Il-22 mutant, preparation method therefor, and use thereof

Abstract

An IL-22 mutant, a preparation method therefor, and a use thereof, relating to the technical field of biology. By mutating IL-22 and modifying the mutation site with PEG, the obtained mutant has good STAT3 / STAT1 preference, significantly reduced STAT1 signal activation, and significantly reduced inflammatory effects. The IL-22 mutant can preferentially act on intestinal target organs in vivo and is not prone to activating skin cells to produce adverse reactions.

Description

IL-22 mutant and its preparation method and application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on September 28, 2023, with application number 202311272942.0 and invention name “IL-22 mutants and their preparation methods and applications”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to an IL-22 mutant and a preparation method and application thereof. Background Art

[0003] IL-22, a member of the interleukin-10 family, has multiple biological functions and plays a key role in maintaining tissue homeostasis during inflammation and infection. IL-22 is produced by various immune cells at physiological barrier interfaces, including CD4+ T cells and ILC3s. It acts on epithelial cells, enhancing tissue integrity and promoting regeneration after injury. IL-22 exerts its effects in multiple organs, including the pancreas, digestive tract, and liver. Research results have shown that IL-22 has significant protective effects in multiple mouse disease models, including IBD, acute liver injury, and intestinal damage caused by GvHD. IL-22 maintains intestinal structural integrity by inducing the expression of genes involved in proliferation, wound healing, and apoptosis. IL-22 also induces innate antimicrobial molecules, including regenerative islet-derived (Reg) proteins, defensins, and S100 proteins, supporting intestinal barrier function. Therefore, the Reg3 molecule (Reg3A in humans and Reg3β in mice) has been widely used as an important pharmacodynamic marker (PD marker) for the treatment of intestinal diseases.

[0004] In addition to its organ-protective function, IL-22 also has potential pathogenic potential. IL-22 can activate inflammatory Th17 cells, exhibiting pro-inflammatory effects in the skin, liver, and digestive tract, leading to the expression of acute phase proteins such as SAA-1 / 2. Both animal and human studies have shown that IL-22 administration can significantly increase serum levels of the acute phase protein SAA. Therefore, SAA is often used as an important inflammation-related marker to evaluate the pro-inflammatory effects of IL-22 in nonclinical and clinical studies.

[0005] IL-22 activates downstream signaling pathways by binding to a heterodimeric receptor complex (IL-22Rα / IL-10Rβ). IL-10Rβ is widely distributed, while the expression of IL-22Rα is mainly limited to epithelial cells, which determines the cell specificity of IL-22. IL-22 binds to its receptor and mainly activates STAT1 and STAT3. Literature shows that the expression of genes related to organ protection and regeneration is mainly mediated by STAT3, and the activation of STAT1 is associated with a variety of pro-inflammatory effects. Therefore, obtaining an activation-preferential IL-22 molecule can significantly reduce its pro-inflammatory effects while maintaining its organ protection activity, which is an important research direction for IL-22-related treatments.

[0006] On the other hand, the expression abundance of IL-10Rβ varies significantly in different tissue cells, leading to differences in the STAT3 activation strength of IL-22 in different tissue cells. Literature reports that IL-22 mutants, through amino acid mutations that alter their affinity for IL-10Rβ, can better retain STAT3 activity in intestinal cells with high IL-10Rβ expression, while reducing STAT3 activity in cells with low IL-10Rβ expression, such as skin cells, thus potentially eliminating skin toxicity.

[0007] Some existing technologies significantly increase the half-life by fusing Fc to the IL22 molecule. However, Fc fusion has no effect on its activation preference, and it can still exhibit significant pro-inflammatory effects and non-target organ toxicity. Literature results show that IL22-Fc (UTTR14147A) has significant pro-inflammatory effects both in vivo and in vitro. Phase I clinical results show that UTTR14147A can cause a significant increase in serum SAA and CRP at low to high doses, and there are obvious adverse skin reactions, manifested as dry skin, desquamation, erythema, etc.

[0008] Other existing technologies use multi-site mutations to reduce STAT1 activation and increase its selectivity. However, the half-life of IL22 mutants is relatively short, similar to that of native IL22, and presents significant limitations in clinical application. A comparison of wt-IL22 and IL22-Fc revealed that the fusion protein was significantly less active than the original protein, suggesting significant uncertainty in the development of long-acting IL22 mutants.

[0009] Therefore, there is still an urgent need in this field to improve the preference of IL22 and reduce adverse reactions.

[0010] Summary of the Invention

[0011] In view of this, the technical problem to be solved by the present invention is to provide an IL22 mutant with activation preference and a preparation method and application thereof.

[0012] The IL-22 mutant provided by the present invention has at least one amino acid at positions 10 to 95 of the amino acid sequence shown in SEQ ID NO: 1 replaced by cysteine-PEG or acetylphenylalanine-PEG.

[0013] In the embodiments of the present invention, the number of mutation sites in the IL-22 mutant is 1, 2, or more, which is not limited by the present invention. In some embodiments, the mutation site is at least one of positions 10, 12, 15, 16, 17, 18, 21, 22, 64, 72, 75, 76, 79, 83, 84, 86, 87, 88, 91, or 95.

[0014] In some embodiments, the mutation site is any one of position 10, position 12, position 15, position 16, position 17, position 18, position 21, position 22, position 64, position 72, position 75, position 76, position 79, position 83, position 84, position 86, position 87, position 88, position 91 or position 95.

[0015] In some specific embodiments, the mutation sites are: D10pAcF, S12pAcF, Q15pAcF, Q16pAcF, P17pAcF, Y18pAcF, N21pAcF, R22pAcF, N64pAcF, F72pAcF, S75pAcF, D76pAcF, Q79pAcF, Q83pAcF, E84pAcF, V86pAcF, P8pAcF, F88pAcF, R91pAcF, R95pAcF. Alternatively, the mutation site is P17C, D76C, P87C or F22C.

[0016] In the present invention, human interleukin-22 (wt-IL22, shown in SEQ ID NO: 1) is used as the starting molecule, and non-natural amino acids (pAcF) or cysteine ​​(Cys) are mutated at different sites, and then polyethylene glycol is modified at the mutated sites. The polyethylene glycol coupling site and PEG molecular weight are further optimized to obtain site-specifically coupled PEGylated interleukin-22 with strong preference and good in vivo efficacy. PEG30 refers to PEG with a molecular weight of 30K.

[0017] Among them, cysteine-PEG refers to cysteine ​​coupled to PEG, where the maleimide of PEG reacts with the sulfhydryl group of cysteine ​​to form a thioether bond; p-acetylphenylalanine-PEG refers to p-acetylphenylalanine coupled to PEG, where the hydroxylamine of PEG reacts with the carbonyl group of p-acetylphenylalanine to form an oxime bond. In the embodiments of the present invention, the molecular weight of PEG is 30kD to 80kD. For example, the molecular weight of PEG is 30kD, 40kD, 50kD, 60kD, 70kD, or 80kD.

[0018] In some embodiments, the mutants of the present invention are:

[0019] The Asp at position 10 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0020] The Ser at position 12 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0021] The Gln at position 15 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0022] The Gln at position 16 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0023] The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0024] The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG40;

[0025] The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG40;

[0026] The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG60;

[0027] The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG80;

[0028] The Tyr at position 18 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0029] The Asn at position 21 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0030] The Arg at position 22 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0031] The Asn at position 64 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0032] The Phe at position 72 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0033] The amino acid sequence shown in SEQ ID NO: 1, wherein Ser at position 75 is replaced with p-acetylphenylalanine-PEG30;

[0034] The Asp at position 76 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0035] The Asp at position 76 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG40;

[0036] The Asp at position 76 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG60;

[0037] The Asp at position 76 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG80;

[0038] The Gln at position 79 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0039] The Gln at position 83 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0040] The Glu at position 84 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0041] The amino acid sequence shown in SEQ ID NO: 1, wherein Val at position 86 is replaced with p-acetylphenylalanine-PEG30;

[0042] The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0043] The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG40;

[0044] The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG40;

[0045] The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG60;

[0046] The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG80;

[0047] The Phe at position 88 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0048] The Phe at position 88 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG40;

[0049] The Arg at position 91 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0050] The Arg at position 95 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by p-acetylphenylalanine-PEG30.

[0051] In some embodiments of the present invention, a tag is further connected to the end of the mutant for ease of separation. For example, the C-terminus of the IL-22 mutant is further connected to a 6×his tag.

[0052] The site-specifically conjugated PEGylated interleukin-22 with activation preference provided by the present invention can enhance STAT3 / STAT1 activation preference compared to IL22-Fc, significantly reducing STAT1 signaling and pro-inflammatory effects. Furthermore, it can enhance tissue activation preference and reduce activation of cells with low IL-10Rβ receptor abundance, such as skin cells, potentially reducing adverse skin reactions.

[0053] The present invention also provides a biomaterial according to any one of the following I) to V):

[0054] 1), a nucleic acid encoding the IL-22 mutant as described above;

[0055] II), an expression unit containing the nucleic acid described in I);

[0056] III), an expression vector containing the nucleic acid described in I) or the expression unit described in II);

[0057] IV), a host containing the expression vector described in III);

[0058] V) A host having the nucleic acid described in I) or the expression unit described in II) integrated into its genome.

[0059] Furthermore, in the present invention, the preparation method of the IL-22 mutant is

[0060] The method comprises: replacing at least one amino acid in positions 10 to 95 of the amino acid sequence shown in SEQ ID NO: 1 with p-acetylphenylalanine, and reacting the mixture with PEG-hydroxylamine to obtain the IL-22 mutant;

[0061] Or it includes: replacing at least one amino acid in positions 10 to 95 of the amino acid sequence shown in SEQ ID NO: 1 with cysteine, and then reacting with PEG-maleimide to obtain the IL-22 mutant.

[0062] In the present invention, the catalysts for the reaction with PEG-hydroxylamine are ammonium chloride and acetylhydrazine.

[0063] In the present invention, the molar ratio of PEG-hydroxylamine to ammonium chloride, acetic hydrazide, and IL-22 mutant is 125:30:5:1; and the molar ratio of PEG-maleimide to IL-22 mutant is 5:1.

[0064] The method for replacing at least one amino acid at positions 10 to 95 of the amino acid sequence of SEQ ID NO: 1 with p-acetylphenylalanine comprises constructing an expression vector for the IL-22 mutant, co-transforming the vector with a helper plasmid capable of introducing pAcF into a host, and then expressing the vector. In the expression vector, the codon at the mutation site in the nucleic acid encoding the IL-22 mutant is replaced with an amber codon.

[0065] The method for replacing at least one amino acid at positions 10 to 95 of the amino acid sequence of SEQ ID NO: 1 with cysteine ​​comprises constructing an expression vector for the IL-22 mutant, transforming the vector into a host, and expressing the mutant. In the expression vector, the codon at the mutation site in the nucleic acid encoding the IL-22 mutant is replaced with a codon for Cys.

[0066] Furthermore, the present invention also provides the use of IL-22 mutants in the preparation of drugs for treating immune-related diseases.

[0067] In the present invention, the immune-related diseases include rheumatoid arthritis, insulin-dependent diabetes mellitus, atherosclerosis, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, hepatitis and fatty liver, multiple sclerosis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, systemic lupus erythematosus, graft-versus-host disease, ulcerative colitis, pancreatitis, psoriatic arthritis and diabetic foot ulcer.

[0068] In the present invention, the treatment includes increasing STAT3 / STAT1 activation preference and / or increasing tissue activation preference. The increasing STAT3 / STAT1 activation preference includes increasing STAT3-mediated physiological activity and / or reducing STAT1-mediated physiological activity, wherein the STAT3-mediated physiological activity includes the expression of organ protection and regeneration-related genes (e.g., Reg3, Muc1), and the STAT1-mediated physiological activity includes a variety of pro-inflammatory effects (e.g., inducing pro-inflammatory CXCL1 expression). The increasing tissue activation preference refers to preferentially exerting tissue protection in target organs such as the intestine and / or not causing or reducing inflammatory effects in non-target organ tissues such as the skin.

[0069] Furthermore, the present invention provides a pharmaceutical composition comprising the IL-22 mutant. The pharmaceutical composition of the present invention also includes a pharmaceutically acceptable excipient. The pharmaceutical dosage form of the present invention is an oral preparation or an injectable formulation, preferably an injectable formulation, including an injectable solution or an injectable powder. The pharmaceutically acceptable excipient includes a pH adjuster, an osmotic pressure regulator, a lyoprotectant, or a solvent.

[0070] The pharmaceutical composition of the present invention may further include other anti-inflammatory agents or drugs with immunomodulatory functions, including but not limited to topical and oral corticosteroids, such as prednisolone, methylprednisolone, budesonide; aminosalicylate, such as mesalamine, olsalazine; immunomodulators, such as azathioprine, 6-mercaptopurine, methotrexate, cyclosporine, IL-10, IL-11, IL-12, CD74 antagonists, CD40 antagonists, OX40L antagonists, GM-CSF, pimecrolimus or rapamycin; anti-TNFα drugs, such as infliximab, adalimumab, onacept, Etanercept; IL-12 / IL-23 antagonists such as ustekinumab, guselkumab, and risankizumab; anti-inflammatory drugs such as PDE-4 inhibitors, TACE inhibitors, ICE inhibitors, and IL-2 receptor antagonists such as daclizumab; selective adhesion molecule antagonists such as natalizumab; analgesics such as, but not limited to, COX-2 inhibitors such as rofecoxib and celecoxib; P / Q-type site-sensitive channel modulators such as gabapentin and pregabalin; NK-1 receptor antagonists; cannabinoid receptor modulators; and anti-cancer, anti-tumor, anti-angiogenic, or chemotherapeutic drugs. Such other modulators may be included in the same composition or administered separately.

[0071] Furthermore, the present invention also provides a method for treating immune-related diseases, which comprises administering the drug of the present invention. The administration method of the drug of the present invention includes oral administration or injection, preferably injection, and the injection includes intramuscular injection or intravenous injection.

[0072] Furthermore, the present invention also provides a method for changing the activation preference of the IL-22 receptor and reducing the toxicity of the IL-22 mutant, comprising:

[0073] Substituting amino acid 76 and / or amino acid 87 of IL-22 shown in SEQ ID NO: 1 with p-acetylphenylalanine or cysteine; and

[0074] The amino acid at position 76 and / or position 87 is site-specifically modified with PEG to obtain an IL-22 mutant.

[0075] In the method, the average molecular weight of the PEG is 30 kD to 80 kD, preferably 30 kD to 60 kD.

[0076] The present invention mutates IL-22 and modifies the mutation site with PEG. The resulting mutant has a favorable STAT3 / STAT1 preference, significantly reducing STAT1 signaling activation and pro-inflammatory effects. Furthermore, it preferentially targets the intestinal target organs in vivo and is less likely to activate skin cells and produce adverse reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] FIG1 shows the activity of PEG-modified IL22 non-natural amino acid mutants in activating STAT3 reporter genes at different concentrations;

[0078] FIG2 shows the ELISA assay for the activation of STAT3 phosphorylation in Colo205 cells by PEG-modified IL22 unnatural amino acid mutants at different concentrations;

[0079] FIG3 shows the ELISA assay for the activation of STAT1 phosphorylation in Colo205 cells by PEG-modified IL22 unnatural amino acid mutants at different concentrations;

[0080] FIG4 shows the FACs assay for the activation of STAT3 phosphorylation in HT29 cells by PEG-modified IL22 unnatural amino acid mutants at different concentrations;

[0081] FIG5 shows the FACs assay for the activation of STAT1 phosphorylation in HT29 cells by PEG-modified IL22 unnatural amino acid mutants at different concentrations;

[0082] Figure 6 shows the FACs assay to detect the activity of PEG-modified IL22 cysteine ​​mutants in activating STAT3 phosphorylation in HT29 cells at different concentrations;

[0083] FIG7 shows the FACs assay to detect the activity of PEG-modified IL22 cysteine ​​mutants in activating STAT1 phosphorylation in HT29 cells at different concentrations;

[0084] FIG8 shows the FACs assay to detect the activity of PEG-modified IL22 mutants in activating STAT3 phosphorylation in HaCaT cells at different concentrations;

[0085] FIG9 shows the concentration-time curve of Reg3β in mouse plasma after a single administration of PEG-modified IL22 mutants;

[0086] FIG10 shows the concentration-time curve of Reg3β in mouse plasma after a single administration of PEG-modified IL22 mutants;

[0087] FIG11 shows the concentration-time curve of SAA in mouse plasma after a single administration of PEG-modified IL22 mutants;

[0088] Figure 12 shows the drug-dose curve of PEG-modified IL22 mutant mice after a single dose;

[0089] FIG13 shows the body weight curve of mice after administration of DSS-induced enteritis model;

[0090] FIG14 shows the DAI scores of the mouse DSS-induced enteritis model after administration;

[0091] FIG15 shows the endpoint colon length after administration in the DSS-induced enteritis model in mice;

[0092] FIG16 shows the colon histological scores at the end point after administration of the DSS-induced enteritis model in mice;

[0093] FIG17 shows the colon pathological section (G1) at the end point after administration of the DSS-induced enteritis model in mice;

[0094] FIG18 shows the colon pathological section (G4) at the end point after administration of the DSS-induced enteritis model in mice;

[0095] FIG19 shows the curve of increasing ear thickness in mice. DETAILED DESCRIPTION

[0096] The present invention provides IL22 mutants and their preparation and use. Those skilled in the art can draw upon the present disclosure and appropriately modify process parameters to achieve these results. It is particularly important to note that all similar substitutions and modifications readily apparent to those skilled in the art are considered encompassed by the present invention. The methods and uses of the present invention have been described using preferred embodiments. It is apparent that those skilled in the art will be able to modify, adapt, and combine these methods and uses to implement and apply the present technology without departing from the present disclosure, spirit, and scope.

[0097] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0098] In this application, the term "and / or" describes the association relationship between associated objects, indicating that three relationships may exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0099] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0100] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that other elements may be present in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0101] The term "nucleic acid" herein refers to a nucleic acid encoding IL-22, which may be single-stranded or double-stranded, and may be DNA, RNA, cDNA, or PNA. The nucleic acid may include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid may be linear or circular in topology. The nucleic acid may be, for example, part of a vector (e.g., an expression or cloning vector), or a fragment or a combination of multiple fragments. The nucleic acid may be obtained directly from a natural source, or may be prepared with the assistance of recombinant, enzymatic, or chemical techniques. The RNA form may be mRNA obtained by gene transcription, etc. The sequence of the nucleic acid described in the present invention may be a wild-type sequence, or may be codon-optimized according to the host, which is not limited by the present invention, and its implementation is within the scope of protection of the present invention.

[0102] The term "expression unit" herein includes the nucleic acid, promoter, and terminator of the present invention. In order to regulate the expression level or protein folding, the expression unit may also include other elements.

[0103] The term "vector" herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors." In the present invention, the vectors are used for amplification and preservation of nucleic acids, or for expression of mutants in a host. The vectors described in the present invention include a backbone and a nucleic acid. Specifically, the backbone may be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, although this is not a limitation of the present invention.

[0104] The term "host cell" herein refers to a cell into which an exogenous nucleic acid is introduced, including the offspring of such a cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as that screened or selected in the initially transformed cells are included herein. In the embodiments of the present invention, a prokaryotic expression system is used, in particular, Escherichia coli is used as an example to express the mutant. In addition, other feasible expression systems, such as yeast expression systems, insect cell expression systems, etc., can also be used for the preparation of the mutant, and the present invention is not limited to this.

[0105] The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as cancer and tumors. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease, as well as subjects susceptible to a condition or disease, or subjects intending to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0106] The subject of the term "administered" herein refers to an organism that receives treatment for a specific disease or condition as described herein. Exemplarily, the subject receiving treatment for a disease or condition is a mammal, such as a human, a primate (e.g., a monkey), or a non-primate mammal.

[0107] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0108] The present invention relates to sequences comprising:

[0109] Amino acid sequence of human IL22:

[0110] The amino acid sequence of the control IL22 mutant hB3-his (underlined amino acids are mutated amino acids):

[0111] Amino acid sequence of IL22-Fc:

[0112] The nucleic acid sequence of wt-IL22 after optimization of E. coli codon preference:

[0113] The nucleic acid sequence of hB3-his after optimization of E. coli codon preference:

[0114] The mutants described in the present invention are mutated based on human IL-22, and the human IL-22 may include a signal peptide or may not include a signal peptide. The test substances involved in the embodiments of the present invention do not contain a signal peptide. The signal peptide contains a total of 33 amino acid residues, and its amino acid sequence is MAALQKSVSSFLMGTLATSCLLLLALLVQGGAA (SEQ ID NO: 7). The coupling sites or mutation sites involved in the claims or description of the present invention are all counted based on the sequence of IL-22 (SEQ ID NO: 1) without a signal peptide. For example, the mutation site D10pAcF refers to replacing the D (i.e., Asp) at position 10 of the amino acid sequence shown in SEQ ID NO: 1 with pAcF.

[0115] To maintain consistency with the original experimental records, the mutants were named using the position numbering within the IL-22 protein containing the signal peptide. That is, if the residue position in the amino acid sequence of SEQ ID NO:1 is designated as X, then the corresponding amino acid residue position in the protein containing the signal peptide of SEQ ID NO:7 is X+33. In the Examples of the present invention, the mutation sites described in Tables 1-18 and Figures 1-19 are numbered based on the corresponding position in SEQ ID NO:1, while the mutant names are numbered based on the corresponding amino acid position in the protein containing the signal peptide. For example, the mutant corresponding to the mutation site D10pAcF is named IL22-hB43-his. This mutation site is located at position 10 in the protein sequence without the signal peptide, and at position 10+33 (i.e., position 43) in the protein sequence containing the signal peptide.

[0116] The naming convention for the names of the PEG-modified mutants in the examples is: PEG MW-PEG-IL-22-amino acid substitution, for example, 80k-PEG-IL-22-D109C means that one PEG molecule with a molecular weight of 80k is conjugated to the substituted cysteine ​​residue at position 76 of the IL-22 molecule (76 is the position of cysteine ​​in the protein sequence without a signal peptide, 76+33=109, so the position is recorded as 109 in the naming).

[0117] The site numbers in the names of the mutants of the present invention are merely for reference and do not indicate that the prepared mutants contain a signal peptide. None of the test substances described in the examples of the present invention contain the signal peptide set forth in SEQ ID NO:7. The test materials used in the present invention are all commercially available products. The present invention will be further described below with reference to the examples:

[0118] Comparative Example 1

[0119] 1) Cloning, expression and purification of native wt-IL22 (SEQ ID NO. 1) in E. coli.

[0120] Based on the protein sequence A34-I179 of mature human IL-22 (Uniprot: Q9GZX6), a cDNA sequence encoding wt-IL-22 (SEQ ID NO: 4) was designed according to the codon preference of E. coli. The start codon ATG and an EcoRI restriction site were added to the 5' end of the cDNA, and three stop codons and a BamHI restriction site were added to the 3' end. The cDNA was prepared by DNA synthesis and then cloned into the expression vector pBV220KR1. The expression vector containing the corresponding cDNA sequence was transformed into DH5α competent cells (TaKaRa) using the calcium chloride method. Positive clones were picked from kanamycin-resistant plates and verified by sequencing as the expression strain for wt-IL-22.

[0121] The wt-IL22 expression strain was inoculated into TB medium and cultured at 35°C, 250 rpm in a shaker until the logarithmic growth phase. The temperature was then raised to 42°C to induce expression for 4 hours. After the incubation period, the cells were harvested by centrifugation at 13,900 g. The cells were washed and resuspended in 50 mM Tris-HCl, 5 mM EDTA, pH 8.0 buffer, disrupted at 700-1000 bar, and centrifuged at 13,900 g to harvest inclusion bodies.

[0122] Wash the inclusion bodies with 50mM Tris, 5mM EDTA, 0.5% DOC, 100mM NaCl, 1mM DTT, pH 8.0, and repeat the wash. Wash once with 50mM Tris, 5mM EDTA, pH 8.0. Resuspend the inclusion bodies in 7M guanidine hydrochloride, 50mM Tris, pH 8.0, disperse at high speed, and stir at 2-8°C for 4 hours. Dilute the protein solution 20-fold with renaturation buffer containing 0.5M arginine, 100mM Tris, 1mM GSH, 0.1mM GSSG, pH 8.0, and incubate at 2-8°C for at least 48 hours. Concentrate the renaturation solution by ultrafiltration and exchange it with 5% trehalose, 10mM NaAc, pH 4.5. Filter and perform ion exchange chromatography using CM Sepharose FF (Cytiva). After equilibration with 3-5 column volumes of 50 mM NaAc, pH 5.0 buffer, the protein solution was loaded onto the chromatography column. After loading, the column was rinsed with 50 mM NaAc, pH 5.0 buffer, pre-washed with 2.5 column volumes of 50 mM NaAc, 250 mM NaCl, pH 5.0, and then equilibrated with 3-5 column volumes of 50 mM NaAc, pH 5.0 buffer, and eluted with 50 mM NaAc, 400 mM NaCl, pH 5.0, and the eluate was collected.

[0123] Comparative Example 2 Cloning of Control IL22 Mutant (hB3-His, SEQ ID NO: 2)

[0124] Referring to the IL22 mutant 22-B3 in the literature (WO2021212056 and The tissue protective functions of interleukin-22 can be decoupled from pro-inflammatory actions through structure-based design. Immunity.2021; 54(4): 660-672.e9.), wt-IL22 was subjected to S12E, Q83A, R91A, and R95A amino acid mutations, and a 6*his tag was added to the C-terminus. According to the codon preference of Escherichia coli, a cDNA sequence encoding hB3-his was designed (SEQ ID NO: 5), and the start codon ATG and NdeI restriction site were added to the 5' end of the cDNA, and the stop codon and EcoRI restriction site were added to the 3' end. The cDNA was prepared by DNA synthesis and then cloned into the expression vector pET30a(+). The expression vector containing the corresponding cDNA sequence was used to transform BL21 (DE3) competent cells (Invitrogen) using the calcium chloride method, and positive clones were picked from the kanamycin-resistant plates and used as expression strains for each mutant protein after sequencing verification.

[0125] The hB3-his expression strain was inoculated into autoinduction medium containing 0.2% lactose and cultured overnight at 37°C, 250 rpm in a shaker to express each mutant protein. The cells were harvested by centrifugation at 13,900 g. The cells were washed and resuspended in 50 mM Tris-HCl, 5 mM EDTA, pH 8.0 buffer, disrupted at 700-1000 bar, and the inclusion bodies were harvested by centrifugation at 13,900 g.

[0126] The inclusion bodies were washed sequentially with 50 mM Tris, 5 mM EDTA, 0.5% DOC, 100 mM NaCl, 1 mM DTT, pH 8.0 buffer and 50 mM Tris, 5 mM EDTA, pH 8.0 buffer, and then solubilized with 7 M guanidine hydrochloride, 50 mM Tris, pH 8.0 buffer. The protein solution was diluted 20-fold with 0.5 M arginine, 100 mM Tris, 1 mM GSH, 0.1 mM GSSG, pH 8.0 refolding buffer and allowed to stand at 2-8°C for at least 48 h. The refolding solution was then concentrated by ultrafiltration to 5% trehalose, 10 mM NaAc, pH 4.5, filtered, and subjected to ion exchange chromatography using CM Sepharose FF (Cytiva). After equilibration with 3-5 column volumes of 40 mM NaAc, pH 5.2 buffer, the protein solution was loaded onto the chromatography column. After loading, the column was rinsed with 40 mM NaAc, pH 5.2 buffer. Elution was performed using a 0-1 M NaCl gradient with 10 column volumes of 40 mM NaAc, 1 M NaCl, pH 5.2, and the eluate was collected. The collected eluate was ultrafiltered and loaded onto a Superdex 75pg (120 ml, Cytiva) for chromatography to collect the target protein peak.

[0127] Comparative Example 3 Cloning, expression and purification of IL22-Fc (code: RG7880, SEQ ID NO: 3).

[0128] The RG7880 plasmid (synthetic gene from Beijing Qingke Biotechnology Co., Ltd.) was transfected into CHO-K1 cells. 24 h after transfection, 10 μg / mL MSX was added for pressure screening. After the cell density and viability were restored, the cells were inoculated for feed-batch expression. The centrifugal supernatant after expression was purified sequentially by protein A, Capto adhere, and Phenyl HP hydrophobic chromatography. The antibody concentration was quantified by the BCA assay.

[0129] Example 1 Cloning of the unnatural amino acid IL22 mutant (6*His) in E. coli

[0130] According to the mutation sites shown in Table 1, the parental amino acid codon at each position in wt-IL22 (SEQ ID NO: 1) was replaced with the amber codon TAG (so that the amino acid at the corresponding position mutated to p-acetylphenylalanine). To add a 6*his tag to the C-terminus, CATCACCACCACCACCAC (SEQ ID NO: 6) was added to the C-terminus of the nucleic acid sequence (SEQ ID NO: 4) optimized based on the codon preference of E. coli. cDNAs encoding the corresponding IL22 mutants with each codon mutation listed in Table 1 were designed. The start codon ATG and NdeI restriction site were added to the 5' end of the cDNA, and the stop codon and XhoI restriction site were added to the 3' end. The cDNAs were prepared by DNA synthesis and then cloned into the expression vector pET30a(+). The calcium chloride method was used to co-transform BL21(DE3) competent cells (Invitrogen) with the expression vector pET30a(+) containing the corresponding cDNA sequence and the helper plasmid pEVOL-pAcF, which can specifically introduce p-acetylphenylalanine (pAcF). Positive clones were picked from kanamycin and chloramphenicol double-resistant plates and used as expression strains for each mutant protein after sequencing verification.

[0131] Table 1 List of IL22 mutants

[0132] The strain expressing each unnatural amino acid mutant protein was inoculated into autoinduction medium containing 1 mM p-acetylphenylalanine, 0.02% arabinose, and 0.2% lactose. The mutant protein was cultured overnight at 37°C in a shaker at 250 rpm to express the mutant protein. The cells were harvested by centrifugation at 13,900 g. The cells were washed and resuspended in 50 mM Tris-HCl, 5 mM EDTA, pH 8.0 buffer, disrupted at 700-1000 bar, and the inclusion bodies were harvested by centrifugation at 13,900 g.

[0133] The inclusion bodies were washed with wash buffer containing 0.5% DOC, and the precipitate was collected by centrifugation. The crude inclusion bodies were dissolved in 50mM MES, 8M Urea, 10mM EDTA, 0.1mM DTT, pH 5.5 buffer, and the supernatant was collected by centrifugation. The supernatant was added to pre-chilled refolding buffer (100mM Tris, 2mM EDTA, 0.5M L-Arg, 1mM GSH, 0.1mM GSSG, pH 8.0) at a ratio of 1:20 (v / v) and allowed to stand at 2-8°C for approximately 72 hours. The refolding buffer was concentrated by ultrafiltration into 10mM NaAc, 5% trehalose, pH 4.5 buffer, filtered, and then subjected to ion exchange chromatography using CM Sepharose FF (Cytiva). The buffers used for CM Sepharose FF ion exchange chromatography are as follows: Buffer A: 20 mM MES, pH 6.0; Buffer B: 20 ​​mM MES, 1 M NaCl, pH 6.0. The column is first equilibrated with 3 to 5 column volumes of Buffer A. The refolding solution is then loaded onto the column. After loading, the column is rinsed with Buffer A and then gradient eluted with 10 column volumes of 0 to 100% Buffer B. The purified sample is then collected.

[0134] Example 2 Unlabeled non-natural amino acid mutants of IL22

[0135] According to the mutation sites shown in Table 2, the parental amino acid codon at each site of wt-IL22 was replaced with the amber codon TAG (so that the amino acid at the corresponding site mutated to p-acetylphenylalanine). According to the codon preference of Escherichia coli, a cDNA encoding the corresponding IL22 mutant with each codon mutation in Table 2 was designed. The start codon ATG and an NdeI restriction site were added to the 5' end of the cDNA, and three stop codons and an XhoI restriction site were added to the 3' end. The cDNA was prepared by DNA synthesis and then cloned into the expression vector pET30a(+). The calcium chloride method was used to co-transform BL21(DE3) competent cells (Invitrogen) with the expression vector pET30a(+) containing the corresponding cDNA sequence and the helper plasmid pEVOL-pAcF, which can specifically introduce p-acetylphenylalanine (pAcF). Positive clones were picked from kanamycin and chloramphenicol double-resistant plates and used as expression strains for each mutant protein after sequencing verification.

[0136] Table 2. List of IL22 unnatural amino acid mutants

[0137] The strain expressing each IL22-pAcF mutant protein was inoculated into autoinduction medium containing 1 mM p-acetylphenylalanine, 0.02% arabinose, and 0.2% lactose. The mutant protein was expressed overnight at 37°C in a shaker at 250 rpm, and the cells were harvested by centrifugation at 13,900 g. The cells were washed and resuspended in 50 mM Tris-HCl, 5 mM EDTA, pH 8.0 buffer, disrupted at 700-1000 bar, and the inclusion bodies were harvested by centrifugation at 13,900 g.

[0138] Inclusion bodies were washed sequentially with 2M urea, 2% Triton X-100, 50mM Tris, 5mM EDTA, pH 8.0 buffer, and then 50mM Tris, 5mM EDTA, pH 8.0 buffer. The inclusion bodies were solubilized with 7M guanidine hydrochloride, 50mM Tris, pH 8.0. The protein solution was diluted 20-fold with renaturation buffer containing 0.5M arginine, 100mM Tris, 1mM GSH, and 0.1mM GSSG, pH 8.0, and allowed to stand at 2-8°C for 48 hours. The renaturation solution was concentrated by ultrafiltration into 10mM NaAc, 5% trehalose, pH 4.5 buffer, filtered, and subjected to ion exchange chromatography using Capto SP ImpRes (Cytiva). After equilibration with 3-5 column volumes of 20 mM NaAc, pH 5.5 buffer, the protein solution was loaded onto the chromatography column. After loading, the column was rinsed with 20 mM NaAc, pH 5.5 buffer, and gradient eluted with 10 column volumes of 20 mM NaAc, 1 M NaCl, pH 5.5 from 0 to 1 M NaC, and the eluate was collected.

[0139] Example 3 Polyethylene glycol modification and purification of IL22 non-natural amino acid mutants.

[0140] The purified proteins from Examples 1 and 2 were ultrafiltrated into 10 mM NaAc, 5% trehalose, pH 4.5, to a final concentration of 5-10 mg / ml. Ammonium chloride was added at a molar ratio of 1:125 (IL22: ammonium chloride); acetylhydrazine was added at a molar ratio of 1:30 (IL22: acetylhydrazine); and PEG-hydroxylamine (e.g., Mw 30k, 40 kDa, Xiamen Sinobond) was added at a molar ratio of 1:5 (IL22: PEG). The reaction was allowed to react at 2-8°C for approximately 48-60 hours to modify the PEG at the mutation site of each mutant. The reaction solution was diluted 10-fold with purified water, the pH adjusted to 5.5, and ion exchange chromatography was performed using Macro Cap SP (Cytiva). After equilibration with 3-5 column volumes of 40 mM NaAc, pH 5.5 buffer, the protein solution was loaded onto the chromatography column. After loading, the column was rinsed with 40 mM NaAc, pH 5.5 buffer and gradient eluted using 0-0.5 M NaCl, 40 mM NaAc, pH 5.5 buffer to collect the modified protein.

[0141] Example 4 Cloning, expression and purification of IL22 cysteine ​​mutants in Escherichia coli.

[0142] According to the mutation sites shown in Table 3 (with SEQ ID NO: 1 as the wild type), the parent amino acid at each site was replaced with cysteine ​​(Cystine, abbreviated as Cys). According to the codon preference of E. coli, cDNAs encoding the corresponding IL22 mutants with each codon mutation in Table 3 were designed. The start codon ATG and an EcoRI restriction site were added to the 5' end of the cDNA, and three stop codons and a BamHI restriction site were added to the 3' end. The cDNA was prepared by DNA synthesis and then cloned into the expression vector pBV220KR1. The expression vector containing the corresponding cDNA sequence was transformed into DH5α competent cells (TaKaRa) using the calcium chloride method. Positive clones were picked from kanamycin-resistant plates and used as expression strains for each mutant protein after sequencing verification.

[0143] Table 3 List of IL22 cysteine ​​mutants

[0144] The strain expressing each IL22 cysteine ​​mutant protein was inoculated into TB medium and cultured at 35°C, 250 rpm in a shaker until the logarithmic growth phase. The temperature was then raised to 42°C to induce expression for 4 hours. After the culture period, the cells were harvested by centrifugation at 13,900 g. The cells were washed and resuspended in 50 mM Tris-HCl, 5 mM EDTA, pH 8.0 buffer, disrupted at 700-1000 bar, and centrifuged at 13,900 g to harvest inclusion bodies.

[0145] Inclusion bodies were washed sequentially with 50 mM Tris, 5 mM EDTA, 2% Triton X-100, pH 8.0 buffer and then 50 mM Tris, 5 mM EDTA, pH 8.0 buffer. After solubilization with 8 M urea, 50 mM MES, 0.1 mM DTT, 10 mM EDTA, pH 5.5, the protein solution was diluted 20-fold with 0.5 M arginine, 100 mM Tris, 1 mM GSH, 0.1 mM GSSG, pH 8.0 refolding buffer and incubated at 2-8°C for 48 h. The refolding solution was concentrated by ultrafiltration into 10 mM NaAc, 5% trehalose, pH 4.5 buffer, filtered, and subjected to ion exchange chromatography using CM Sepharose FF (Cytiva). After equilibration with 3-5 column volumes of 40 mM NaAc, pH 5.1 buffer, the protein solution was loaded onto the chromatography column. After loading, the column was rinsed with 40 mM NaAc, pH 5.1 buffer, and gradient eluted with 10 column volumes of 40 mM NaAc, 1 M NaCl, pH 5.1 at a rate of 0-100%, and the eluate was collected.

[0146] Example 5 Polyethylene glycol modification and purification of IL22 cysteine ​​mutants

[0147] Each IL-22 cysteine ​​mutant obtained in Example 4 was modified with polyethylene glycol. The PEG used in this example contains a maleimide group, which can react specifically with the cysteine ​​residue at the specific mutation site of the mutant, thereby allowing PEG to site-specifically modify the corresponding IL-2 mutant.

[0148] The purified IL-22 cysteine ​​mutant protein was adjusted to pH 6.0. PEG-maleimide (e.g., PEG with Mw 40k, 60k, or 80kDa, available from Xiamen Sinobond) was weighed at a molar ratio of 1:5. The PEG powder was dissolved in purified water to a final concentration of 100 mg / ml and then added to the protein solution. The mixture was mixed and allowed to react overnight at 2-8°C.

[0149] The reaction solution was diluted 10-fold with purified water, the pH adjusted to 5.1, and ion exchange chromatography was performed using a Macro Cap SP (Cytiva). After equilibration with 3-5 column volumes of 40 mM NaAc, pH 5.1 buffer, the protein solution was loaded onto the column. After loading, the column was rinsed with 40 mM NaAc, pH 5.1 buffer and gradient eluted using 0-0.5 M NaCl, 40 mM NaAc, pH 5.1 buffer to collect the modified protein.

[0150] Table 4 List of test substance information of PEG-IL2 cysteine ​​mutants

[0151] Effect verification

[0152] 1. Evaluation of STAT3 activation activity of PEG-IL22 mutants at different modification sites using reporter gene assay

[0153] COLO205-SIE cells were constructed to test the activation effect of each test substance on STAT3.

[0154] Specific method: COLO205 cells were cultured in RPMI1640 + 10% FBS in a culture dish until sufficient volume was obtained. TM COLO205 cells were transfected with the pGL4.47[luc2P / SIE / Hygro] (Promega) plasmid using an electroporation transfection system (Invitrogen) to obtain COLO205 / SIE cells, which were then resuspended in RPMI1640 + 10% FBS to a volume of 1 × 10 6 50 μl / well of the IL22 sample was added to a 96-well plate and incubated overnight at 37°C, 5% CO2 for approximately 16 hours. IL22 samples were serially diluted and added at 50 μl / well. After incubation at 37°C, 5% CO2 for 6 hours, 100 μl / well of the Bio-Lite Luciferase Assay System (Vazyme, Cat: DD1201-02) was added and chemiluminescence was measured. The logarithmic value of the final IL22 sample concentration was plotted on the X-axis, and the assay results for each well were plotted on the Y-axis. A regression curve for IL22-stimulated STAT3 phosphorylation reporter gene activity in COLO205 / SIE cells was obtained using a four-parameter regression fit in GraphPad Prism 9 software.

[0155] According to the results of STAT3 reporter gene assay in COLO205-SIE cells, the cells with better STAT3 phosphorylation activity (EC 50 The molecules with lower STAT3 / STAT1 preference were then used for subsequent STAT3 / STAT1 preference studies.

[0156] Table 5 Reporter gene assay to detect STAT3 activity of PEG-IL22 mutants in EC 50

[0157] The activity of the PEG-modified IL22 non-natural amino acid mutants prepared in Example 3 in activating the STAT3 reporter gene at different concentrations is shown in FIG1 .

[0158] 2. COLO205STAT3 / STAT1 Preference

[0159] The ELISA method was used to screen molecules with potential preferential advantages in the activation ability of p-STAT1 / p-STAT3 in COLO205 cells.

[0160] COLO205 cells were cultured in RPMI1640 + 10% FBS in a culture dish until sufficient volume was obtained. An appropriate amount of cell suspension was centrifuged, washed twice with PBS, and resuspended to 8 × 10 5 Cells / ml, 50 μl / well were added to a 96-well plate, incubated at 37°C, 5% CO2 for 6 hours, IL22 samples were serially diluted, 50 μl / well were added thereto, incubated at 37°C, 5% CO2 for 0.5 hour, and the 96-well plate was placed on ice for 5-10 minutes. The cell lysis buffer in the kit was prepared into 2×, 100 μl / well were added to the above 96-well plate, and placed at 4°C or on ice for 5-10 minutes. The 96-well plate was then transferred to -80°C, stored for not less than 30 minutes, and then thawed at room temperature.

[0161] STAT1 kit ( Phospho-Stat1 (Tyr701) Sandwich ELISA Kit, Manufacturer: CST, Cat: 73582), STAT3 kit ( After the microwell strips (Phospho-Stat3 (Tyr705) Sandwich ELISA Antibody Pair, Manufacturer: CST, Cat: 7300) have reached room temperature, break off the desired microwells and place them in the strip rack. Unused microwells must be resealed and immediately stored at 4°C.

[0162] Add 90 μl of cell lysate from each well to the appropriate microplate wells provided with the STAT1 or STAT3 kit. Seal the wells with sealing film and press firmly onto the top of the wells. Incubate the plate at 37°C for 2 hours, or overnight at 4°C.

[0163] Discard the contents of the plate and wash four times with 1× wash buffer, 200 μl per well. Between washes, tap the plate against unused absorbent paper to remove any residual solution from each well, but never allow the wells to dry completely. Add 100 μl of the reconstituted detection antibody to each well. Seal the plate with sealing film and incubate at 37°C for 1 hour, repeating the wash procedure. Add 100 μl of the reconstituted HRP-conjugated secondary antibody to each well. Seal with tape and incubate the plate at 37°C for 30 minutes, repeating the wash procedure. Add 100 μl of TMB substrate to each well. Seal the plate with sealing film and incubate the plate at 37°C for 10 minutes or at 25°C for 30 minutes. Add 100 μl of STOP solution to each well and shake gently for a few seconds. Read at 450 nm on a microplate reader within 30 minutes of adding the STOP solution. The final concentration of IL22 sample was taken as the Log value on the X-axis, and the detection results of each well were taken as the Y-axis. The regression curve of STAT1 and STAT3 phosphorylation activity of COLO205 cells stimulated by IL22 was obtained by four-parameter regression fitting in GraphPad Prism9 software.

[0164] ECs using activated STAT3 and STAT1 phosphorylation 50 The STAT3 / STAT1 preference was evaluated by the ratio of 30K-PEG-IL22-hB50 and 30K-PEG-IL22-hB120. The results of COLO205 STAT3 / STAT1 preference showed that among all the tested substances, 30K-PEG-IL22-hB50 and 30K-PEG-IL22-hB120 had a better preference for STAT3 / STAT1 activation than wt-IL22. 50 The preference is lower than the first two molecules, but its STAT1 phosphorylation activation Emax is lower, suggesting that it has low activation ability even at high concentrations.

[0165] Table 6 ELISA detection of STAT1 / STAT3 activity of PEG-IL22 mutants in EC 50

[0166] In addition, the ELISA assay for the activation of STAT3 phosphorylation in COLO205 cells by the polyethylene glycol-modified IL22 non-natural amino acid mutants at different concentrations is shown in Figure 2 , and the ELISA assay for the activation of STAT1 phosphorylation in COLO205 cells by the polyethylene glycol-modified IL22 non-natural amino acid mutants at different concentrations is shown in Figure 3 .

[0167] 3. Effects of different molecular weight polyethylene glycol modifications on IL22 receptor preference

[0168] Using the untagged IL22 non-natural amino acid mutants prepared in Example 5, PEG-modified IL22 mutant products with different molecular weights were prepared according to the method of Example 6, as shown in the following table:

[0169] Table 7 PEG-IL22 non-natural amino acid mutant test substance information

[0170] FACs were used to examine the preferential phosphorylation of pSTAT1 and pSTAT3 receptors by PEG-modified IL22 in adherent human colon cancer cell line HT-29. hB3-his and IL22-Fc were used as control molecules.

[0171] The day before the experiment, 2 × 10 5 Cells were incubated overnight at 37°C, 5% CO2 to allow attachment. Different test substances were serially diluted in McCoy's cell culture medium containing 10% FBS, and 100 μL / well was added. The cells were incubated at 37°C, 5% CO2 for 30 minutes. The cells were washed once with 100 μL / well PBS, discarded by aspiration, and digested with 25 μL / well trypsin (Gibco, 25200-560) at 37°C for approximately 5 minutes to form a suspended cell suspension. The cells were gently tapped to disperse and neutralized with 80 μL / well McCoy's cell culture medium containing 10% FBS. The suspension was then homogenized by vortexing to form a single-cell suspension. 100 μL of the suspension was transferred to a V-bottom plate and centrifuged. Resuspend the cells in 100 μL / well of preheated 1× fixative (BD bioscience, 558049) or 4% paraformaldehyde (Sigma-Aldrich, 158127-5G) at 37°C for 15–20 minutes to fix them. After centrifugation, aspirate the fixative and disperse the cells with the remaining solution. Then, thoroughly resuspend the cells in 80 μL / well of -20°C pre-chilled permeabilization solution (BD bioscience, 558050). Permeabilize the cells on ice for 0.5–1 hour, gently tapping 2–3 times to prevent cell clumping. Wash the cells thoroughly with PBS and centrifuge them. Flow cytometry antibodies (anti-STAT1 pY701, BD bioscience 612596 and anti-STAT3 pY705, BD bioscience 557815) were added and incubated in the dark for 30–60 minutes. After washing again, the cells were analyzed by flow cytometry. The MFI values ​​corresponding to FITC and APC were determined for each sample.

[0172] The final concentration of each sample was taken as the Log value on the X-axis, and the MFI result of each well was taken as the Y-axis. The regression curve of STAT1 / STAT3 phosphorylation activity of HT-29 cells stimulated by IL22-related test substances was obtained by four-parameter regression fitting in GraphPad Prism 9 software.

[0173] The FACs assay was used to detect the activity of PEG-modified IL22 non-natural amino acid mutants in activating STAT3 phosphorylation in HT29 cells at different concentrations, as shown in Figure 4 . The FACs assay was used to detect the activity of PEG-modified IL22 non-natural amino acid mutants in activating STAT1 phosphorylation in HT29 cells at different concentrations, as shown in Figure 5 .

[0174] Compared to IL22-Fc and hB3-His molecules, all 40k-PEG-modified IL-22 mutants significantly reduced STAT3 phosphorylation activity, far below that of IL22-Fc. 30k-PEG-modified IL-22 mutants retained significant STAT3 phosphorylation activity and exhibited enhanced preference for IL22-Fc. Compared to IL22-Fc, 30k-PEG-IL22-hB120 increased STAT3 phosphorylation activity by approximately 20-fold while maintaining comparable STAT1 phosphorylation activity. However, 30k-PEG-IL22-hB50 and 30k-PEG-IL22-hB121 significantly reduced STAT1 phosphorylation activity while maintaining comparable STAT3 activity.

[0175] Table 8 FACs assay for the activation of STAT1 / STAT3 phosphorylation preference by PEG-IL22 unnatural amino acid mutants

[0176] 4. FACs detection of pSTAT1 and pSTAT3 activation preference

[0177] Following the same method as in item 3 above, the receptor preference of PEG-modified IL22 was tested using the same FACs. hB3-his and IL22-Fc were used as control molecules.

[0178] Table 9 FACs assay for the phosphorylation preference of PEG-IL22 cysteine ​​mutants in activating STAT1 / STAT3

[0179] The FACs assay was used to detect the activity of PEG-modified IL22 cysteine ​​mutants in activating STAT3 phosphorylation in HT29 cells at different concentrations, as shown in Figure 6 . The FACs assay was used to detect the activity of PEG-modified IL22 cysteine ​​mutants in activating STAT1 phosphorylation in HT29 cells at different concentrations, as shown in Figure 7 .

[0180] Among the IL22 molecules conjugated to cysteine ​​polyethylene glycol, PEG-IL22-D109C and PEG-IL22-P120C molecules showed a preference advantage over IL22-Fc. 40k / 60k-PEG-IL22-D109C activated STAT1 / STAT3 EC in HT29 cells.50 The ratio was significantly higher than that of IL22-Fc and the control molecule hB3-his, while PEG-IL22-P120C not only 50 The ratio shows an advantage, and its STAT1 activity curve has a lower Emax than that of IL22-Fc, suggesting that the level of STAT1 activation is weaker at high concentrations.

[0181] 5. Tissue Activation Preference

[0182] Human immortalized epidermal HaCaT cells were revived and cultured in MEM / EBSS medium (Cytiva, SH30024.01) containing 15% FBS and 1% P / S (Cytiva, SV30010) until the cell confluence reached 70%-80%. The cells were then digested with 0.25% Trypsin-EDTA (Gibco, 25200-056) and terminated with complete medium. The cells were then resuspended to 1.25×10 6 Cells were plated at a density of 1.5 × 10 cells / ml. 120 μl / well of the mixed cells were plated into a 96-well plate (Corning, 3599). 5 The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0183] The working concentration of wt-IL22 was 1000nM, and the working concentration of the other samples was 10000nM in the first well. Except for 60K-PEG-IL22-D109C which was diluted 5-fold in a complete medium, the other samples were diluted 4-fold in a complete medium. 120μl / well of each gradient dilution sample was added to the cell plate after overnight culture and the medium was aspirated, and placed in a 37°C, 5% CO2 incubator for stimulation for 30 minutes. After the effect was over, the sample was aspirated and washed once with 1×PBS (HyClone, SH30256.01) preheated to 37°C. After drying, 25μl / well of 0.25% Trypsin-EDTA was added for digestion, and 75μl / well of complete medium was added to terminate the reaction and the cells were pipetted and mixed until single cells were obtained. 80 μl of the single-cell suspension was pipetted and mixed into a 96-well V-bottom plate (NEST, 701201) containing 80 μl / well of 4% paraformaldehyde (Sigma-Aldrich, 158127-5G) solution. The cells were placed at room temperature for 20 min, centrifuged at 1800 rpm for 5 min, and the supernatant was removed. 100 μl / well of pre-chilled methanol (Merck, 11.06007.4008) was added to permeabilize the membrane. The cells were placed at 4°C for 1 h, centrifuged to remove the supernatant, and washed once with 1× PBS. The prepared STAT3 antibody (Alex Fluor647 mouse anti-Stat3 (pY705), BD, 557815) was added for staining. After staining for 1 h at room temperature in the dark, the cells were washed twice with 1× PBS, and the mean fluorescence intensity (MFI) of the channel was measured by flow cytometry.

[0184] The tissue activation preferences of the different test compounds were assessed by comparing their STAT3 activation activity in intestinal HT29 cells and skin-derived HaCaT cells. The results show that compared to IL22-Fc, 30k-PEG-IL22-hB120, 60k-PEG-IL22-D109C, and 60k-PEG-IL22-P120C all exhibited superior tissue preference, preferentially activating intestinal HT29 cells while relatively weakly activating skin-derived HaCaT cells. This suggests that they preferentially target the intestinal tract in vivo and are less likely to activate skin cells and cause adverse reactions. The FACs assay for STAT3 phosphorylation activation in HaCaT cells at different concentrations of PEG-modified IL22 mutants is shown in Figure 8.

[0185] Table 10 STAT3 phosphorylation preference of PEG-IL22 mutants in activating human intestinal cell lines and human skin cell lines detected by FACs assay

[0186] 6. Mouse PD (Reg3β)

[0187] The ELISA method was used to determine the baseline and post-dose change trends of the biomarker Reg3β in plasma samples before and after administration of different test substances.

[0188] For the preferred test substances, one mouse was injected subcutaneously at each dose of 0.5 / 1 / 3 mg / kg. The control molecules were IL22-Fc, injected subcutaneously at 0.3 and 1 mg / kg, and wt IL-22, injected intraperitoneally at the corresponding doses.

[0189] Approximately 40 μL of orbital blood was collected one day before administration and 4, 24, 48, 96, 144, 192, 264, 336, and 408 hours after administration, and the blood was anticoagulated and the plasma separated. ELISA plates were coated with 1 μg / mL anti-Reg3β (Sino Biological, 51153-R065) dissolved in PBS overnight at 4°C. A serial dilution of mouse Reg3β (Sino Biological, 51153-M08H) was used as a positive control standard curve. Plasma samples were diluted appropriately to fit within the linear range of the control. After blocking, the plates were loaded and incubated for 2 h. Secondary antibodies, sheep anti-mouse Reg3β (R&D systems, AF5110-SP) and HRP-conjugated antibodies (HRP-Rabbit anti-sheep IgG H&L, Abcam, Ab97130) were then incubated for coupling. After a final wash, tetramethylbenzidine peroxidase substrate (TMB, Surmodics, TMBS-1000-01) was added for color development for 10 min. The reaction was terminated with 2 M sulfuric acid and the plate was read at 450 nm using a microplate reader with a reference wavelength of 630 nm. The initial Reg3β concentration in each sample was calculated. The concentration was plotted against time (Figure 9).

[0190] The results showed that all test substances activated Reg3β expression to a certain extent. Among them, wt-IL-22 at different doses had a weaker activation effect on Reg3β, with no significant dose-dependent enhancement. Compared with IL22-Fc at similar (0.5 mg / kg vs. 0.3 mg / kg) or the same concentration (1 mg / kg), 60k-PEG-IL22-D109C, 60k-PEG-IL22-P120C, and 40k-PEG-IL22-hB120 exhibited higher peak stimulatory concentrations for Reg3β.

[0191] Table 11 Peak plasma Reg3β concentrations in mice after single administration of PEG-modified IL22 mutants

[0192] 7. Mouse PD Preference Study (Reg3β and SAA)

[0193] Based on the in vitro preference results and preliminary PD screening results, the molecules 60k-PEG-IL22-D109C and 60k-PEG-IL22-P120C were selected. PD testing was performed in three mice at different doses, using the same method as in Example 14. The pharmacodynamically relevant molecule Reg3β and the inflammation-associated molecule SAA were also tested. IL22-Fc was used as a control. Blood samples were collected before dosing and at 4, 24, 48, 72, 96, 144, 192, 264, and 336 hours post-dose, followed by anticoagulation and plasma separation. For the control molecules wt-IL22 and hB3-his, blood samples were collected only up to 96 hours.

[0194] The Reg3β detection method was carried out according to Example 14, and the initial concentration of Reg3β in each sample was calculated. The concentration was plotted against time.

[0195] The specific SAA detection method is as follows: SAA concentrations in appropriately diluted plasma samples were determined using a kit containing a detection antibody pair and a positive control (Abcam, Ab216059). The procedure was performed according to the kit instructions. Sa-HRP (Pierce 21126) was used for coupling. After washing, tetramethylbenzidine peroxidase substrate (TMB, Surmodics, TMBS-1000-01) was added for color development for 10 minutes. The reaction was terminated with 2M sulfuric acid, and the plate was read at 450 nm with a 630 nm reference using a microplate reader. SAA detection was performed before administration and at 24, 48, 72, 96, and 144 hours after administration. The initial SAA concentration in each sample was calculated. Concentration was plotted against time (Figures 10-11).

[0196] Results of a mouse PD preference study demonstrated that wt-IL22 and hB3-his stimulated mice to produce significantly lower levels of Reg3β than the other test compounds. For different doses of candidate test compounds, under conditions of equivalent or superior Reg3β Cmax (e.g., 60K-PEG-IL22-D109C 0.1mg / kg vs. 60K-PEG-IL22-P120C 0.3mg / kg vs. IL22-Fc 1mg / kg), the site-specifically conjugated PEG-IL22 test compound activated SAA at a significantly lower Cmax than IL22-Fc, suggesting a superior non-inflammatory preference, capable of maintaining organ protective activity while significantly reducing proinflammatory effects.

[0197] Table 12 Dosing information for mouse PD preference study (Reg3β and SAA)

[0198] 8. Mouse PK

[0199] Balb / C male mice (4 mice per group) received a single subcutaneous injection of 1 mg / kg. Blood samples were collected before dosing and 4 (not for 60k-PEG-IL22-D109C and 60k-PEG-IL22-P120C), 8, 24, 24, 48, 72, 96, and 120 hours after dosing for anticoagulation and plasma separation.

[0200] The ELISA method was used to quantify each test substance in plasma. Anti-IL22 Antibody (Sino Biological, 13059-MM24) 2 μg / mL dissolved in PBS was used for coating. The standard curves of each test substance and plasma samples were diluted to the linear range of detection, and detection was performed using Recombinant Anti-IL22 Antibody (Sino Biological, 13059-R034-H). The original concentration of the test substance in each sample was calculated from the four-parameter fit of the standard curve of each test substance. The plasma pharmacokinetic parameters of each group of test substance mice were analyzed (Figure 12).

[0201] Table 13 Dosing information for mouse PK study

[0202] The pharmacokinetic parameters of each test substance are analyzed as follows

[0203] Table 14 PK pharmacokinetic parameters of each test substance in mice

[0204] 9. Mouse DSS-induced enteritis efficacy model

[0205] Balb / C mice were used to test the efficacy of the drug in the DSS mouse acute colitis model. DSS (Dextran Sulfate Sodium Salt, MW: 36,000-50,000) can damage intestinal epithelial cells and induce acute inflammatory bowel disease (IBD) in mice when added to drinking water at a certain ratio. Therefore, the potential pharmacological effects of the test compound can be evaluated using the 4% DSS-induced mouse IBD model.

[0206] Balb / C male mice were allowed to freely drink drinking water containing 4% DSS until the average body weight of the model group dropped to about 15%. Fresh drinking water was then replaced and the recovery period was 2 days. The body weight of the mice and the severity of individual loose stools / bloody stools were monitored throughout the process (Figure 13), and a comprehensive DAI (disease activity index) assessment was performed (Figure 14). At the endpoint, the length of the colon of each mouse was measured (Figure 15), photographed, rolled into a Swiss roll, and fixed with 4% paraformaldehyde. After dehydration and embedding, the sections were processed into paraffin and stained with HE (Figures 17-18), and the tissue morphology was histologically scored (Figure 16).

[0207] The dosing schedule for the test substance is as follows (the day of modeling is D1, the day before is D-1, and so on).

[0208] Table 15 Dosing information for the study of DSS-induced enteritis model in mice

[0209] Table 16 DAI scoring criteria

[0210] Paraffin sections of colonic tissue were stained with hematoxylin and eosin (HE) and pathologically analyzed. Scoring criteria for the severity of lesions were as follows: very mild (1 point), mild (2 points), moderate (3 points), or severe (4 points). Lesion area percentage (%) was scored as ≥50% (4 points), 25%-50% (3 points), 10%-25% (2 points), and ≤10% (1 point). Inflammation depth was scored as follows: epithelium (1 point), lamina propria (2 points), and muscularis mucosa (3 points). No lesion was scored as "-." The sum of the scores for these three dimensions was used.

[0211] Tissue sections in the model group (G1) showed severe and extensive structural destruction of the colonic mucosa, loss of intestinal epithelium and crypts, and severe and extensive inflammatory cell infiltration and fibrosis in the mucosa and submucosa. In the 0.1 mg / kg 60k-PEG-IL22-P120C group (G4), colonic tissue morphology was relatively intact, with minimal structural destruction of the colonic mucosa and no significant inflammatory cell infiltration or fibrosis.

[0212] The results of the study showed that the test substance 60k-PEG-IL22-P120C had a clear efficacy in the mouse DSS acute enteritis model. Based on the analysis of body weight, DAI, colon length and histological scores, 0.1 mg / kg 60k-PEG-IL22-P120C (G4 group) was more effective than 0.03 mg / kg IL22-Fc (G6 group).

[0213] 10. Mouse ear injection swelling model

[0214] Balb / C mice were used for the ear skin inflammation induction test. The test substance was injected into the ear continuously and the changes in ear swelling thickness after injection were measured to reflect the induction of local inflammatory response of the test substance on the skin.

[0215] On days 0, 1, 2, and 4, 5 μL of PBS solution containing the test substances at the following concentrations was injected into each ear of the mice. Ear thickness was measured daily using a micrometer screw, and the delta thickness increase on each day after injection relative to the ear thickness on day 0 was calculated.

[0216] Table 17 Dosing information for mouse ear swelling model

[0217] Table 18 Peak increase in ear thickness of mice after administration

[0218] The results showed that intraauricular injection of various doses of each test substance in mice resulted in a dose-dependent increase in ear thickness (Figure 18). The equivalent dose of 60k-PEG-IL22-P120C to induce an increase in overall ear thickness was approximately 8 times that of IL22-Fc (1.6 μg vs. 0.2 μg). Combined with the results of the mouse efficacy dose (3-fold, 0.1 mg / kg vs. 0.03 mg / kg), this suggests that 60k-PEG-IL22-P120C has a lower potential risk of skin toxicity than IL22-Fc.

[0219] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An IL-22 mutant, wherein at least one of the amino acids at positions 10 to 95 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with cysteine-PEG or p-acetylphenylalanine-PEG.

2. The IL-22 mutant according to claim 1, characterized in that The mutation site is selected from at least one of position 10, position 12, position 15, position 16, position 17, position 18, position 21, position 22, position 64, position 72, position 75, position 76, position 79, position 83, position 84, position 86, position 87, position 88, position 91 or position 95.

3. The IL-22 mutant according to claim 1, characterized in that The average molecular weight of PEG is 30 kD to 80 kD, preferably 30 kD to 60 kD.

4. The IL-22 mutant according to claim 1, characterized in that Having at least one of the following mutations: The Asp at position 10 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Ser at position 12 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Gln at position 15 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Gln at position 16 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG40; The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG40; The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG60; The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG80; The Tyr at position 18 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Asn at position 21 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Arg at position 22 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Asn at position 64 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Phe at position 72 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Ser at position 75 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Asp at position 76 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Asp at position 76 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG40; The Asp at position 76 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG60; The Asp at position 76 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG80; The Gln at position 79 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Gln at position 83 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Glu at position 84 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Val at position 86 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG40; The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG40; The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG60; The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG80; The Phe at position 88 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Phe at position 88 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG40; The Arg at position 91 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30; The Arg at position 95 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by p-acetylphenylalanine-PEG30.

5. A biological material according to any one of the following I) to V): I), a nucleic acid encoding the IL-22 mutant as described above; II), an expression unit containing the nucleic acid described in I); III), an expression vector containing the nucleic acid described in I) or the expression unit described in II); IV), a host containing the expression vector described in III); V) A host having the nucleic acid described in I) or the expression unit described in II) integrated into its genome.

6. The method for preparing the IL-22 mutant according to any one of claims 1 to 4, include: Substituting at least one amino acid in positions 10 to 95 of the amino acid sequence shown in SEQ ID NO: 1 with p-acetylphenylalanine, and then reacting with PEG-hydroxylamine to obtain the mutant according to any one of claims 1 to 4; Or comprising: replacing at least one amino acid in positions 10 to 95 of the amino acid sequence shown in SEQ ID NO: 1 with cysteine, and then reacting with PEG-maleimide to obtain the mutant according to any one of claims 1 to 4.

7. The preparation method according to claim 6, characterized in that: The catalysts for the reaction with PEG-hydroxylamine are ammonium chloride and acetylhydrazine.

8. A pharmaceutical composition comprising the IL-22 mutant according to any one of claims 1 to 4.

9. Use of the IL-22 mutant according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 8 in the preparation of a drug for treating immune-related diseases.

10. The use according to claim 9, characterized in that: The immune-related diseases include at least one of rheumatoid arthritis, psoriasis and psoriatic arthritis, vitiligo, alopecia areata, dystrophic epidermolysis bullosa, systemic lupus erythematosus, insulin-dependent diabetes mellitus, diabetic foot ulcers, Crohn's disease, ulcerative colitis, atherosclerosis, hemolytic anemia, rheumatic fever, thyroiditis, myasthenia gravis, glomerulonephritis, pancreatitis, hepatitis and fatty liver, multiple sclerosis or graft-versus-host disease.

11. A method for changing the activation preference of IL-22 receptor and reducing the toxicity of IL-22 mutants, comprising: Substituting the amino acid at position 76 and / or position 87 of IL-22 shown in SEQ ID NO: 1 with acetylphenylalanine or cysteine; as well as The substituted amino acids were site-specifically modified using PEG to obtain IL-22 mutants.

12. The method according to claim 11, characterized in that The average molecular weight of the PEG is 30 kD to 80 kD, preferably 30 kD to 60 kD.

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