 5% CO for 20–28 h.

[0209] After incubation, all medium was removed from each well, and 100 μl / well of serially diluted reference standard (i.e., a fixed batch of derivative 1), assay control, and sample was added. Assay controls were independent dilutions of the reference standard. Plates were incubated under a cover at 37°C, 5% CO for 3-5 hours.

[0210] After incubation, 100 μl / well of One-Glo® solution was added, and the plate was incubated in the dark for 5 to 20 minutes. The reaction solution was mixed at least four times by inversion pipetting. 100 μl / well of the reaction solution was then transferred from the culture plate to a 96-well white plate. The plate was placed in a plate reader (Molecular Devices SpectroMax M5e) and luminescence was measured.

[0211] Illustratively, binding to IL-22 and IL-10 receptors located on the surface of BHK IL22 cells activated an intracellular luciferase reporter gene under the control of a STAT3-driven promoter. The potency of Derivative 1 was determined by monitoring the activation of the luciferase reporter in BHK IL22 cells using the One-Glo® Luciferase Assay System. The intensity of luminescence was proportional to the potency of Derivative 1 in each well.

[0212] Assays were repeated to ensure accuracy and validity.

[0213] The main PD parameters measured were EC 99 , the slope coefficient of the curve (Hill slope), the peak (minimum) effect (E min ) and peak (maximum) effect (E max ) was.

[0214] The dose-response curves (emission vs. concentration of Derivative 1) were fitted with a 4PL model. The results were compared to the EC 50 The values ​​were reported as relative potency calculated using the

[0215] result Figure 8 shows in vitro STAT3 activation in the IL-22 receptor reporter BHK cell line. As increasing concentrations of Derivative 1 were administered to the cells, the luminescence of the luciferase reporter increased in a sigmoidal fashion. Two replicates of the assay showed virtually identical results, demonstrating precision and validity.

[0216] Table 10 shows the main PD parameters from the in vitro STAT3 curve for Derivative 1.

[0217] [Table 12]

[0218] The mean EC for two technical replicates of the duplicate determinations is shown in Table 10. 99was found to be 883 ng / mL, indicating that derivative 1 was active in the nanomolar range in this sensitive reporter cell line for IL-22 receptor activation.

[0219] conclusion Derivative 1 was shown to be potent in binding to IL10RB and IL22RA on the surface of BHK cells in vitro, and thus demonstrated potent and fully active activity at average exposures predicted to be reached in humans after subcutaneous and intravenous administration (as described in Example 9).

[0220] Example 6 - PD Effect in Diabetic Mouse Model This study aimed to evaluate the effect of Derivative 1 on blood glucose in db / db mice. These mice are used to model phases 1 to 3 of type 2 diabetes and obesity.

[0221] method (i)Animals Twenty 7- to 8-week-old C57BKS db / db mice were obtained from Charles River (Germany) and acclimatized in group housing (10 mice per cage) for 18 days prior to the start of the study. Animals were housed according to standard procedures. Two weeks after arrival, each mouse was microchipped and blood glucose was measured. Three days prior to the start of the study, mice were randomized into two groups (n = 10 mice per group), individually caged, and blood glucose was measured again. Food intake was measured daily until the start of the study.

[0222] (ii) Preparation of dosage formulations Derivative 1 was prepared as a 0.125 mg / mL dosing solution in PBS. PBS vehicle (pH 7.4 with 70 ppm polysorbate 20) was used as a control and was prepared according to standard procedures.

[0223] (iii) Treatment Mice were administered 4 mL / kg of derivative 1 or PBS subcutaneously on days 0 to 16 of the study as detailed in Table 11. This was done at 10 AM every day except for day 16, which was done at 7 AM.

[0224] [Table 13]

[0225] Body weight (pre-dose) and food intake were measured daily. Blood glucose was also measured pre-dose (t=0 min) and then daily post-dose (t=1, 2, and 6 h).

[0226] result Figure 9 compares blood glucose levels over time for mice in groups 1 and 2. Mice in group 1 (administered only the PBS vehicle) were characterized by high blood glucose levels, which persisted over time. A progressive and significant decrease in blood glucose levels over time was observed with treatment in group 2 (administered 0.5 mg / kg of derivative 1).

[0227] conclusion Lowering of blood glucose was achieved in murine models of diabetes (type 2) and obesity after subcutaneous administration of Derivative 1. This supports the use of Derivative 1 and other derivatives described herein in the management of these conditions. This is important because type 2 diabetes is by far the most common type of diabetes.

[0228] Patients with type 2 diabetes either do not produce enough insulin or do not produce insulin that the body can use properly. Cells in the body become resistant to insulin, which requires more insulin to keep blood glucose levels within normal ranges. Eventually, the pancreas can become exhausted by producing excess insulin and begin to produce less and less insulin. Type 2 diabetes can usually be managed through diet, exercise, and self-monitoring of blood glucose, at least for the first few years after diagnosis. However, type 2 diabetes is a progressive condition, and most people require pills and / or insulin injections after living with type 2 diabetes for 5 to 10 years.

[0229] Given the findings in the studies at hand, Derivative 1 and other derivatives described herein may offer a new treatment regimen for this condition.

[0230] Example 7 - Therapeutic effects in a diet-induced obese mouse model the purpose This study aimed to evaluate the effect of 4 weeks of treatment with derivative 6 on body weight in male diet-induced obese mice. A parallel study aimed to evaluate the effect of 4 weeks of treatment with derivative 1 on body weight and fasting plasma insulin in male diet-induced obese mice.

[0231] method (i)Animals Seventy male 6-week-old C57Bl / 6JRj mice were obtained from Janvier (France), housed according to standard procedures, and fed a 60% high-fat diet (SSNIFF, Germany) for 29 weeks prior to the start of the study to achieve rapid weight gain. Two weeks prior to the start of the study, mice were singly housed. One week prior to the start of the study, mice were randomized according to body weight into seven groups (n = 10 per group), and baseline measurements, including body weight, were taken. The high-fat diet was maintained throughout the study period.

[0232] (ii) Preparation of dosage formulations Derivative 1 was prepared as dosing solutions of 0.01, 0.03, and 0.06 mg / mL in PBS. Derivative 6 was prepared as dosing solutions of 0.0025, 0.01, and 0.03 mg / mL in PBS. These were stored at 4°C for up to 3 days until needed. PBS was also used as a control vehicle and was prepared according to standard procedures.

[0233] (iii) Treatment Mice were subcutaneously administered 5 mL / kg of Derivative 1, Derivative 6 or PBS in the afternoon of each of days 0 to 28 of the study, as detailed in Table 12. Body weight was measured daily.

[0234] [Table 14]

[0235] (iv) Blood sample collection On day 24, all mice were fasted for 4 hours, and 50 μl blood samples were collected from each mouse via the tail vein into heparinized tubes. Plasma was separated (yielding a sample volume of 20 μl) and stored at −80°C until analysis. Insulin was measured using a commercially available MSD platform (Meso Scale Diagnostics) according to the manufacturer's instructions.

[0236] result Figure 10 compares the body weight of mice in groups 1 to 4 over time. Mice in group 1 (administered only the PBS vehicle) were characterized by high body weight throughout the time course. A dose-dependent decrease in body weight was observed over time with treatment in groups 2 to 4 (administered 0.0125 mg / kg, 0.05 mg / kg, or 0.15 mg / kg of derivative 6).

[0237] Figure 11A compares the body weight of mice in groups 1 and 5-7 over time. As noted above, mice in group 1 (administered only the PBS vehicle) were characterized by high body weight throughout the time course. Again, a dose-dependent decrease in body weight was observed over time with treatment in groups 5-7 (administered 0.05 mg / kg, 0.15 mg / kg, or 0.3 mg / kg of derivative 1).

[0238] Figure 11B compares fasting plasma insulin over time in mice from groups 1 and 5-7. Mice from group 1 (administered only the PBS vehicle) were characterized by high plasma insulin throughout the time course. A dose-dependent decrease in plasma insulin was observed over time with treatment in groups 5-7 (administered 0.05 mg / kg, 0.15 mg / kg, or 0.3 mg / kg of derivative 1).

[0239] conclusion Weight loss was achieved in a murine model of obesity after subcutaneous administration of derivative 1 or derivative 6. A reduction in plasma insulin was simultaneously achieved in mice treated with derivative 1. This supports the use of derivatives 1, 6, and other derivatives described herein in the treatment of obesity in humans. Steps are now needed to address obesity in the world's population, as obesity not only causes obvious physical changes but can also lead to a number of serious and potentially life-threatening conditions, such as type 2 diabetes, coronary heart disease, and some types of cancer, e.g., breast and intestinal cancer.

[0240] Example 8 - Therapeutic effect in a mouse model of liver injury the purpose This study aimed to evaluate the effect of treatment with Derivative 1 in a murine model of concanavalin A-induced liver immune-mediated injury.

[0241] method (i)Animals Fifty-five 6-week-old, pathogen-free male C57Bl / 6J mice were obtained from Japan SLC and housed according to standard procedures (up to five mice per cage) with sterilized normal chow and drinking water ad libitum. Body weights were recorded daily before treatment. One day before the start of the study, mice were randomized according to body weight into one group of five mice (Group 1) and five groups of 10 mice (Groups 2-6). Mice in Group 1 served as controls and remained untreated throughout the study.

[0242] (ii) Preparation of dosage formulations Derivative 1 was prepared as a 9.48 mg / mL stock solution in PBS. Dose formulations were prepared by diluting the stock solution to the required concentration in sterile-filtered PBS. PBS was used as a control vehicle and was prepared according to standard procedures.

[0243] (iii) Treatment 5 mL / kg of Derivative 1 or PBS was administered subcutaneously to mice in groups 2 to 6 at 72, 48, 24 and 1 hour before concanavalin A injection, as detailed in Table 13.

[0244] [Table 15]

[0245] (iv) Induction of hepatitis Liver immune-mediated injury (hepatitis) was induced in mice in groups 2 to 6 by a single intravenous injection of 20 mg / kg of concanavalin A (Sigma-Aldrich Co. LLC, USA) dissolved in saline at a concentration of 2.5 mg / mL.

[0246] Survival, clinical signs (lethargy, convulsions, respiratory distress) and behavior were monitored daily.

[0247] Mice were sacrificed 48 hours after concanavalin A injection by exsanguination via direct cardiac puncture under isoflurane anesthesia.

[0248] (v) Blood sample collection At the time of sacrifice, blood was collected into pre-chilled polypropylene tubes containing anticoagulant and stored on ice until centrifugation. Blood samples were centrifuged at 1000 g for 15 minutes at 4°C.

[0249] (vi) Bioanalysis Plasma ALT levels were measured using a FUJI DRI-CHEM 7000 (Fujifilm).

[0250] Statistical analysis was performed using Prism Software 6 (GraphPad Software, USA). Bonferroni multiple comparisons were performed between Group 2 (vehicle) and the remaining groups. A P value of <0.05 was considered statistically significant. Results were expressed as mean ± standard deviation.

[0251] result Figure 12 compares plasma ALT levels in mice from groups 2 to 6. Mice from group 2 (administered only the PBS vehicle) were characterized by plasma ALT. A dose-dependent decrease in plasma ALT was observed with treatment in groups 3 to 6 (administered 0.05, 0.15, 0.30 or 0.60 mg / kg of derivative 1).

[0252] conclusion Reduction of plasma ALT levels was achieved in a murine model of liver immune-mediated injury after subcutaneous administration of Derivative 1. Beneficial effects were observed at a dose of only 0.05 mg / kg. Thus, the data support the use of Derivative 1 and other derivatives described herein to prevent or alleviate liver immune-mediated injury (hepatitis).

[0253] Example 9 - Extension of Animal Data to Humans the purpose This study was based on the PK measurements in minipigs and mice obtained in Examples 1, 2 and 4 and aimed to determine the exposure to Derivative 1 after subcutaneous injection and intravenous infusion in humans.

[0254] method An allometrically scaled cross-species population PK model of Derivative 1 exposure as a function of dose, time, and body weight was developed based on the 1-week single-dose data in minipigs described in Examples 1 and 2. The allometric expansion principle was applied with parameter values ​​normalized to a body weight of 70 kg.

[0255] result Table 14 expands on the PK data provided in Examples 1 and 2 and shows the mean predicted PK parameters for a 70 kg human from subcutaneous and intravenous administration of Derivative 1 up to 1 week after a single dose. The PK parameters are the mean concentration (C average168 ) and AUC at t = 168 hours (AUC 168 ) is included.

[0256] [Table 16]

[0257] The lower limit of the dose range in humans was found to be 1 μg / kg for both subcutaneous injection and intravenous infusion. This was based on the initial therapeutic effect observed in a murine acute DSS-induced colitis model where derivative 1 was administered subcutaneously at a dose of 0.05 mg / kg (see Figure 7 and Example 3). Based on a single-dose mouse PK study (Example 4), this dose resulted in a C of 1.12 ng / mL in mice. max and 22.7h * AUC in ng / mL INF Since 1 μg / kg is the lowest practical dose of derivative 1, it is also the lowest therapeutic dose.

[0258] The upper end of the dose range was found to be 400 μg / kg for subcutaneous injection and 300 μg / kg for intravenous infusion, based on in vitro STAT3 activation in the IL-22 receptor reporter BHK cell line (Figure 8). Here, the mean EC for two technical replicates of duplicate determinations was99 were found to be 883 ng / mL, respectively (Table 10). The doses given are the highest doses for each route of administration, at which the mean exposure (C average ) does not exceed that observed in in vitro assays. Therefore, higher dosing is not expected to result in improved therapeutic efficacy.

[0259] conclusion The animal PK data were extended to humans to predict exposure after subcutaneous or intravenous administration of derivative 1, allowing for the establishment of a therapeutic range.

[0260] Example 10 - Clinical Trial Protocol the purpose To investigate the safety, tolerability, PK, immunogenicity and exploratory PD of Derivative 1 after subcutaneous or subcutaneous and intravenous administration in healthy participants.

[0261] overview A phase I, randomized, double-blind, placebo-controlled, single-center, single- and multiple-ascending-dose study of Derivative 1 in healthy adult participants. The primary route of administration was subcutaneous, with the possibility of switching to a 30-minute intravenous infusion in case of unacceptable local tolerability at the injection site.

[0262] method (i) Study Design The study population consisted of healthy applicants aged 18 to 55 years (inclusive) at screening, with a weight range of 50 kg to 100 kg (inclusive), and a body mass index range of 18.5 kg / m² to 27.0 kg / m² (inclusive) at screening.

[0263] A maximum of 96 participants (excluding replacements) received Derivative 1 or placebo at different ascending doses in two study parts, with up to 64 participants in Part 1 (single ascending dose) and up to 32 participants in Part 2 (multiple ascending doses). Participants were not allowed to participate in more than one cohort.

[0264] Participants were screened for up to 28 days. Eligible participants visited the clinical site on the morning of Day -1 for evaluation of clinical laboratory tests, urinalysis, oral temperature, and coronavirus disease 2019 (COVID-19) testing. Patients were admitted to the clinical site on the evening of Day -1. Participants were discharged after final evaluations on Day 4 in Part 1 and Day 22 in Part 2, at the investigator's discretion. Participants may return to the clinical site for outpatient visits on Days 29, 36, 43, 50, 57, and 71.

[0265] Screened during follow-up, the total duration of involvement for each participant was approximately 12 weeks for Part 1 and 14 weeks for Part 2.

[0266] No interim analyses were performed.

[0267] Part 1 (single ascending dose) evaluated up to seven dose levels in up to eight cohorts. Each cohort consisted of eight healthy participants, each receiving a single dose of either Derivative 1 (n=6) or placebo (n=2). The interval between participants receiving medication in subsequent cohorts was at least 14 days.

[0268] In each cohort, two sentinel participants received either Derivative 1 or placebo on the same day. The remaining participants in the cohort received only one dose after reviewing safety data at least 48 hours after dosing for the sentinel participants. Two sentinel participants were randomized in a 1:1 ratio (Derivative 1:placebo), and the remaining six participants were randomized in a 5:1 ratio.

[0269] In cohort 1, a single dose of 3 μg / kg of derivative 1 or placebo was administered by subcutaneous injection in the abdomen or thigh. Subsequent cohorts received derivative 1 or placebo either subcutaneously (10 μg / kg to 400 μg / kg) or intravenously (3 μg / kg to 300 μg / kg). In the unexpected event that an adverse skin reaction at the injection site led to the decision to switch to intravenous administration, derivative 1 or placebo was administered as a 30-minute continuous intravenous infusion.

[0270] Table 15 provides an overview of the dose levels for each cohort in Part 1 with and without a switch to 30-minute intravenous infusion.

[0271] [Table 17]

[0272] The first intravenous dose after switching from subcutaneous is the expected exposure of the next scheduled subcutaneous dose (C max and AUC 0-168h The test was designed to achieve an exposure not exceeding 100 mg / kg / day (taking into account both the

[0273] The initiation of Part 2 and the dose of the first cohort of Part 2 (including the loading dose) were determined based on a review of available blinded safety and tolerability data from Cohort 5 of Part 1 through at least Day 8, and blinded PK data from Cohorts 1-4 of Part 1 through at least Day 8. If Part 2 was administered intravenously, PK data from at least one intravenous single ascending dose cohort in Part 1 through at least Day 8 were included. All available biomarker data were also taken into account, and the decision was supported by modeling and simulation predictions.

[0274] Part 2 (multiple ascending doses) evaluated up to four dose levels in up to four cohorts. Each cohort consisted of eight healthy participants, who received three doses of either Derivative 1 or placebo on days 1, 8, and 15, respectively. Participants were randomized in a 6:2 ratio (Derivative 1:placebo). The interval between the first participants dosed in Cohort 1 and Cohort 2 was at least 35 days. The interval between the first participants dosed in subsequent cohorts was at least 28 days.

[0275] For each cohort, the first dose on day 1 was a loading dose higher than the doses administered on days 8 and 15, with the intention of achieving steady state after the first dose of Derivative 1. The dose did not exceed a maximum dose of 400 μg / kg subcutaneously or 300 μg / kg intravenously. Specifically, the dosing schedule was as follows: Cohort 1: a loading dose of 2.5 μg / kg body weight, followed by a weekly dose of 1.25 μg / kg body weight (the loading dose was given in week 1, and weekly doses were given for the remainder of the treatment period). Cohort 2: a loading dose of 5 μg / kg, followed by a weekly dose of 2.5 μg / kg. Cohort 3: a loading dose of 10 μg / kg, followed by a weekly dose of 5 μg / kg. Cohort 4: a loading dose of 15 μg / kg, followed by a weekly dose of 7.5 μg / kg. Administration of Derivative 1 or placebo was either subcutaneous or intravenous, depending on whether a decision was made to switch to intravenous administration in part 1.

[0276] Furthermore, the 3 μg / kg body weight subcutaneous dose level was found to be more potent than expected in this study, and therefore an additional 1 μg / kg body weight subcutaneous dose level was included.

[0277] (ii) Dosage Formulation Derivative 1 was provided in 2 mL vials with an extractable volume of 1 mL for single use. The unit dose strength was 10.1 mg / mL.

[0278] A placebo control was used to determine the frequency and magnitude of changes in clinical endpoints that could occur in the absence of an active test product. The placebo comparator for subcutaneous administration was a non-preserved, sterile, ready-to-use liquid formulation with pH=7.0 and no active product, provided in a 2 mL vial with an extractable volume of 1 mL for single use. The placebo comparator for intravenous use was saline (0.9 g / L sodium chloride) provided in a bottle for intravenous infusion.

[0279] (iii) Evaluation Blood samples were collected for PK analysis of Derivative 1 in serum.

[0280] The following PK parameters were determined for Derivative 1 by non-compartmental analysis using individual serum concentration-time profiles at actual sampling times.

[0281] In Part 1: max , t max , AUC 0-168h , AUC last , AUC inf , apparent first-order terminal rate constant (λz), and t 1 / 2 For intravenous administration, CL, Vz, and estimated volume of distribution at steady state (Vss) were also calculated.

[0282] In Part 2 (after each dose): C max , t max , mean serum concentration over the dosing interval (C avg ), the observed trough serum concentration at the end of the dosing interval (C trough ), AUC 0-168h (i.e., AUC at steady state tau ), AUC inf (only after the last dose), λz, t 1 / 2 and cumulative ratio (C max and AUC 0-168h (Rac calculated from dose-corrected values ​​for both the second and third doses relative to the first dose). For intravenous administration, CL, Vz, and Vss (after the last dose only) were also calculated.

[0283] Serum and plasma samples for the 7-plex cytokine panel and determination of various biomarkers were obtained from blood collected at the following time points during part 1 of the study:

[0284] [Table 18]

[0285] Serum and plasma samples for determination of the same cytokines and biomarkers were obtained from blood collected at the following time points during part 2 of the study:

[0286] [Table 19]

[0287] Blood samples were kept frozen during transport and stored at −75°C ± 10°C until analysis.

[0288] PD parameters measured were serum levels of the biomarkers REG3a and hsCRP, and SAA; plasma levels of the biomarker fibrinogen-C; and absolute values ​​and changes from baseline in serum levels of a cytokine panel (including interferon [IFN]-γ, tumor necrosis factor [TNF]-α, IL-1β, IL-2, IL-6, IL-8, and IL-10).

[0289] Analysis of serum cytokines and SAA was performed using electrochemiluminescence immunoassays (SGS France) with commercially available kits (Meso Scale Discovery) according to the manufacturer's instructions. Analysis of serum samples for REG3A determination was performed using the Ella platform with a commercially available ProteinSimple Human REG3A Simple Plex assay cartridge according to the manufacturer's instructions (Bio-Techne, USA). Analysis of serum samples for hsCRP determination was performed by immunoturbidimetric assay for latex particles using a commercially available Cardiac C-reactive Protein (Latex) High Sensitive pack according to the manufacturer's instructions (Roche Diagnostics GmbH, USA). All samples were assayed directly on the Cobas® 6000 platform, with calibration standards assayed in duplicate and control and clinical samples assayed singly. Analysis of plasma samples for fibrinogen C determination was performed using an ELISA assay with a commercially available Human Fibrinogen ELISA kit according to the manufacturer's instructions (ELISA Systems Biology, USA).

[0290] The immunogenicity of Derivative 1 was evaluated by assessing the presence, specificity and titer of anti-drug antibodies.

[0291] The study assessed safety and tolerability by evaluation of adverse events, skin and injection site examinations, clinical laboratory tests, 12-lead electrocardiograms, vital signs, and physical examinations.

[0292] Statistical evaluation used standard statistical tools (e.g., arithmetic mean, standard deviation, median, minimum, maximum, coefficient of variation [CV%], geometric mean, and geometric CV%).

[0293] result Figure 13A compares REG3A levels over time following a single subcutaneous administration of Derivative 1 in humans. Data were pooled from the cohort that received placebo (a non-preserved, sterile, ready-to-use liquid formulation with a pH of 7.0 and no active product). These subjects demonstrated baseline levels of REG3A over the time course, including a peak response around 100 hours. A dose-dependent increase in REG3A levels was observed with treatment with Derivative 1 (dosed at 1 μg / kg to 30 μg / kg). The peak response progressively increased with increasing dose and lasted longer than baseline. All doses achieved REG3A responses above baseline levels.

[0294] Figure 13B compares hsCRP levels over time after a single subcutaneous dose of Derivative 1 in humans. Data from the cohort receiving placebo were pooled. These subjects exhibited baseline levels of hsCRP over time. A dose-dependent increase in hsCRP levels was observed with treatment with Derivative 1 (dosed at 1 μg / kg to 30 μg / kg). Peak responses progressively increased with increasing dose and lasted longer than baseline. All doses achieved hsCRP responses above baseline levels.

[0295] conclusion Increases in REG3A and hsCRP were achieved in humans after subcutaneous administration of Derivative 1 (dosed at 1 μg / kg to 30 μg / kg). REG3A and hsCRP are biomarkers indicative of IL-22 target engagement in the liver and intestine. Thus, the data indicate that Derivative 1 was able to bind to the IL-22 receptor and engage in different activities / pathways that contribute to IL-22-mediated beneficial effects. Such biomarker responses were not demonstrated by competing products. Furthermore, all tested doses achieved REG3A and hsCRP responses above baseline levels. Thus, only 1 μg / kg of Derivative 1 was shown to have beneficial effects. This efficacy of Derivative 1 in humans was quite surprising. Thus, the data support the therapeutic use of the derivatives described herein in humans, even at low doses.

[0296] While certain features of the invention have been illustrated and described herein, many modifications and equivalents will occur to those skilled in the art. It is, therefore, to be understood that the claims are intended to cover all such modifications and equivalents as fall within the true spirit of the invention.

[0297] Example 11 - Clinical Trial Protocol 2.0 the purpose To investigate the safety, tolerability, PK, immunogenicity and exploratory PD of Derivative 1 after subcutaneous administration in healthy participants and otherwise healthy obese participants.

[0298] overview Part 1 (single ascending dose) of a Phase 1 randomized, double-blind, placebo-controlled, single-center, single and multiple ascending dose study of Derivative 1 in healthy adult participants, and Part 2 (multiple ascending doses) in healthy participants and otherwise healthy obese participants.

[0299] The primary route of administration was subcutaneous, with the possibility of switching to a 30-minute intravenous infusion in the event of unacceptable local tolerance at the injection site.

[0300] method (i) Study Design Part 1 was designed as a single ascending dose (SAD) study, and the study population consisted of healthy applicants aged 18 to 55 years (inclusive) at screening, with a weight range of 50 to 100 kg (inclusive), and a body mass index range of 18.5 kg / m² to 27.0 kg / m² (inclusive) at screening.

[0301] Part 2 was designed as a multiple ascending dose (MAD) study, and the study population consisted of healthy Part 1 applicants and "otherwise healthy obese participants." "Otherwise healthy obese participants" were defined as participants with a BMI of ≥ 30 kg / m², who did not have comorbidities (exclusion criteria #1 and #2 below), did not have obesity induced by known endocrine or genetic disorders (e.g., Cushing's syndrome, hypothyroidism, Prader-Willi syndrome; see exclusion criteria #3), and had relatively unchanged body weight during the period leading up to screening. Furthermore, "otherwise healthy obese participants" were not participants who had used prescription or nonprescription medications for weight loss or had undergone surgical or medical device procedures for obesity (liposuction was permitted if performed > 1 year prior to screening). Finally, morbidly obese participants were not included in the "otherwise healthy obese participants" participant category. Because obese participants do not require treatment, they are more likely to have clinically insignificant out-of-normal laboratory values ​​for lipids, glucose, and liver enzymes. Therefore, the acceptable values ​​for the eligibility assessment of these parameters were adjusted for participants in Part 2 of the study (exclusion criterion #1). Similarly, the upper accepted thresholds for systolic and diastolic blood pressure and for QTcF were slightly relaxed.

[0302] [Table 20]

[0303] A maximum of 88 participants will receive derivative 1 or placebo at different escalating doses in two study parts: up to 40 participants in part 1 (SAD) and up to 48 participants in part 2 (MAD). Participants were not allowed to participate in more than one cohort.

[0304] Participants were screened for up to 28 days.

[0305] In Part 1, eligible participants presented to the clinical site on the morning of Day -1 for clinical laboratory tests, urinalysis, oral temperature, and coronavirus disease 2019 (COVID-19)-19 study assessments, were admitted to the clinical site on the evening of Day -1, and were then discharged after a final assessment on Day 4 at the investigator's discretion.

[0306] In Part 2, eligible participants visited the clinical site on the morning of Day -1 for clinical laboratory tests, urinalysis, and weight assessment (oral temperature and COVID-19 testing will be performed as needed and in accordance with local regulations), were admitted to the clinical site the evening before each dose of Derivative 1, and were discharged after a final assessment 2 days after each dose of Derivative 1 at the investigator's discretion. Participants returned to the clinical site for outpatient visits.

[0307] Screened during follow-up, the total duration of each participant's involvement lasted approximately 12 weeks for Part 1 and 4 months for Part 2.

[0308] No interim analyses will be performed.

[0309] Part 1 (single ascending dose) Part 1 - Evaluation of the safety, tolerability, PK, immunogenicity and exploratory PD of SAD of Derivative 1 at up to five dose levels (see Table 16) in up to five cohorts in healthy participants.

[0310] Each cohort consisted of up to eight participants and received a single dose of either Derivative 1 (n=6) or placebo (n=2) administered by SC injection in the abdomen or thigh.

[0311] The interval between participants receiving medication in subsequent cohorts was at least 14 days.

[0312] In each cohort, two sentinel participants received derivative 1 on the same day. The remainder of the participants in the cohort were dosed only once progression criteria were met and all available safety data from the sentinel patients had been reviewed by the principal investigator or co-investigators.

[0313] Two sentinel participants were randomized in a 1:1 ratio (one participant receiving derivative 1: one participant receiving placebo), and the remaining six participants were randomized in a 5:1 ratio (five participants receiving derivative 1: one participant receiving placebo).

[0314] [Table 21]

[0315] Part 2 (multiple ascending doses) Each cohort of 12 participants received six doses of either Derivative 1 or placebo SC on days 1, 8, 15, 22, 29, and 36. Participants were randomized in a 9:3 ratio (9 participants receiving Derivative 1: 3 participants receiving placebo). The interval between the first participants dosed in subsequent cohorts was at least 42 days.

[0316] For each cohort, the first dose on day 1 was a loading dose higher than the maintenance doses administered on days 8, 15, 22, 29 and 36 with the intention of achieving steady state after the first dose of Derivative 1 (see Table 20).

[0317] The loading and maintenance doses applied during the study are shown in Table 20.

[0318] [Table 22]

[0319] (ii) Dosage formulation was carried out according to Example 10(ii) Dosage formulation.

[0320] (iii) Evaluation was carried out generally according to Example 10(iii) Evaluation.

[0321] The timescale and events for the multiple ascending dose protocol are summarized in Figure 16 with the following reference footnotes. (a) IP is prepared by a pharmacist and should be used at room temperature within 4 hours of preparation. IP is administered by SC injection into the abdomen or thigh. The injection site is rotated with each SC administration. (b) PK samples should be collected at the following time points: Dose 1: Pre-dose and 1, 6, 12 hours (Day 1), 24 and 36 hours (Day 2), 48 hours (Day 3), 72 hours (Day 4), 96 hours (Day 5), 120 hours (Day 6), and 144 hours (Day 7) after IP administration. Doses 2-6: Pre-dose on Days 8, 15, 22, 29, and 36. Dose 6: 1, 6, 12 hours (Day 36), 24 and 36 hours (Day 37), 48 hours (Day 38), 72 hours (Day 39), 96 hours (Day 40), 120 hours (Day 41), 144 hours (Day 42), and 168 hours (Day 43) after IP administration. Additionally, samples will be collected as indicated in the table. (c) Pre-dose samples should be collected within 5 minutes of IP administration. (d) Post-dose blood samples for biomarkers measured in serum should be collected 12 hours (Day 1 / 36), 24 hours (Day 2 / 37), 48 hours (Day 3 / 38), and 96 hours (Day 5 / 40) after the first and sixth IP doses. Additionally, blood samples will be collected as indicated in the table. (e) Evaluation should be performed prior to administration of IP. (f) Body weight should be measured after an overnight fast (at least 8 hours of fasting). (g) Screening is from day -28 to day -2 before the first dose of IP. (h) A follow-up visit should be conducted on Day 92 (± 3 days). All participants who discontinue early from the study, except for participants who withdraw consent, will be seen with a Withdrawal Visit at the time of study discontinuation and a Follow-up Visit on Day 92 (± 3 days). At the Withdrawal Visit, participants should complete as many procedures as possible that were scheduled on the day the participant terminated early. In the case of an AE, appropriate follow-up will be conducted.

[0322] The protocol includes evaluation of the following clinical laboratory tests:

[0323] [Table 23-1]

[0324] [Table 23-2]

[0325] result Selected results are included herein, although additional data may become available after this application is filed.

[0326] FIG. 14 illustrates the dose-dependent increase in the target engagement marker REG3a after SC administration of Derivative 1 in a multiple ascending dose study in humans.

[0327] Figure 15 illustrates the dose-dependent reduction of total cholesterol in blinded data from a multiple ascending dose study of SC administration of Derivative 1 in humans. Each cohort consisted of nine subjects receiving active treatment with Derivative 1 and three subjects receiving placebo treatment. During the first week of treatment, subjects received a loading dose of twice the maintenance dose (Cohorts 1 and 2) or 150% (Cohort 3). Thus, the data in Figure 15 highlight that all loading doses in Cohorts 1, 2, and 3 had a cholesterol-lowering effect. Even when the data were blinded, dose-dependence could be suggested, as there was a greater cholesterol reduction in two of the cohorts (Cohorts 2 and 3) compared to Cohort 1. The cholesterol-lowering effect declined after approximately 43 days, which can be explained by the last dose of Derivative 1 administered on Day 36. Therefore, it can be assumed that the effect of Derivative 1 subsequently declines according to its half-life of approximately one week in humans.

[0328] conclusion A stable increase in REG3A was achieved in humans after subcutaneous administration of Derivative 1 (dosed at 1 μg / kg to 15 μg / kg). REG3A is a biomarker indicating IL-22 target engagement in the liver and intestine. Thus, the data indicate that Derivative 1 was able to bind to the IL-22 receptor and engage in different activities / pathways that contribute to IL-22-mediated beneficial effects. Such a stable biomarker response was not demonstrated by competing products. In comparison, a stable and significant decrease in cholesterol was observed, even in blinded data. This may be beneficial for many patients, especially obese patients, and administration of Derivative 1 may provide a protective effect against cardiovascular disease.

[0329] Furthermore, all tested doses achieved REG3A response above baseline level. Therefore, it was shown that only 1 μg / kg of derivative 1 has beneficial effect. It was also shown that multiple doses have stable effect not only on REG3A but also on cholesterol lowering.

[0330] This potency and exposure level of derivative 1 in humans is quite surprising, and the significant effect on cholesterol is equally unexpected but highly rewarding. Thus, the data support the therapeutic use of the derivatives described herein in humans, even at low doses in single and / or multiple doses.

[0331] While certain features of the invention have been illustrated and described herein, many modifications and equivalents will occur to those skilled in the art. It is, therefore, to be understood that the claims are intended to cover all such modifications and equivalents as fall within the true spirit of the invention.

Claims

1. A derivative of IL-22 comprising a fatty acid covalently attached to the IL-22 protein for use in a method of treating a metabolic, intestinal and / or hepatic disease, disorder or condition, said method comprising subcutaneously administering said derivative of IL-22.

2. A derivative of IL-22 comprising a fatty acid covalently attached to the IL-22 protein for use in a method of treating a metabolic, intestinal and / or hepatic disease, disorder or condition, said method comprising intravenously administering said derivative of IL-22.

3. The fatty acid (i) a C12, C14, C16, C18 or C20 diacid; (ii) a C14, C16, or C18 diacid, and / or (iii) C18 diacid 3. A derivative for use in the method according to claim 1 or 2, wherein

4. A derivative for use in the method according to any one of claims 1 to 3, wherein said IL-22 protein is native mature human IL-22 (hIL-22, SEQ ID NO: 1) or a variant thereof.

5. The mutant is (i) substituted at positions 1, 6, 33, 35, 64, 95, and / or 106 of hIL-22; (ii) comprising a substitution in hIL-22 selected from the group consisting of A1C, H6C, A33C, N35Q, N64Q, R95C, and L106C; (iii) contains a Cys residue at position 1 of hIL-22; (iv) contains a Cys residue at position 95 of hIL-22; (v) containing a Cys residue at position 106 of hIL-22; (vi) has at least 10% sequence identity with hIL-22; and / or (vii) comprising one, two, three, four, five or more mutations in hIL-22, wherein said mutations are independently selected from the group consisting of deletions, substitutions and insertions. A derivative for use in the method of claim 4.

6. The mutant is (i) comprises an N-terminal peptide; (ii) comprises an N-terminal trimer; (iii) contains an N-terminal G; (iv) contains an N-terminal G-P-G; (v) comprising an N-terminal peptide of up to 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids; A derivative for use in the method of claim 4 or claim 5.

7. the fatty acid is covalently attached to the IL-22 protein by a linker, the linker comprising: (i) one or more amino acids, optionally including Glu and / or Lys; (ii) one or more oligo(ethylene glycol) (OEG) residues; (iii) ethylenediamine (C ２ D-A) group, (iv) an acetamide (Ac) group; (v) γGlu-OEG-OEG-C ２ DA-Ac, and / or (vi) γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc A derivative for use in the method according to any one of claims 1 to 6, comprising:

8. The linker is (i) a Cys residue in hIL-22 or a variant thereof; (ii) a substituted Cys residue at position 1, 6, 33, 95, or 106 of hIL-22; (iii) a Cys residue at position −7 relative to hIL-22; (iv) a substituted Cys residue at position 1 of hIL-22; (v) a substituted Cys residue at position 95 of hIL-22, and / or (vi) a substituted Cys residue at position 106 of hIL-22 8. A derivative for use in the method of claim 7, which is a Cys-reactive linker attached to

9. 9. A derivative for use in the method of any one of claims 1 to 8, wherein the derivative comprises a C18 diacid covalently attached by a linker to a variant of hIL-22, the variant comprising an N-terminal G-P-G and a Cys residue substituted at position 1 of hIL-22, the linker being attached to the Cys residue.

10. 9. A derivative for use in the method of any one of claims 1 to 8, wherein the derivative comprises a C18 diacid covalently attached by a linker to a variant of hIL-22, the variant comprising a substituted Cys residue at position 95 or 106 of hIL-22, and the linker is attached to the Cys residue.

11. The linker and fatty acid together form a compound of the following formula 1A, 1B or 1C, wherein: ＊ A derivative for use in the method of any one of claims 1 to 10, having a structure selected from: 【Chemistry 1】 【Chemistry 2】

12. (i) the variant has the sequence set forth in SEQ ID NO: 23, the linker is attached at position 1, and the linker and fatty acid together conform to formula 1A (derivative 1); (ii) the variant has the sequence set forth in SEQ ID NO: 24, the linker is attached at position -7, and the linker and fatty acid together conform to formula 1A (derivative 2); (iii) the variant has the sequence set forth in SEQ ID NO: 23, the linker is attached at position 1, and the linker and fatty acid together conform to formula 1B (derivative 3); (iv) the variant has the sequence set forth in SEQ ID NO: 24, the linker is attached at position -7, and the linker and fatty acid together conform to formula 1B (derivative 4); (v) the variant has the sequence set forth in SEQ ID NO: 23, the linker is attached at position 1, and the linker and fatty acid together conform to formula 1C (derivative 5); (vi) the variant has the sequence set forth in SEQ ID NO: 25, the linker is attached at position 1, and the linker and fatty acid together conform to formula 1A (derivative 6); (vii) the variant has the sequence set forth in SEQ ID NO: 26, the linker is attached at position 1, and the linker and fatty acid together conform to formula 1A (derivative 7); (viii) the variant has the sequence set forth in SEQ ID NO: 27, the linker is attached at position 6, and the linker and fatty acid together conform to formula 1A (derivative 8); (ix) the variant has the sequence set forth in SEQ ID NO: 28, the linker is attached at position 33, and the linker and fatty acid together conform to formula 1A (derivative 9); (x) the variant has the sequence set forth in SEQ ID NO: 25, the linker is attached at position 1, and the linker and fatty acid together conform to formula 1B (derivative 10); (xi) the variant has the sequence set forth in SEQ ID NO: 29, the linker is attached at position 106, and the linker and fatty acid together comply with formula 1A (derivative 11); (xii) the variant has the sequence set forth in SEQ ID NO: 30, the linker is attached at position 95, and the linker and fatty acid together comply with formula 1A (derivative 12); (xiii) said variant has the sequence set forth in SEQ ID NO: 31, said linker being attached at position 106, and said linker and fatty acid together conform to formula 1A (derivative 13), or (xiv) the variant has the sequence set forth in SEQ ID NO: 32, the linker is attached at position 95, and the linker and the fatty acid together comply with formula 1A (derivative 14); A derivative for use in the method of claim 11.

13. The derivative is 【Transformation 3】 【Chemistry 4】 12. A derivative for use in the method of claim 11, wherein:

14. (i) the metabolic disease, disorder, or condition is obesity, type 1 diabetes, type 2 diabetes, hyperlipidemia, hyperglycemia, or hyperinsulinemia; (ii) the liver disease, disorder, or condition is nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), cirrhosis, alcoholic hepatitis, acute liver failure, chronic liver failure, acute exacerbation of chronic liver failure (ACLF), acetaminophen-induced hepatotoxicity, acute liver injury, sclerosing cholangitis, biliary cirrhosis, or a condition caused by surgery or transplantation; or (iii) the intestinal disease, disorder or condition is inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, graft-versus-host disease (GvHD), chemical injury, viral infection, bacterial infection or short bowel disease; A derivative for use in the method according to any one of claims 1 to 13.

15. A derivative for use in the method according to any one of claims 1 to 14, wherein the recipient of said derivative of IL-22 has cardiovascular disease (CVD).

16. A derivative for use in the method according to any one of claims 1 to 15, wherein said method comprises administering said derivative of IL-22 subcutaneously by injection or intravenously by infusion.

17. The method comprises administering the derivative of IL-22 to a subject comprising: (i) 1 μg / kg to 500 μg / kg body weight, (ii) 1 μg / kg to 15 μg / kg body weight, (iii) 1 μg / kg to 10 μg / kg body weight, (iv) 1 μg / kg to 5 μg / kg body weight, (v) 1.25 μg / kg to 3 μg / kg body weight, (vi) 3 μg / kg to 100 μg / kg body weight, (vii) 5 μg / kg to 80 μg / kg body weight, (viii) 5 μg / kg to 15 μg / kg body weight, (ix) 10 μg / kg to 150 μg / kg body weight, (x) 20 μg / kg to 150 μg / kg body weight, (xi) 25 μg / kg to 300 μg / kg body weight, (xii) 30 μg / kg to 150 μg / kg body weight, (xiii) 50 μg / kg to 350 μg / kg body weight, (xiv) 100 μg / kg to 400 μg / kg body weight, and / or (xv) 200 μg / kg to 400 μg / kg body weight, wherein the weekly dose is about 2.5 μg / kg. A derivative for use in the method according to any one of claims 1 and 3 to 16.

18. The method comprises administering the derivative of IL-22 to a subject comprising: (i) 1 μg / kg to 400 μg / kg body weight, (ii) 1 μg / kg to 15 μg / kg body weight, (iii) 1 μg / kg to 10 μg / kg body weight, (iv) 1 μg / kg to 5 μg / kg body weight, (v) 1.25 μg / kg to 3 μg / kg body weight, (vi) 3 μg / kg to 100 μg / kg body weight, (vii) 5 μg / kg to 80 μg / kg body weight, (viii) 5 μg / kg to 15 μg / kg body weight, (ix) 10 μg / kg to 150 μg / kg body weight, (x) 20 μg / kg to 150 μg / kg body weight, (xi) 25 μg / kg to 300 μg / kg body weight, (xii) 30 μg / kg to 150 μg / kg body weight, (xiii) 50 μg / kg to 350 μg / kg body weight, (xiv) 100 μg / kg to 300 μg / kg body weight, and / or (xv) 150 μg / kg to 300 μg / kg body weight, wherein the weekly dose is about 2.5 μg / kg. A derivative for use in the method according to any one of claims 2 to 16.

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