IL-22 mutant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an IL22 mutant as well as a preparation method and application thereof. According to the invention, IL-22 is mutated, and the mutation site is modified with PEG, so that the obtained mutant has good STAT3 / STAT1 preference, the STAT1 signal activation is significantly reduced, and the proinflammatory effect is significantly reduced. In addition, the compound can preferentially act on target organs of intestinal tracts in vivo, and does not easily activate skin cells to generate adverse reactions.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to IL-22 mutants and preparation methods and applications thereof. Background Art

[0002] IL-22 is a member of the interleukin 10 family and has multiple biological functions. It plays a key role in maintaining tissue homeostasis during inflammatory infection. IL-22 is produced by a variety of immune cells at the physiological barrier interface, including CD4+T cells, ILC3s, etc. It acts on epithelial cells, enhancing tissue integrity and promoting injury regeneration. IL-22 can exert effects in multiple organs, including the pancreas, digestive tract, liver, etc. Research results show that IL-22 has a significant protective effect in multiple mouse disease models, including IBD models, acute liver injury, and intestinal damage caused by GvHD. IL-22 maintains the structural integrity of the intestine by inducing the expression of genes related to proliferation, wound healing, and apoptosis. At the same time, IL-22 can also induce innate antibacterial molecules, including regenerative islet-derived (Reg) proteins, defensins, and S100 proteins, to support intestinal barrier function. Therefore, Reg3 molecules (Reg3A in humans and Reg3β in mice) are widely used in various studies as important pharmacodynamic markers (PD markers) of IL-22 in the treatment of intestinal diseases.

[0003] In addition to its organ protection function, IL-22 also has some potential pathogenic potential. IL-22 can activate inflammatory Th17 cells, exhibit pro-inflammatory effects in the skin, liver, and digestive tract, and cause the expression of acute phase proteins such as SAA-1 / 2. Both animal experiments and human studies have shown that IL-22 administration can significantly increase the concentration of serum acute phase protein SAA, so SAA is often used as an important inflammation-related marker to evaluate the pro-inflammatory effect of IL-22 in non-clinical and clinical studies.

[0004] IL-22 activates downstream signaling pathways by binding to heterodimeric receptor complexes (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 the 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 activation-preferential IL-22 molecules can significantly reduce their pro-inflammatory effects while maintaining their organ protection activity, which is an important research direction for IL-22-related treatments.

[0005] On the other hand, the expression abundance of IL-10Rβ in different tissue cells is significantly different, resulting in differences in the intensity of STAT3 activation by IL-22 in different tissue cells. It has been reported that IL-22 mutants change their affinity for IL-10Rβ through amino acid mutations, which can better retain STAT3 activity in intestinal cells with high IL-10Rβ expression abundance, while reducing STAT3 activity in skin cells with low IL-10Rβ abundance, thus having the potential to eliminate skin toxicity.

[0006] 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 show obvious pro-inflammatory effects and non-target organ toxicity. Literature results show that IL22-Fc (UTTR14147A) has obvious pro-inflammatory effects 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.

[0007] Other existing technologies use multi-site mutations to reduce the STAT1 activation effect and increase preference. However, the half-life of IL22 mutants is relatively short, similar to natural IL22, and there are obvious defects in clinical application. By comparing wt-IL22 and IL22-Fc, it was found that the activity of the fusion protein was significantly lower than that of the prototype protein, suggesting that there is obvious uncertainty in the long-term transformation of IL22 mutants.

[0008] Therefore, there is still an urgent need in this field to improve the preference of IL22 and reduce adverse reactions. Summary of the invention

[0009] 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.

[0010] 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.

[0011] In an embodiment of the present invention, the number of mutation sites of the IL-22 mutant is 1, 2 or more, and the present invention is not limited to this. In some embodiments, the mutation site is at least one of the 10th, 12th, 15th, 16th, 17th, 18th, 21st, 22nd, 64th, 72nd, 75th, 76th, 79th, 83rd, 84th, 86th, 87th, 88th, 91st or 95th positions.

[0012] In some embodiments, the mutation site is any one of position 10, 12, 15, 16, 17, 18, 21, 22, 64, 72, 75, 76, 79, 83, 84, 86, 87, 88, 91 or 95.

[0013] In some specific embodiments, the mutation site is: D10pAcF, S12pAcF, Q15pAcF, Q16pAcF, P17pAcF, Y18pAcF, N21pAcF, R22pAcF, N64pAcF, F72pAcF, S75pAcF, D76pAcF, Q79pAcF, Q83pAcF, E84pAcF, V86pAcF, P8 pAcF, F88pAcF, R91pAcF, R95pAcF. Alternatively, the mutation site is P17C, D76C, P87C or F22C.

[0014] 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. And further by optimizing the polyethylene glycol coupling site and PEG molecular weight, it is confirmed that the site-specific coupling PEGylated interleukin-22 with strong preference and good in vivo efficacy is obtained. PEG30 refers to PEG with a molecular weight of 30K

[0015] Among them, cysteine-PEG refers to cysteine ​​coupled to PEG, and the maleimide of PEG reacts with the sulfhydryl group of cysteine ​​and is connected through a thioether bond; p-acetylphenylalanine-PEG refers to p-acetylphenylalanine coupled to PEG, and the hydroxylamine of PEG reacts with the carbonyl group of p-acetylphenylalanine and is connected through an oxime bond. In an embodiment 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.

[0016] In some embodiments, the mutants described in the present invention are:

[0017] The Asp at position 10 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0018] The Ser at position 12 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0019] The Gln at position 15 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0020] The Gln at position 16 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0021] The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0022] The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG40;

[0023] The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG40;

[0024] The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG60;

[0025] The Pro at position 17 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG80;

[0026] The Tyr at position 18 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0027] The Asn at position 21 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0028] The Arg at position 22 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0029] The Asn at position 64 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0030] The Phe at position 72 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0031] The Ser at position 75 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0032] The Asp at position 76 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0033] The Asp at position 76 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG40;

[0034] The Asp at position 76 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG60;

[0035] The Asp at position 76 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG80;

[0036] The Gln at position 79 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0037] The Gln at position 83 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0038] The Glu at position 84 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0039] The Val at position 86 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0040] The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0041] The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG40;

[0042] The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG40;

[0043] The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG60;

[0044] The Pro at position 87 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by cysteine-PEG80;

[0045] The Phe at position 88 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0046] The Phe at position 88 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG40;

[0047] The Arg at position 91 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with p-acetylphenylalanine-PEG30;

[0048] The Arg at position 95 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by p-acetylphenylalanine-PEG30.

[0049] In some embodiments of the present invention, a tag is also connected to the end of the mutant for the convenience of separation. For example, the C-terminus of the IL-22 mutant is also connected to a 6×his tag.

[0050] The activation-preferred site-specific coupled PEGylated interleukin-22 provided by the present invention can improve the activation preference of STAT3 / STAT1 compared with IL22-Fc, significantly reduce the activation of STAT1 signal, and significantly reduce the pro-inflammatory effect. In addition, it can improve the tissue activation preference, reduce the activation ability of cells with low abundance of IL-10Rβ receptors, such as skin cells, and has the potential to reduce adverse skin reactions.

[0051] The present invention also provides a biomaterial according to any one of the following I) to V):

[0052] I), a nucleic acid encoding the IL-22 mutant as described above;

[0053] II), an expression unit containing the nucleic acid described in I);

[0054] III), an expression vector containing the nucleic acid described in I) or the expression unit described in II);

[0055] IV), a host containing the expression vector described in III);

[0056] V) A host having the nucleic acid described in I) or the expression unit described in II) integrated into its genome.

[0057] Furthermore, in the present invention, the method for preparing the IL-22 mutant is:

[0058] 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 with PEG-hydroxylamine to obtain the IL-22 mutant;

[0059] 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.

[0060] In the present invention, the catalysts for the reaction with PEG-hydroxylamine are ammonium chloride and acetylhydrazine.

[0061] In the present invention, the molar ratio of PEG-hydroxylamine to ammonium chloride, acetic hydrazide and IL-22 mutant is 125:30:5:1; the molar ratio of PEG-maleimide to IL-22 mutant is 5:1.

[0062] The method for replacing at least one amino acid at positions 10 to 95 of the amino acid sequence shown in SEQ ID NO: 1 with p-acetylphenylalanine comprises: constructing an expression vector of the IL-22 mutant, co-transforming the host with an auxiliary plasmid capable of introducing pAcF, and then expressing the vector. In the expression vector, in the nucleic acid encoding the IL-22 mutant, the codon at the mutation site is replaced with an amber codon.

[0063] The method for replacing at least one amino acid at positions 10 to 95 of the amino acid sequence shown in SEQ ID NO: 1 with cysteine ​​comprises: constructing an expression vector of the IL-22 mutant, transforming the host and expressing the mutant. In the expression vector, in the nucleic acid encoding the IL-22 mutant, the codon at the mutation site is replaced with a codon for Cys.

[0064] Furthermore, the present invention also provides the use of IL-22 mutants in the preparation of drugs for treating immune-related diseases.

[0065] 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.

[0066] 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.

[0067] Furthermore, the present invention also provides a pharmaceutical composition, which includes 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 injection, preferably an injection, which includes an injection liquid or an injection powder. The pharmaceutically acceptable excipient includes a pH regulator, an osmotic pressure regulator, a lyophilization protectant or a solvent.

[0068] The pharmaceutical composition of the present invention may further include other anti-inflammatory agents or drugs with immunomodulatory function, including but not limited to topical and oral corticosteroids, such as prednisolone, methylprednisolone, budesonide; aminosalicylate, such as mesalazine, 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, liscentizumab, 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, 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 agents or chemotherapeutic drugs. Such other modulators can be included in the same composition or administered separately.

[0069] Furthermore, the present invention also provides a method for treating immune-related diseases, which is to administer 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.

[0070] Furthermore, the present invention also provides a method for changing the activation preference of IL-22 receptor and reducing the toxicity of IL-22 mutants, comprising:

[0071] Substituting the amino acid at position 76 and / or position 87 of IL-22 shown in SEQ ID NO: 1 with acetylphenylalanine or cysteine; and

[0072] The amino acid at position 76 and / or position 87 is site-specifically modified with PEG to obtain an IL-22 mutant.

[0073] In the method, the average molecular weight of the PEG is 30 kD to 80 kD, preferably 30 kD to 60 kD.

[0074] The present invention mutates IL-22 and modifies PEG at the mutation site, and the obtained mutant has good STAT3 / STAT1 preference, significantly reduces STAT1 signal activation, and significantly reduces the pro-inflammatory effect. In addition, it can preferentially act on intestinal target organs in vivo and is not easy to activate skin cells and produce adverse reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 The results show that the PEG-modified IL22 non-natural amino acid mutants can activate the STAT3 reporter gene at different concentrations;

[0076] Figure 2 The ELISA method was used to detect the activity of PEG-modified IL22 non-natural amino acid mutants in activating STAT3 phosphorylation in Colo205 cells at different concentrations;

[0077] Figure 3 The ELISA method was used to detect the activity of PEG-modified IL22 non-natural amino acid mutants in activating STAT1 phosphorylation in Colo205 cells at different concentrations;

[0078] Figure 4 The FACs method 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;

[0079] Figure 5 The FACs method 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;

[0080] Figure 6 The FACs method was used to detect the activity of PEG-modified IL22 cysteine ​​mutants in activating STAT3 phosphorylation in HT29 cells at different concentrations;

[0081] Figure 7 The FACs method was used to detect the activity of PEG-modified IL22 cysteine ​​mutants in activating STAT1 phosphorylation in HT29 cells at different concentrations;

[0082] Figure 8 FACs method was used to detect the activity of PEG-modified IL22 mutants in activating STAT3 phosphorylation in HaCaT cells at different concentrations;

[0083] Fig. 9 The concentration-time curve of mouse plasma Reg3β after a single administration of PEG-modified IL22 mutant is shown;

[0084] Fig.10 The concentration-time curve of mouse plasma Reg3β after a single administration of PEG-modified IL22 mutant is shown;

[0085] Fig.11 The concentration-time curve of SAA in mouse plasma after a single administration of PEG-modified IL22 mutants is shown;

[0086] Fig.12 Shown is the drug-time curve of PEG-modified IL22 mutant mice after single administration;

[0087] Fig.13 Shows the body weight curve of the mouse DSS-induced enteritis drug efficacy model after administration;

[0088] Fig.14 Shows the DAI score after administration of the drug efficacy model of DSS-induced enteritis in mice;

[0089] Fig.15 The endpoint colon length after administration of the DSS-induced enteritis drug efficacy model in mice;

[0090] Fig.16 The end-point colon histology score of the mouse DSS-induced enteritis pharmacodynamic model after administration is shown;

[0091] Fig.17 The colon pathological sections (G1) of the mouse DSS-induced enteritis model after administration are shown;

[0092] Fig.18 The colon pathological sections (G4) of the mouse DSS-induced enteritis model after administration are shown;

[0093] Fig.19 The curve of increasing ear thickness of mice is shown. DETAILED DESCRIPTION

[0094] The present invention provides IL22 mutants and preparation methods and applications thereof. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0095] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0096] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0097] In this application, "at least one" means one or more, "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.

[0098] The terms "include", "comprising" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein also provides a "consisting of..." solution.

[0099] 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, a part of a vector (e.g., an expression or cloning vector), or a combination of one or more fragments. The nucleic acid may be obtained directly from a natural source, or may be prepared with the assistance of recombination, enzymatic or chemical techniques. The RNA form is 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.

[0100] The term "expression unit" herein includes the nucleic acid, promoter and terminator described in the present invention. In order to regulate the expression level or protein folding, the expression unit may also include other elements.

[0101] The term "vector" herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is connected. The term includes vectors that are self-replicating nucleic acid structures and vectors that are integrated 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 connected. 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 is derived from plants, animals, bacteria, fungi, bacteriophages or viruses, and the present invention is not limited to this.

[0102] The term "host cell" herein refers to a cell into which 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 parent cells in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initially transformed cells are included herein. In the embodiments of the present invention, a prokaryotic expression system is used, particularly Escherichia coli is used as an example to express mutants. 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 mutants, and the present invention is not limited to this.

[0103] The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions 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 deterioration), 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 who already have a disorder or disease, as well as subjects who are susceptible to a disorder or disease or subjects who intend to prevent a disorder 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.

[0104] 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.

[0105] 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, and 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.

[0106] The present invention relates to sequences comprising:

[0107] Amino acid sequence of human IL22:

[0108] APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI (denoted as wt-IL22 in the embodiment, the sequence is shown in SEQ ID NO.1)

[0109] The amino acid sequence of the control IL22 mutant hB3-his (the underlined amino acids are the mutated amino acids):

[0110] APISSHCRLDK E NFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYM A EVVPFLA A LSN A LSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACIHHHHHH (denoted as hB3-his in the examples, the sequence is shown in SEQ ID NO: 2)

[0111] Amino acid sequence of IL22-Fc:

[0112] APISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACIRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:3)

[0113] The nucleic acid sequence of wt-IL22 after optimization of E. coli codon preference:

[0114] GCTCCCATAAGTTCACACTGTAGGCTAGATAAGTCCAACTTCCAACAGCCGTACATTACCAATCGCACGTTTATGCTGGCGAAAGAAGCGAGCTTGGCGGACAACAACACCGATGTTCGTCTTATCGGCGAGAAGTTGTTTCATGGTGTGAGCATGTCTGAACGCTGCTATCTGATGAAACAGGTGTTGAATTTCACTCTGGAAGAAGTTCTGTTCCCGCA GAGCGATCGTTTCAACCATACATGCAAGAAGTCGTGCCGTTTCTGGCTCGTTTATCTAACCGCCTGTCGACCTGTCATATCGAGGGCGACGACCTGCATATCCAGCGTAATGTTCAGAAACTGAAGGACACCGTAAAAAAGCTGGGTGAGAGCGGTGAGATCAAAGCAATTGGCGAGCTGGATTTGCTGTTCATGTCGCTGCGTAACGCCTGCATT(SEQ ID NO: 4).

[0115] The nucleic acid sequence of hB3-his after optimization of E. coli codon preference:

[0116] GCTCCCATAAGTTCACACTGTAGGCTAGATAAGGAGAACTTCCAACAACCATACATCACGAACCGTACCTTTATGCTGGCGAAAGAAGCTAGCTTGGCGGATAACAACACCGATGTTCGTCTGATCGGCGAGAAATTATTCCACGGCGTGAGCATGTCTGAACGCTGCTATCTGATGAAACAGGTTTTGAATTTCACCCTCGAAGAGGTCCTGTTTCCGCAAAGCGATCG TTTTCAGCCGTACATGGCAGAAGTGGTGCCGTTTCTGGCGGCACTGTCAAATGCTCTGTCGACCTGTCATATTGAAGGCGACGACCTGCATATCCAGCGTAATGTTCAGAAGCTTAAGGACACTGTAAAAAAGCTGGGTGAGAGCGGTGAGATCAAAGCGATTGGTGAGCTGGACTTGCTGTTCATGTCCTTGCGCAACGCCTGCATTCATCACCATCACCACCAC(SEQ ID NO:5)

[0117] The mutant described in the present invention is mutated on the basis of 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 (SEQID NO: 1) without a signal peptide. For example, the mutation site D10 pAcF refers to replacing the D (i.e., Asp) at position 10 of the amino acid sequence shown in SEQ ID NO: 1 with pAcF.

[0118] In order to be consistent with the original experimental records, the mutants were named using the sites in the IL-22 protein containing the signal peptide. That is, the residue position of the amino acid sequence shown in SEQ ID NO: 1 is marked as X, and the position of the corresponding amino acid residue in the protein containing the signal peptide shown in SEQ ID NO: 7 is X+33. In the embodiments of the present invention, Tables 1 to 18 and Figures 1 to 19The mutation sites are counted according to the corresponding positions in SEQ ID NO: 1, and the mutant names are counted according to the positions of the corresponding amino acids in the protein containing the signal peptide. For example, the mutant name corresponding to the mutation site D10pAcF is IL22-hB43-his, which is located at position 10 in the protein sequence without a signal peptide and at position 10+33, i.e., position 43, in the protein sequence containing a signal peptide.

[0119] The naming convention for the names of the mutants modified with PEG 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 76th substituted cysteine ​​residue of the IL-22 molecule (76 is the position of cysteine ​​in the protein sequence without a signal peptide, 76+33=109, so the site is recorded as 109 when naming).

[0120] The numbers of the sites in the names of the mutants of the present invention are only used as marks, but do not mean that the prepared mutants contain signal peptides. The test materials involved in the examples of the present invention do not contain the signal peptide shown in SEQ ID NO:7. The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described in conjunction with the examples below: Comparative Example 1

[0121] 1) Cloning, expression and purification of natural wt-IL22 (SEQ ID NO. 1) in E. coli.

[0122] Based on the protein sequence A34-I179 (Uniprot: Q9GZX6) of human mature IL22, the cDNA sequence encoding wt-IL22 (SEQ ID NO: 4) was designed according to the codon preference of Escherichia coli, and the start codon ATG and EcoRI restriction site were added to the 5' end of the cDNA, and three stop codons and 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, and the positive clones were picked from the kanamycin resistance plate and used as the expression strain of wt-IL22 after sequencing verification.

[0123] The expression strain of wt-IL22 was inoculated into TB medium, cultured in a shaker at 35°C and 250 rpm until the logarithmic growth phase, and the temperature was raised to 42°C to induce expression for 4 hours. After the culture was completed, the bacterial cells were harvested by centrifugation at 13900g. The cells were washed and resuspended with 50mMTris-HCl, 5mM EDTA, pH 8.0 buffer, and the cells were broken at a pressure of 700-1000 bar, and the inclusion bodies were harvested by centrifugation at 13900g.

[0124] Wash the inclusion bodies with 50mM Tris, 5mM EDTA, 0.5% DOC, 100mM NaCl, 1mM DTT, pH8.0, and repeat the wash once. Wash once with 50mM Tris, 5mM EDTA, pH8.0. Resuspend the inclusion bodies with 7M guanidine hydrochloride, 50mM Tris, pH8.0, and stir at 2-8℃ for 4h after high-speed dispersion. Dilute the protein solution 20 times with 0.5M arginine, 100mM Tris, 1mM GSH, 0.1mM GSSG, pH8.0 refolding buffer and let stand at 2-8℃ for at least 48h. Ultrafiltration and concentration of the refolding solution to 5% trehalose, 10mM NaAc, pH4.5, filter and use CM Sepharose FF (Cytiva) ion exchange for chromatography. After equilibration with 3-5 column volumes of 50mM NaAc, pH5.0 buffer, load the protein solution onto the chromatography column. After loading, rinse the chromatography column with 50mM NaAc, pH5.0 buffer, pre-wash with 2.5 column volumes of 50mM NaAc, 250mM NaCl, pH5.0, equilibrate with 3-5 column volumes of 50mM NaAc, pH5.0 buffer, elute with 50mM NaAc, 400mM NaCl, pH5.0, and collect the eluate.

[0125] Comparative Example 2 Cloning of control IL22 mutant (hB3-His, SEQ ID NO: 2)

[0126] Referring to the IL22 mutant 22-B3 in (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, a 6*his tag was added to the C-terminus, and a cDNA sequence encoding hB3-his (SEQ ID NO: 5) was designed according to the codon preference of Escherichia coli. 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 the positive clones were picked from the kanamycin resistance plate and used as the expression strains of each mutant protein after sequencing verification.

[0127] The hB3-his expression strain was inoculated into an autoinduction medium containing 0.2% lactose, cultured overnight at 37°C, 250 rpm in a shaker to express each mutant protein, and the bacterial cell was harvested by centrifugation at 13900 g. The bacterial cell was washed and resuspended with a buffer solution of 50 mM Tris-HCl, 5 mM EDTA, pH 8.0, and the bacterial cell was broken at a pressure of 700-1000 bar, and the inclusion body was harvested by centrifugation at 13900 g.

[0128] The inclusion bodies were washed with 50mM Tris, 5mM EDTA, 0.5% DOC, 100mM NaCl, 1mM DTT, pH8.0 buffer and 50mM Tris, 5mM EDTA, pH8.0 buffer, respectively, and then dissolved with 7M guanidine hydrochloride, 50mM Tris, pH8.0 buffer. The protein solution was diluted 20 times with 0.5M arginine, 100mM Tris, 1mM GSH, 0.1mM GSSG, pH8.0 refolding buffer, and allowed to stand at 2-8°C for at least 48h. The refolding solution was ultrafiltered and concentrated to 5% trehalose, 10mM NaAc, pH4.5, and then ion exchanged with CM Sepharose FF (Cytiva) for chromatography. After balancing with 3-5 column volumes of 40mM NaAc, pH5.2 buffer, the protein solution was loaded onto the chromatography column. After loading, the chromatography column was rinsed with 40mM NaAc, pH5.2 buffer, and gradient eluted with 0-1M NaCl with 10 column volumes of 40mM NaAc, 1M NaCl, pH5.2, and the eluate was collected. The collected eluate was ultrafiltered with an ultrafiltration tube and loaded onto Superdex 75pg (120ml, Cytiva) for chromatography, and the target protein peak was collected. Comparative Example 3 Cloning, expression, and purification of IL22-Fc (code: RG7880, SEQ ID NO: 3).

[0129] The RG7880 (synthetic gene of Beijing Qingke Biotechnology Co., Ltd.) plasmid was transfected into CHO-K1 cells. 10 μg / mL MSX was added for pressure screening 24 hours after transfection. After the cell density and viability were restored, the cells were inoculated for feed-batch expression. The centrifugal supernatant after expression was purified by protein A, Capto adhere and Phenyl HP hydrophobic chromatography in sequence, and the antibody concentration was quantified by BCA method.

[0130] Example 1 Cloning of non-natural amino acid IL22 mutant (6*His) in E. coli

[0131] According to the mutation sites shown in Table 1, the parent amino acid codons of each site of wt-IL22 (SEQ ID NO: 1) were replaced with amber codons TAG (so that the amino acid at the corresponding site was mutated to p-acetylphenylalanine). In order to add a 6*his tag at 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 Escherichia coli, and cDNA encoding the corresponding IL22 mutants with each codon mutation in Table 1 was designed, and 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 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 auxiliary plasmid pEVOL-pAcF that can specifically introduce p-Acetyl-L-phenylalanine (pAcF). Positive clones were picked from the kanamycin and chloramphenicol double-resistant plates and used as expression strains for each mutant protein after sequencing verification.

[0132] Table 1 List of IL22 mutants

[0133]

[0134] The expression strains of each non-natural amino acid mutant protein were inoculated into an autoinduction medium containing 1 mM p-acetylphenylalanine, 0.02% arabinose and 0.2% lactose, and cultured overnight at 37°C, 250 rpm in a shaker to express each mutant protein, and the bacterial cells were harvested by centrifugation at 13900 g. The bacterial cells were washed and resuspended with a buffer solution of 50 mM Tris-HCl, 5 mM EDTA, pH 8.0, and the bacterial cells were broken at a pressure of 700-1000 bar, and the inclusion bodies were harvested by centrifugation at 13900 g.

[0135] The inclusion bodies were washed with a washing buffer containing 0.5% DOC, and the precipitate was collected by centrifugation. The crude inclusion bodies were dissolved with 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 the pre-cooled refolding solution (100mM Tris, 2mM EDTA, 0.5M L-Arg, 1mM GSH, 0.1mM GSSSG, pH 8.0) at a ratio of 1:20 (v / v) and allowed to stand at 2-8°C for about 72h. The refolding solution was ultrafiltered and concentrated to 10mM NaAc, 5% trehalose, pH 4.5 buffer, filtered, and chromatographed using CM Sepharose FF (Cytiva) ion exchange. The buffer used for CM SepharoseFF ion exchange chromatography is as follows: Buffer A: 20mM MES, pH6.0, Buffer B: 20mM MES, 1M NaCl, pH6.0. The column is first equilibrated with 3 to 5 column volumes of Buffer A, and then the refolded solution is 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, and the purified sample is collected.

[0136] Example 2 Unlabeled non-natural amino acid mutants of IL22

[0137] According to the mutation sites shown in Table 2, the parent amino acid codons at each site of wt-IL22 were replaced with amber codons TAG (so that the amino acid at the corresponding site was mutated to acetylphenylalanine), and according to the codon preference of Escherichia coli, cDNAs encoding IL22 mutants corresponding to each codon mutation in Table 2 were designed, and the start codon ATG and NdeI restriction site were added to the 5' end of the cDNA, and three stop codons and 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 auxiliary plasmid pEVOL-pAcF that can specifically introduce p-Acetyl-L-phenylalanine (pAcF). Positive clones were picked from the kanamycin and chloramphenicol double-resistant plates and used as expression strains for each mutant protein after sequencing verification.

[0138] Table 2. List of IL22 unnatural amino acid mutants

[0139] Mutant name Mutation site IL22-hB50 P17 pAcF IL22-hB120 P87 pF IL22-hB121 F88

[0140] The expression strains of each IL22-pAcF mutant protein were inoculated into an autoinduction medium containing 1 mM p-acetylphenylalanine, 0.02% arabinose and 0.2% lactose, and cultured overnight at 37°C, 250 rpm in a shaker to express each mutant protein, and the bacterial cells were harvested by centrifugation at 13900 g. The bacterial cells were washed and resuspended with a buffer solution of 50 mM Tris-HCl, 5 mM EDTA, pH 8.0, and the bacterial cells were broken at a pressure of 700-1000 bar, and the inclusion bodies were harvested by centrifugation at 13900 g.

[0141] The inclusion bodies were washed with 2M urea, 2% Triton X-100, 50mM Tris, 5mM EDTA, pH 8.0 buffer and 50mMTris, 5mM EDTA, pH 8.0 buffer in sequence. The inclusion bodies were dissolved with 7M guanidine hydrochloride, 50mM Tris, pH 8.0, and the protein solution was diluted 20 times with 0.5M arginine, 100mM Tris, 1mM GSH, 0.1mM GSSG, pH 8.0 refolding buffer and allowed to stand at 2-8°C for 48h. The refolding solution was ultrafiltered and concentrated into 10mM NaAc, 5% trehalose, pH 4.5 buffer, filtered, and chromatographed using Capto SPImpRes (Cytiva) ion exchange. The protein solution was loaded onto the chromatography column after equilibration with 3-5 column volumes of 20 mM NaAc, pH 5.5 buffer. After loading, the chromatography 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 with 0-1 M NaC, and the eluate was collected.

[0142] Example 3 Polyethylene glycol modification and purification of IL22 non-natural amino acid mutants.

[0143] Take the purified protein of Example 1 and Example 2, and replace the solution with 10mM NaAc, 5% trehalose, pH 4.5 by ultrafiltration, with a final concentration of 5-10mg / ml. Add ammonium chloride at a molar ratio of 1:125 (IL22: ammonium chloride); add acetylhydrazine at a molar ratio of 1:30 (IL22: acetylhydrazine); add PEG-hydroxylamine (e.g., Mw30k, 40kDa, Xiamen Sinobond) at a molar ratio of 1:5 (IL22: PEG), react at 2-8°C for about 48-60h, and modify PEG to the mutation site of each mutant. The reaction solution was diluted 10 times with purified water, the pH value was 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 chromatography column was rinsed with 40 mM NaAc, pH 5.5 buffer, and gradient eluted with 0-0.5 M NaCl, 40 mM NaAc, pH 5.5 buffer to collect the modified protein. Example 4 Cloning, expression, and purification of IL22 cysteine ​​mutants in Escherichia coli.

[0144] According to the mutation sites shown in Table 3 (with SEQ ID NO: 1 as the wild type), the parent amino acid of each site was replaced with cysteine ​​(Cystine, referred to as Cys), and the cDNA encoding the corresponding IL22 mutant with each codon mutation in Table 3 was designed according to the codon preference of Escherichia coli, and the start codon ATG and EcoRI restriction site were added to the 5' end of the cDNA, and 3 stop codons and 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, and the positive clones were picked from the kanamycin resistance plate, and the expression strains of each mutant protein were used after sequencing verification.

[0145] Table 3 List of IL22 cysteine ​​mutants

[0146]

[0147] The expression strains of each IL22 cysteine ​​mutant protein were inoculated into TB medium, cultured in a shaker at 35°C and 250 rpm until the logarithmic growth phase, and the temperature was raised to 42°C to induce expression for 4 hours. After the culture was completed, the bacterial cells were harvested by centrifugation at 13900g. The bacterial cells were washed and resuspended with a buffer solution of 50mM Tris-HCl, 5mM EDTA, pH 8.0, and the bacterial cells were broken at a pressure of 700-1000 bar, and the inclusion bodies were harvested by centrifugation at 13900g.

[0148] The inclusion bodies were washed with 50mM Tris, 5mM EDTA, 2% Triton X-100, pH 8.0 buffer and 50mM Tris, 5mM EDTA, pH 8.0 buffer in sequence. After the inclusion bodies were dissolved with 8M urea, 50mM MES, 0.1mM DTT, 10mM EDTA, pH 5.5, the protein solution was diluted 20 times with 0.5M arginine, 100mM Tris, 1mM GSH, 0.1mM GSSG, pH 8.0 refolding buffer and allowed to stand at 2-8°C for 48h. The refolding solution was ultrafiltered and concentrated into 10mM NaAc, 5% trehalose, pH 4.5 buffer, filtered and chromatographed using CM Sepharose FF (Cytiva) ion exchange. 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 chromatography 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 0-100%, and the eluate was collected.

[0149] Example 5 Polyethylene glycol modification and purification of IL22 cysteine ​​mutants

[0150] Each IL22 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 a specific mutation site of the above mutant, so that the PEG site-specifically modifies the corresponding IL-2 mutant.

[0151] Take the purified IL-22 cysteine ​​mutant protein, adjust its pH value to 6.0, weigh PEG-maleimide (e.g., PEG with Mw40k, 60k, 80kDa, Xiamen Sinobond) at a molar ratio of 1:5 between IL-22 mutant and PEG, dissolve PEG powder in purified water to a final concentration of 100 mg / ml, and add to the protein solution. Mix well and react at 2-8°C overnight.

[0152] The reaction solution was diluted 10 times with purified water, the pH value was adjusted to 5.1, and ion exchange chromatography was performed using Macro Cap SP (Cytiva). The protein solution was loaded onto the chromatography column after equilibration with 3-5 column volumes of 40mM NaAc, pH5.1 buffer. After loading, the chromatography column was rinsed with 40mM NaAc, pH5.1 buffer, and gradient elution was performed using 0-0.5M NaCl, 40mM NaAc, pH5.1 buffer to collect the modified protein.

[0153] Table 4 List of test substance information of PEG-IL2 cysteine ​​mutants

[0154]

[0155]

[0156] Effect verification

[0157] 1. Evaluation of STAT3 activation activity of PEG-IL22 mutants at different modification sites using reporter gene assay

[0158] COLO205-SIE cells were constructed to test the activation effect of each test substance on STAT3.

[0159] 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 pGL4.47[luc2P / SIE / Hygro](Promega,) plasmid using electroporation transfection system (Invitrogen) to obtain COLO205 / SIE cells, which were resuspended in RPMI1640+10% FBS to 1×10 6 pcs / ml, 50μl / well was added to a 96-well plate, and incubated at 37°C, 5% CO2 overnight, about 16h; the IL22 sample was diluted in gradients, 50μl / well was added thereto, incubated at 37°C, 5% CO2 for 6h, and then Bio-Lite Luciferase Assay System (Vazyme, Cat: DD1201-02) was added, 100μl / well, and the chemiluminescence value was detected. The final concentration of the 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 the STAT3 phosphorylation reporter gene activity of COLO205 / SIE cells stimulated by IL22 was obtained by four-parameter regression fitting in GraphPadPrism 9 software.

[0160] According to the results of STAT3 reporter gene detection 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.

[0161] Table 5 Detection of STAT3 activation activity of PEG-IL22 mutants by reporter gene assay 50

[0162]

[0163]

[0164] 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 as follows: Figure 1 .

[0165] 2. COLO205 STAT3 / STAT1 preference

[0166] The ELISA method was used to screen for molecules with potential preferential advantages in the activation ability of p-STAT1 / p-STAT3 in COLO205 cells.

[0167] COLO205 cells were cultured in a culture dish with RPMI1640 + 10% FBS until sufficient volume was obtained. An appropriate amount of cell suspension was centrifuged, washed twice with PBS, and resuspended to 8 × 10 5 / ml, 50μl / well was added to a 96-well plate, incubated at 37℃, 5% CO2 for 6 hours, IL22 sample was gradient diluted, 50μl / well was added thereto, incubated at 37℃, 5% CO2 for 0.5 hour, the 96-well plate was placed on ice for 5-10 minutes, the cell lysis buffer in the kit was prepared into 2×, 100μl / well was added to the above 96-well plate, placed at 4℃ or on ice for 5-10 minutes, the 96-well plate was transferred to -80℃, stored for not less than 30 minutes, and then thawed at room temperature.

[0168] STAT1 kit ( Phospho-Stat1(Tyr701)Sandwich ELISA Kit, Manufacturer: CST, Cat:73582), STAT3 kit ( After the microwell strips equipped with Phospho-Stat3 (Tyr705) Sandwich ELISA Antibody Pair, manufacturer: CST, Cat: 7300) have reached room temperature, break off the required microwells. Place the microwells in the strip rack. Unused microwells must be resealed and immediately stored at 4°C.

[0169] Take out 90 μl of the cell lysate from each well and add it to the appropriate microplate wells provided with the STAT1 kit and STAT3 kit, seal it with sealing film and press it firmly on the top of the microplate. Incubate the plate at 37°C for 2 hours, or incubate the plate at 4°C overnight.

[0170] Discard the contents of the plate and wash 4 times with 1× Wash Buffer, 200 μl per well. Between washes, tap the plate heavily on 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, repeat 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, repeat the wash procedure. Add 100 μl of TMB substrate to each well. Seal 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 on a microplate reader at 450 nm within 30 minutes of adding STOP solution. The final concentration of IL22 sample was taken as the Log value on the X-axis, and the detection result of each well was 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 Prism 9 software.

[0171] Using ECs that activate STAT3 and STAT1 phosphorylation 50 The STAT3 / STAT1 preference of COLO205 was evaluated by the ratio of 1:1. 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 not as high as the first two molecules, but its STAT1 phosphorylation activation Emax is lower, suggesting that it has a low activation ability even at high concentrations.

[0172] Table 6 ELISA detection of STAT1 / STAT3 activity of PEG-IL22 mutants in EC 50

[0173]

[0174]

[0175] In addition, ELISA was used to detect the activity of PEG-modified IL22 non-natural amino acid mutants in activating STAT3 phosphorylation in COLO205 cells at different concentrations. Figure 2 ELISA was used to detect the activity of PEG-modified IL22 non-natural amino acid mutants in activating STAT1 phosphorylation in COLO205 cells at different concentrations. Figure 3 .

[0176] 3. Effects of different molecular weight polyethylene glycol modifications on IL22 receptor preference

[0177] The untagged IL22 non-natural amino acid mutants prepared in Example 5 were used to prepare PEG-modified IL22 mutant products with different molecular weights according to the method of Example 6, as shown in the following table:

[0178] Table 7 Information on the test substances of PEG-IL22 non-natural amino acid mutants

[0179] Mutant name (test substance) coupling site PEG molecular weight 30K-PEG-IL22-hB50 17 30kD 40K-PEG-IL22-hB50 17 40kD 30K-PEG-IL22-hB120 87 30kD 40K-PEG-IL22-hB120 87 40kD 30K-PEG-IL22-hB121 88 30kD 40K-PEG-IL22-hB121 88 40kD

[0180] Human colon cancer cell line HT-29 adherent cells were selected and FACs were used to detect the preference of PEG-modified IL22 for phosphorylation activation of pSTAT1 and pSTAT3 receptors. hB3-his and IL22-Fc were used as control molecules.

[0181] The day before the experiment, 2 × 10 5 Cells were incubated at 37°C, 5% CO2 overnight to adhere to the wall. Different test substances were diluted in a gradient manner using McCoy's cell culture medium containing 10% FBS, and 100 μL / well was added thereto. The cells were incubated at 37°C, 5% CO2 for 30 minutes. The cells were washed once with 100 μL / well PBS, discarded by suction, and 25 μL / well trypsin (Gibco, 25200-560) was added to digest at 37°C for about 5 minutes to make suspended cells. The cells were gently tapped to disperse, and 80 μL / well McCoy's culture medium containing 10% FBS was added to neutralize, and the suspension was blown evenly to form a single cell suspension. 100 μL of each was transferred to a V-bottom plate for centrifugation. Resuspend the cells with 100 μL / well preheated 1× fixative (BD bioscience, 558049) or 4% paraformaldehyde (Sigma-Aldrich, 158127-5G) at 37°C for 15-20 minutes to fix the cells. After centrifugation, discard the fixative and use the remaining liquid to disperse the cells. Then add 80 μL / well -20°C precooled permeabilization solution (BD bioscience, 558050) to fully resuspend the cells. Let them stand on ice for 0.5-1h, tap 2-3 times in the middle to avoid cell clumping. Wash thoroughly with PBS and centrifuge. Add flow cytometry antibodies (anti-STAT1 pY701, BD bioscience 612596 and anti-STAT3 pY705 BD bioscience 557815) and incubate in the dark for 30-60 minutes. Wash again and perform flow cytometry detection on the machine to determine the MFI values ​​corresponding to FITC and APC in each sample.

[0182] The final concentration of each sample was taken as the Log value of 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.

[0183] FACs method 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. Figure 4 The FACs method was used to detect the activity of PEG-modified IL22 non-natural amino acid mutants in activating HT29 cell STAT1 phosphorylation at different concentrations. Figure 5 .

[0184] Compared with IL22-Fc and hB3-His molecules, 40k-PEG modified IL-22 mutants significantly reduced STAT3 phosphorylation activity, which was far lower than IL22-Fc. 30k-PEG modified IL-22 mutants retained good STAT3 phosphorylation activity, and all showed enhanced preference relative to IL22-Fc. Compared with IL22-Fc, 30k-PEG-IL22-hB120 increased STAT3 phosphorylation activity by about 20 times when STAT1 phosphorylation activity was equivalent. However, 30k-PEG-IL22-hB50 and 30k-PEG-IL22-hB121 significantly reduced STAT1 phosphorylation activity when STAT3 activity was equivalent.

[0185] Table 8 FACs method to detect the phosphorylation preference of PEG-IL22 non-natural amino acid mutants in activating STAT1 / STAT3

[0186]

[0187] 4. FACs detection of pSTAT1 and pSTAT3 activation preference

[0188] The same FACs were used to detect the preference of PEG-modified IL22 for receptor activation according to the method in item 3 above. hB3-his and IL22-Fc were used as control molecules.

[0189] Table 9 FACs method to detect the phosphorylation preference of PEG-IL22 cysteine ​​mutants in activating STAT1 / STAT3

[0190]

[0191] FACs method was used to detect the activity of PEG-modified IL22 cysteine ​​mutants in activating STAT3 phosphorylation in HT29 cells at different concentrations. Figure 6The FACs method was used to detect the activity of PEG-modified IL22 cysteine ​​mutants in activating STAT1 phosphorylation in HT29 cells at different concentrations. Figure 7 .

[0192] Among the IL22 molecules conjugated to cysteine ​​polyethylene glycol, PEG-IL22-D109C and PEG-IL22-P120C molecules showed preference for IL22-Fc. 40k / 60k-PEG-IL22-D109C activated HT29 cell STAT1 / STAT3 EC 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.

[0193] 5. Tissue Activation Preference

[0194] Human immortalized epidermal cells 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 and resuspended to 1.25×10 6 120 μl / well of the mixed cells were plated in a 96-well plate (Corning, 3599) at a cell plating density of 1.5×10 5 The cells were placed in a 37°C, 5% CO2 incubator and cultured overnight.

[0195] 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 times in a series with complete medium, the other samples were diluted 4 times in a series with 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 absorbing dryness, 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 blown and mixed until single cells. Take 80 μl of the single-cell suspension mixed by pipetting and transfer it to a 96-well V-bottom plate (NEST, 701201) containing 80 μl / well of 4% paraformaldehyde (Sigma-Aldrich, 158127-5G) solution, pipette and mix, place at room temperature for 20 min, centrifuge at 1800 rpm for 5 min, remove the supernatant, add 100 μl / well of pre-cooled methanol (Merck, 11.06007.4008) to break the membrane, place at 4°C for 1 h, centrifuge to remove the supernatant, wash once with 1×PBS, add the prepared STAT3 antibody (Alex Fluor647 mouse anti-Stat3 (pY705), BD, 557815) for staining, stain at room temperature in the dark for 1 h, wash twice with 1×PBS, and detect the mean fluorescence intensity (MFI) of the channel by flow cytometry.

[0196] The tissue activation preference of different test substances was evaluated by comparing the activity of STAT3 in intestinal HT29 cells and skin-derived HaCaT cells. The results show that compared with IL22-Fc, 30k-PEG-IL22-hB120, 60K-PEG-IL22-D109C, and 60K-PEG-IL22-P120C all show more advantageous tissue preference, preferentially activating intestinal HT29 cells and relatively weaker activation of skin-derived HaCaT cells, indicating that they can preferentially act on intestinal target organs in vivo and are not easy to activate skin cells and produce adverse reactions. The FACs method was used to detect the activity of polyethylene glycol-modified IL22 mutants in activating STAT3 phosphorylation in HaCaT cells at different concentrations. Figure 8 .

[0197] Table 10 FACs method to detect STAT3 phosphorylation preference of PEG-IL22 mutants in activating human intestinal cell lines and human skin cell lines

[0198]

[0199] 6. Mouse PD (Reg3β)

[0200] 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.

[0201] 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 wtIL-22, and the corresponding doses were injected intraperitoneally.

[0202] About 40 μL of orbital blood was drawn 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 at 4°C overnight. Mouse Reg3β (sino Biological, 51153-M08H) was used as a positive control standard curve in gradient dilution. Plasma samples were appropriately diluted to fit the linear range of the control. After blocking, the samples 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 used for incubation and coupling. After the final wash, tetramethylbenzidine peroxidase substrate (TMB, Surmodics, TMBS-1000-01) was added for color development for 10 minutes. The reaction was terminated with 2M sulfuric acid. The plate was read at 450 nm using an ELISA reader with 630 nm as the reference. The original concentration of Reg3β in each sample was calculated. Plot concentration versus time ( Fig. 9 ).

[0203] The results showed that all the test substances could activate the expression of Reg3β molecules to a certain extent, among which different doses of wt-IL-22 had a weaker activation effect on Reg3β, and there was no obvious dose-dependent enhancement. At similar (0.5mg / kg vs. 0.3mg / kg) or the same concentration (1mg / kg), compared with IL22-Fc, 60k-PEG-IL22-D109C, 60k-PEG-IL22-P120C, and 40k-PEG-IL22-hB120 in the test substances showed a higher peak concentration of stimulation for Reg3β.

[0204] Table 11 Peak plasma Reg3β concentrations in mice after single administration of PEG-modified IL22 mutants

[0205]

[0206] 7. Mouse PD preference study (Reg3β and SAA)

[0207] Based on the in vitro preference results and the preliminary PD screening results, the molecules 60k-PEG-IL22-D109C and 60k-PEG-IL22-P120C were selected, and the PD test of 3 mice at different doses was performed according to the method of Example 14, including the efficacy-related molecule Reg3β and the inflammation-related molecule SAA. The control was IL22-Fc. Blood was collected and anticoagulated and plasma was separated at 4, 24, 48, 72, 96, 144, 192, 264, and 336 hours before and after administration. For the control molecules wt-IL22 and hB3-his, only up to 96 hours were taken.

[0208] The Reg3β detection method was carried out according to Example 14, and the original concentration of Reg3β in each sample was calculated. The concentration was plotted against time.

[0209] The specific SAA detection method is as follows: Use a kit containing a detection antibody pair and a positive control (Abcam, Ab216059) to detect the SAA concentration in each appropriately diluted plasma sample, operate according to the kit instructions, use Sa-HRP (Pierce 21126) for coupling, add tetramethylbenzidine peroxidase substrate (TMB, Surmodics, TMBS-1000-01) for color development for 10 minutes after washing, terminate the reaction with 2M sulfuric acid, and use an enzyme reader to read the plate at 450nm with 630nm as a reference. The SAA detection time points are before administration and 24, 48, 72, 96, and 144 hours after administration. Calculate the original SAA concentration in each sample. Plot the concentration against time ( Figures 10-11 ).

[0210] The results of the mouse PD preference study showed that the level of Reg3β produced by wt-IL22 and hB3-his-stimulated mice was significantly lower than that of the other test substances. For candidate test substances at different doses, under equivalent or superior conditions of Reg3βCmax (such as 60K-PEG-IL22-D109C 0.1mg / kg vs 60K-PEG-IL22-P120C 0.3mg / kg vs IL22-Fc 1mg / kg), the site-specific conjugated PEG-IL22 test substance activated SAA Cmax significantly lower than IL22-Fc, indicating that it has better non-inflammatory preference and can significantly reduce the pro-inflammatory effect while maintaining organ protection activity.

[0211] Table 12 Dosing information for mouse PD preference study (Reg3β and SAA)

[0212] Serial number Mutant name (test substance) ROA Dosage (mg / kg) 1 60K-PEG-IL22-P120C sc 1 / 0.3 / 0.1 2 60K-PEG-IL22-D109C sc 0.5 / 0.1 / 0.02 3 IL22-Fc sc 1 / 0.3 4 hB3-his ip 3 5 wt-IL22 ip 3

[0213] 8. Mouse PK

[0214] Balb / C male mice were used, and 4 mice in each group were injected subcutaneously once, and the dosage was 1 mg / kg. Blood was collected before administration and 4 (not for 60k-PEG-IL22-D109C and 60k-PEG-IL22-P120C), 8, 24, 24, 48, 72, 96, and 120 hours after administration, and plasma was separated by anticoagulation.

[0215] 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, and the standard curve of each test substance and plasma samples were diluted to the linear range of detection, and Recombinant Anti-IL22 Antibody (Sino Biological, 13059-R034-H) was used for detection. The original concentration of the test substance in each sample was calculated from the four-parameter fitting of the standard curve of each test substance. The plasma pharmacokinetic parameters of each group of test substance mice were analyzed ( Fig.12 ).

[0216] Table 13 Dosing information for mouse PK study

[0217] Mutant name (test substance) dose Dosage 30k-PEG-IL22-hB50 1mg / kg sc 40k-PEG-IL22-hB50 1mg / kg sc 30k-PEG-IL22-hB120 1mg / kg sc 40k-PEG-IL22-hB121 1mg / kg sc 60k-PEG-IL22-P120C 1mg / kg sc 60k-PEG-IL22-D109C 1mg / kg sc

[0218] The pharmacokinetic parameters of each test substance are analyzed as follows

[0219] Table 14 PK pharmacokinetic parameters of each test substance in mice

[0220]

[0221] 9. Mouse DSS-induced enteritis efficacy model

[0222] Balb / C mice were used to test the efficacy of the DSS mouse acute colitis model. DSS is dextran sulfate sodium salt (MW: 36000-50000). When added to drinking water at a certain ratio, it can destroy intestinal epithelial cells and cause acute inflammatory bowel disease (IBD) in mice. Therefore, the 4% DSS-induced mouse IBD model can be used to evaluate the potential pharmacological effects of the test substance.

[0223] Balb / C male mice were allowed to drink freely the drinking water containing 4% DSS until the average body weight of the model group dropped to about 15%, and then fresh drinking water was replaced. The recovery period was 2 days. The body weight of mice and the severity of loose stools / bloody stools of individuals were monitored throughout the whole process. Fig.13) and conduct a comprehensive DAI (disease activity index) assessment ( Fig.14 The colon length of each mouse was measured at the end point ( Fig.15 ), photographed and rolled into a Swiss roll, fixed with 4% paraformaldehyde, dehydrated and embedded, and processed into paraffin sections for HE staining ( Figures 17-18 ) and histological scoring of tissue morphology ( Fig.16 ).

[0224] 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).

[0225] Table 15 Drug administration information for the study of DSS-induced enteritis model in mice

[0226]

[0227] Table 16 DAI scoring criteria

[0228]

[0229] HE staining and pathological analysis were performed on paraffin sections of colon tissue. According to the actual degree of lesion reaction, the following scores were used: very mild (1 point), mild (2 points), moderate (3 points), severe (4 points); lesion area percentage (%) scoring criteria: ≥50% (4 points), 25%-50% (3 points), 10%-25% (2 points), ≤10% (1 point); inflammation depth scoring criteria: epithelial layer (1 point), lamina propria (2 points), muscularis mucosa (3 points). No lesion was recorded as "-". The scores were added together based on the above three dimensional indicators.

[0230] In the model group (G1), the tissue sections showed severe and large-scale structural destruction of the colon mucosa layer, disappearance of intestinal epithelium and crypts, and severe and large-scale inflammatory cell infiltration and fibrosis in the mucosa layer and submucosa. In the 0.1mg / kg 60k-PEG-IL22-P120C group (G4), the colon tissue morphology was relatively complete, the structural destruction of the colon mucosa layer was not severe, and no significant inflammatory cell infiltration and fibrosis were observed.

[0231] 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, the efficacy of 0.1 mg / kg 60k-PEG-IL22-P120C (G4 group) was better than that of 0.03 mg / kg IL22-Fc (G6 group).

[0232] 10. Mouse ear injection swelling model

[0233] 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 the thickness of ear swelling after injection were detected to reflect the induction effect of the test substance on the local inflammatory response of the skin.

[0234] On days 0, 1, 2, and 4, 5 μL of PBS solution containing the test substance at the following concentrations was injected into the left and right ears of the mice. The thickness of the mouse ears was measured every day using a micrometer, and the increase in ear thickness (deltathickness) on each day after injection relative to day 0 was calculated.

[0235] Table 17 Dosing information for mouse ear swelling model

[0236]

[0237] Table 18 Peak increase in ear thickness of mice after administration

[0238]

[0239] The results showed that different doses of the test substances injected into the ears of mice could cause a dose-dependent increase in ear thickness ( Fig.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 times, 0.1 mg / kg vs 0.03 mg / kg), it suggests that compared with IL22-Fc, 60k-PEG-IL22-P120C has a lower potential risk of causing skin toxicity.

[0240] 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: Asp at position 10 of the amino acid sequence shown in SEQ ID NO: 1 is replaced by 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 of the amino acids at 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 5; 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 5.

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

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

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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