Interleukin-2 Fusion Protein and Its Applications in IBD

Abstract

A fusion protein of interleukin 2 characterized by comprising human interleukin 2 or a variant thereof, and human serum albumin or a variant thereof, and its application for the treatment or prevention of inflammatory bowel disease are disclosed.

Description

Technology Field

[0001] The present invention belongs to the field of biopharmaceutical technology, and specifically, the present invention relates to a fusion protein of interleukin 2 and its use in the treatment or prevention of inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) is a type of chronic, nonspecific intestinal inflammatory disease with an unclear etiology, and clinical symptoms include diarrhea, abdominal pain, and bloody stools. Although the pathogenesis has not yet been fully elucidated, previous studies have shown a close association with host genetic susceptibility, gut microbiome imbalance, damage to the intestinal mucosal barrier, mucosal immune abnormalities, and environmental and psychological factors. IBD is incurable, recurs and progresses throughout a lifetime, and can cause disability, thereby inflicting severe physical suffering and mental burden on patients.

[0003] The treatment goals for IBD are to induce and maintain clinical remission and mucosal healing, prevent and treat complications, improve the patient's quality of life, and enhance long-term management. The primary drugs currently used to treat active IBD are still employed to block or disrupt the inflammatory chain reaction; however, this can only partially alleviate symptoms and cannot completely cure the disease. Current treatments for IBD include non-targeted therapies, targeted biological therapies, induction therapies, and other novel treatments. These therapies are described in detail below.

[0004] Non-targeted therapies include aminosalicylates, corticosteroids, and immunosuppressants. Among these, aminosalicylates are primarily effective for mild to moderate ulcerative colitis (UC), while corticosteroids and immunosuppressants have a wide range of side effects.

[0005] Targeted biological therapies primarily utilize biological agents for treatment, and their mechanisms of action are as follows: 1. Targeting and blocking the biological effects of various pro-inflammatory cytokines associated with intestinal mucosal inflammatory responses, such as anti-TNF-α antibodies (infliximab, adalimumab, golimumab, cetozumab) and anti-IL12 / 23p40 antibodies (ustekinumab); 2. Inhibiting the migration of activated leukocytes to the intestinal mucosa, such as anti-integrin antibodies (vedolizumab, natalizumab). Targeted biological therapies improve clinical remission rates in IBD patients to varying degrees and reduce relapse and surgical rates, thereby providing more treatment options for IBD patients. However, primary non-response to biological agents occurs in up to 30% of patients and secondary non-response occurs in up to 50% of patients.

[0006] Induction therapy primarily utilizes oral small molecule drugs such as selective JAK inhibitors (tofacitinib) and sphingosin-1-phosphate (SIP) receptor modulators (ozanimod). Small molecule drugs can provide more treatment options for IBD patients by improving clinical remission rates to some extent and reducing relapse and surgical rates. While small molecule drugs have overcome the immunogenicity of biological agents and patient compliance issues associated with parenteral administration routes, they can induce drug interactions and cause drug toxicity during IBD treatment, potentially leading to treatment failure. Currently available JAK inhibitors carry risks of serious infection, malignancy, and thrombosis, while ozanimod carries a risk of cardiovascular disease.

[0007] Other new treatments include stem cell regeneration, barrier repair agents, and fecal microbiota transplantation.

[0008] Regulatory T cells (T reg T is a subgroup of T cells expressing Foxp3, CD25, and CD4, which suppresses the activation and proliferation of potential autoreactive T cells in a normal body through activity regulation and is involved in maintaining the body's immunological tolerance. reg It has been confirmed that cells play a crucial role in the development of not only IBD but also other autoimmune diseases. In normal intestinal mucosa, effector T cells (T eff ) and T reg Cells maintain a dynamic balance and preserve the stability of the intestinal environment; however, if this balance is disrupted due to excessive inflammatory effects or reduced regulatory effects, IBD can occur.

[0009] Currently, through various routes T reg Reducing intestinal inflammation in IBD by improving cell quantity and / or function has become a hot spot for research. This is generally T reg Microbiota that promotes cell differentiation, low-dose interleukin-2 (IL-2)(T reg Since cells inherently express high-affinity IL-2 receptors, low doses of IL-2 T reg The number of cells can be selectively increased, but T eff (does not increase cells), a relatively stable IL-2-anti-IL-2 antibody complex in vivo, engineered IL-2 variants (called mutant proteins, capable of selectively binding to high-affinity IL-2 receptors), and adoptive T reg It includes cell therapy, etc.

[0010] Despite improvements in IBD treatment, the rate of long-term colectomy in patients with ulcerative colitis has not decreased over the past decade. In other words, since the clinical demand for IBD remains high and unmet, new therapeutic drugs or approaches are needed to improve the quality of life for patients with inflammatory bowel disease. The problem to be solved

[0011] The object of the present invention is to provide an interleukin 2 fusion protein and its use in the treatment or prevention of inflammatory bowel disease (IBD), characterized by extended administration intervals, relief of patient pain, reduced treatment costs, and improved patient compliance and quality of life. means of solving the problem

[0012] In a first aspect, the present invention provides a fusion protein of interleukin 2. The fusion protein comprises human interleukin 2 or a variant thereof, and human serum albumin or a variant thereof;

[0013] The above human interleukin 2 or its variant comprises an amino acid sequence represented by SEQ ID NO. 1; or an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO. 1;

[0014] The above human serum albumin or its variant comprises an amino acid sequence represented by SEQ ID NO. 2; or an amino acid sequence having 90% identity with the amino acid sequence represented by SEQ ID NO. 2.

[0015] In the present invention, the amino acid sequence represented by SEQ ID NO. 1 is human interleukin 2 consisting of 133 amino acid residues, and the amino acid sequence represented by SEQ ID NO. 2 is human serum albumin consisting of 585 amino acid residues.

[0016] In some embodiments, the fusion protein of interleukin 2 comprises an amino acid sequence represented by SEQ ID NO. 1 and an amino acid sequence represented by SEQ ID NO. 2.

[0017] In some embodiments, the amino acid at position 125 of the amino acid sequence represented by SEQ ID NO. 1 is not cysteine. Human interleukin 2 (IL-2) has an amino acid residue at position 125 of the mature protein and an intrachain disulfide bond; if the amino acid residue is cysteine, it is likely to form a mismatch disulfide bond with the other two cystes, which can lead to a loss of IL-2 activity.

[0018] In some embodiments, the amino acid at position 125 of the amino acid sequence represented by SEQ ID NO. 1 is substituted with serine or alanine. Mutating the amino acid residue at position 125 of human interleukin 2 with serine or alanine prevents mismatch of disulfide bonds and maintains the activity of human interleukin 2.

[0019] In some embodiments, the human interleukin 2 or its variant is directly connected to the human serum albumin or its variant, or the human interleukin 2 or its variant is connected to the human serum albumin or its variant through a linking peptide.

[0020] In some embodiments, a linking peptide is provided between human interleukin 2 or a variant thereof and human serum albumin or a variant thereof to increase the gap between the two parts of the fusion protein and to maximize the possibility of the human interleukin 2 part binding to the interleukin 2 receptor.

[0021] In some embodiments, the general formula of the linking peptide is (G n S) m and, among them, n and m are integers from 1 to 10, respectively.

[0022] In some embodiments, the general formula of the linking peptide is (G n S) m and, among them, n is an integer from 1 to 4, and m is an integer from 0 to 3.

[0023] In some embodiments, the general formula of the linking peptide is (G n S) m and, among them, n is an integer from 1 to 4, and m is an integer from 1 to 3.

[0024] In some embodiments, the amino acid sequence of the linking peptide is GGGGSGGGGS (Sequence No. 6).

[0025] In some embodiments, the amino acid sequence of the linking peptide is GGGGS (Sequence No. 7).

[0026] In some embodiments, the N-terminus of the fusion protein carries a signal peptide. Adding the signal peptide to the N-terminus of the fusion protein can further increase the expression level of the fusion protein.

[0027] In some embodiments, the signal peptide is secretory signal peptide CD33.

[0028] In some embodiments, the nucleic acid sequence encoding the signal peptide may be represented by SEQ ID NO. 5.

[0029] In some embodiments, the fusion protein has an amino acid sequence represented by SEQ ID NO. 4.

[0030] In some embodiments, the fusion protein comprises, in order from the N-terminus, human interleukin 2-linked peptide-human serum albumin, or human serum albumin-linked peptide-human interleukin 2.

[0031] In some embodiments, SEQ ID NO. 4 is a protein composed of a sequence of 728 amino acid residues, wherein amino acids 1 to 133 are human interleukin 2, amino acids 134 to 143 are linked peptide GGGGSGGGGS (SEQ ID NO. 6), and amino acids 144 to 728 are human serum albumin.

[0032] In a second aspect, the present invention provides an isolated nucleic acid molecule. The nucleic acid molecule codes for a fusion protein according to the first aspect of the present invention.

[0033] In some embodiments, the fusion protein is encoded by a nucleotide sequence represented by SEQ ID NO. 3.

[0034] In the present invention, based on the gene sequences of human interleukin 2 and human serum albumin disclosed by Genbank, the nucleotide sequence can be optimized according to the codon preference of mammals, the optimized nucleotide sequence of the fusion protein can be constructed on an expression vector, transfected into a host cell to be expressed in the host cell, and the target protein, namely the fusion protein of interleukin 2, can be isolated and purified.

[0035] The interleukin 2 and human serum albumin fusion protein expression system obtained a CHO monoclonal cell line that expresses a human-derived recombinant protein stably and highly efficiently by transfecting CHO cells with a plasmid carrying a fusion gene of interleukin 2 and human serum albumin (e.g., by electroporation injection), and the monoclonal cell line of the present invention can secrete a fusion protein expressing interleukin 2 and human serum albumin (i.e., a fusion protein of interleukin 2), can significantly extend the plasma half-life of human interleukin 2, and can be used in the manufacture of drugs related to human interleukin 2 expression.

[0036] In some embodiments, the expression vector is selected from pEE14.4, pcDNA3.1, and pEE6.4, and preferably pEE14.4.

[0037] In a third aspect, the present invention provides an expression system. The expression system comprises a CHO cell, and the CHO cell comprises a nucleic acid molecule according to the second aspect of the present invention;

[0038] The above expression system expresses a fusion protein according to the first aspect of the present invention.

[0039] The present invention unexpectedly discovered that when using CHO cells, particularly when using the CHO-K1 cell line, the cell expression level is very high, reaching 4 g / L; the expressed interleukin 2 fusion protein has high activity and can strongly promote the proliferation of specific cells such as NK cells and / or T cells and / or Treg cells in vitro.

[0040] In some embodiments, the expression system is a CHO-K1 cell, and the CHO-K1 cell was deposited on May 26, 2022, at the General Microbiology Center of the Chinese Microbial Culture Collection Committee under accession number CGMCC No. 45173.

[0041] In a fourth aspect, the present invention provides a pharmaceutical composition. The pharmaceutical composition comprises a fusion protein according to the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0042] In some embodiments, the formulation of the pharmaceutical composition is selected from injectables, tablets, or capsules.

[0043] In some embodiments, the formulation of the pharmaceutical composition is selected from a liquid injectable or a lyophilized powder injectable.

[0044] In some embodiments, the drug comprises a pharmaceutically acceptable carrier.

[0045] In some embodiments, the carrier is selected from the group consisting of excipients, diluents, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and / or stabilizers.

[0046] In a fifth aspect, the present invention provides a method for treating or preventing inflammatory bowel disease. The method comprises administering an effective amount of a fusion protein according to the first aspect of the present invention, or a pharmaceutical composition according to the fourth aspect of the present invention, to a subject.

[0047] In some embodiments, the inflammatory bowel disease is selected from ulcerative colitis, Crohn's disease, or indeterminate colitis.

[0048] In some embodiments, the fusion protein or the pharmaceutical composition is administered by injection.

[0049] In some embodiments, the administration is selected from subcutaneous injection or intravenous injection.

[0050] In some embodiments, the administration is administered by subcutaneous injection.

[0051] In some embodiments, the dosage of the fusion protein is 3×10 4 IU / ses ~1X10 6 It is IU / time.

[0052] In some embodiments, based on the fusion protein in the pharmaceutical composition, the dosage of the pharmaceutical composition is 3×10 4 IU / ses ~1X10 6 It is IU / time.

[0053] In some embodiments, the fusion protein is administered once every 7 to 28 days.

[0054] In some embodiments, the fusion protein is administered once every 14 to 28 days.

[0055] In some embodiments, the pharmaceutical composition is administered once every 7 to 28 days.

[0056] In some embodiments, the pharmaceutical composition is administered once every 14 to 28 days.

[0057] In a sixth aspect, the present invention provides a use of the fusion protein according to the first aspect of the present invention in treating or preventing inflammatory bowel disease.

[0058] In a seventh aspect, the present invention provides a use of a pharmaceutical composition according to the fourth aspect of the present invention for treating or preventing inflammatory bowel disease.

[0059] In the eighth aspect, the present invention provides a use of the fusion protein according to the first aspect of the present invention in the manufacture of a drug for treating or preventing inflammatory bowel disease.

[0060] In a ninth aspect, the present invention provides a use of a pharmaceutical composition according to the fourth aspect of the present invention in the manufacture of a drug for treating or preventing inflammatory bowel disease. Effects of the invention

[0061] The interleukin 2 fusion protein provided by the present invention has high biological activity and a longer plasma half-life.

[0062] The interleukin 2 fusion protein provided by the present invention can be used to treat or prevent inflammatory bowel disease.

[0063] The interleukin 2 fusion protein provided by the present invention enhances the therapeutic effect of inflammatory bowel disease and further improves diarrhea and colon lesions.

[0064] The interleukin 2 fusion protein provided in the present invention can significantly extend the interval between drug administrations by slowing down hydrolysis in the patient's body, enhance therapeutic effects, and reduce the total amount of drug administered during treatment, thereby reducing patient pain and treatment costs, as well as reducing the occurrence of side effects, improving patient compliance and quality of life, and bringing hope to the recovery of health of patients with inflammatory bowel disease. Brief explanation of the drawing

[0065] Figure 1 shows the results of reduction electrophoresis analysis of the supernatant of well plate cultures screened for the IL-2-HSA / CHO-K1 clone, with a loading amount of 20 μL of supernatant. Figure 2 shows the results of non-reducing electrophoresis analysis of the supernatant of fed-batch culture D13 screened for the IL-2-HSA / CHO-K1 clone and the protein expression results of each cell line, with a loading amount of 5 μL of supernatant. Figure 2A shows the results of non-reducing electrophoresis analysis of the supernatant of fed-batch culture D13 screened for the IL-2-HSA / CHO-K1 clone; Figure 2B shows the protein expression results of each cell line. Figure 3 shows the results of non-reducing electrophoresis analysis of the supernatant of fed-batch culture D13, which was subjected to monoclonal screening for IL-2-HSA / CHO-K1 clone #9, and the protein expression results of each cell line, with a loading amount of 3 μL of supernatant. Figure 3A shows the results of non-reducing electrophoresis analysis of the supernatant of fed-batch culture D13, which was subjected to monoclonal screening for clone #9; Figure 3B shows the protein expression results of each cell line. Figure 4 shows the results of non-reducing electrophoresis analysis of the supernatant of fed-batch culture D13, which was subjected to monoclonal screening for IL-2-HSA / CHO-K1 clones #9-6, and the protein expression results of each cell line, with a supernatant loading amount of 2 μL. Figure 4A shows the results of non-reducing electrophoresis analysis of the supernatant of fed-batch culture D13, which was subjected to monoclonal screening for IL-2-HSA / CHO-K1 clones #9-6; Figure 4B shows the protein expression results of each cell line. Figure 5 is the IL-2-HSA / CHO-K1 cell culture kinetics curve. Fed-fed culture was performed in a 125 mL shaking flask, and the initial cell density was 0.3 x 10⁻⁶ 6 The cell count is 1 cell / mL, the initial culture volume is 25 mL, and the maximum viable cell density is 19.3 x 10⁻⁶ 6 Reached cell / mL. Figure 6 shows the protein expression kinetic curves of supernatant D7~D13 obtained from IL-2-HSA / CHO-K1 cells through a low-density inoculation culture process. Figure 7 shows the results of non-reducing electrophoresis of the supernatant of IL-2-HSA / CHO-K1 cell expression, with a loading amount of 1 μL of supernatant. Figure 8 is the IL-2-HSA / CHO-K1 cell culture kinetics curve. Fed-fed culture was performed in a 1L shaking flask, and the initial cell density was 2×10 6 The cell count is 1 cell / mL, the initial culture volume is 300 mL, and the maximum viable cell density is 16×10⁻¹⁰ 6 Reached cell / mL. Figure 9 shows the protein expression kinetic curves of supernatant D0~D10 obtained from IL-2-HSA / CHO-K1 cells through a high-density inoculation culture process. Figure 10 shows the results of non-reducing electrophoresis analysis of the supernatant of IL-2-HSA / CHO-K1 cell expression, with a loading amount of 5 μL of supernatant. Figure 11 shows the NK-92 proliferation curve induced by IL-2-HSA stimulation. Figure 12 shows the CTLL-2 proliferation curve induced by IL-2-HSA stimulation. Figure 13 shows the effect of IL-2-HSA fusion protein on the survival rate of a DSS-induced C57BL / 6 mouse colitis model during the recovery period (D8–D15). Figure 14 shows the effect of IL-2-HSA fusion protein on body weight change in a DSS-induced C57BL / 6 mouse colitis model. Figure 15 shows the effect of IL-2-HSA fusion protein on fecal characteristics of a DSS-induced C57BL / 6 mouse colitis model. Figure 16 shows the effect of IL-2-HSA fusion protein on the degree of hematochezia in a DSS-induced C57BL / 6 mouse colitis model. Figure 17 is a photograph of a colon sample from a C57BL / 6 mouse euthanized at D8. Figure 18 shows the effect of IL-2-HSA fusion protein on colon length in a DSS-induced C57BL / 6 mouse colitis model. Figure 19 shows the effect of IL-2-HSA fusion protein on colon weight in a DSS-induced C57BL / 6 mouse colitis model. Figure 20 shows the effect of IL-2-HSA fusion protein on spleen weight and organ counts in a DSS-induced C57BL / 6 mouse colitis model. Figure 21 shows the effect of IL-2-HSA fusion protein on the splenic lymphocyte subpopulation in a DSS-induced C57BL / 6 mouse colitis model. Figure 22 shows the spleen T in a colitis model mouse. reg (CD4 + CD25 + Foxp3 + This shows the representative flow cytometry detection results of the cell (D8). Figure 23 shows the spleen T in a colitis model mouse. reg (CD4 + CD25 + Foxp3 + This shows the representative flow cytometry detection results of ) cells (D15). Figure 24 shows the results of measuring the binding ability of IL-2-HSA fusion protein to IL-2R in various species (human, dog, rat, mouse). Figure 25 shows T in human PBMC reg (CD3 + CD4 + CD25 + CD127 low / - This shows the results of the in vitro proliferation effects of IL-2-HSA fusion protein and rhIL-2 on subgroups. Figure 26 shows T in human PBMC reg (CD3 + CD4 + CD25 + CD127 low / - This shows the results of flow cytometry analysis for the subgroup. Specific details for implementing the invention

[0066] The present invention will be further explained below through examples. It should be understood that the examples of the present invention are intended only to explain the invention and not to limit it, and that all simple improvements to the invention based on the concept of the present invention fall within the scope of protection of the present invention.

[0067] The inventors [discuss] regulatory T cells (T reg We discovered that a decrease in the level of ) is associated with increased disease severity and can predict disease progression and survival, suggesting that they could be potential therapeutic targets. In addition, T reg The production, activation, and survival of depend entirely on Interleukin 2 (IL-2). Based on this, the inventors conducted the following experiments.

[0068] definition

[0069] As used in this invention, the term "fusion protein" refers to a biologically active polypeptide (usually a TCR or antibody) covalently linked (i.e. fused) to an effector molecule (usually a protein or peptide sequence). If desired, the fusion molecule may be fused at one or multiple locations via a linking peptide sequence. Alternatively, a linking peptide may be used to aid in the construction of the fusion molecule. A particularly preferred fusion molecule is a fusion protein. Generally, fusion molecules may also include conjugated molecules.

[0070] The terms "expression vector" and "expression structure" as used in this invention may be used interchangeably. When the isolated nucleic acid molecule is connected to the vector, the nucleic acid sequence may be directly or indirectly connected to a regulatory element on the vector, provided that such a regulatory element can regulate the translation or expression of the nucleic acid molecule. Such a regulatory element may originate directly from the vector itself or may be exogenous, that is, not originate from the vector itself. In other words, the nucleic acid molecule is operably connected to the regulatory element. In this specification, "operably connected" means that a foreign gene is connected to the vector so that a regulatory element within the vector, such as a transcriptional regulatory sequence and a translational regulatory sequence, can perform the intended function of regulating the transcription and translation of the foreign gene. Of course, polynucleotides encoding the heavy and light chains of antibodies may be inserted independently into different vectors, but are generally inserted into the same vector. Commonly used vectors may include plasmids, phages, etc.

[0071] Compared to the sequence, the sequence used in the present invention and "at least 90% sequence identity" may include having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity and having the same or similar functionality.

[0072] The term "pharmaceuticalally acceptable carrier" as used in the present invention includes any biocompatible solvent, dispersion medium, coating agent, antimicrobial agent, antifungal agent, isotonic agent, and absorption retardant, etc. Specific examples may be one or more of water, saline solution, phosphate-buffered saline solution, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, the pharmaceutically acceptable carrier may include sugars, polyols (e.g., mannitol, sorbitol), or isotonic agents such as sodium chloride. Of course, the pharmaceutically acceptable carrier may include trace amounts of auxiliary substances, such as humectants, emulsifiers, preservatives, or buffers, to extend the shelf life or efficacy of the protein.

[0073] The term "subject" as used in the present invention refers to a subject who suffers from inflammatory bowel disease or is at risk of developing inflammatory bowel disease. The subject may be an animal, preferably a mammal, and more preferably a human.

[0074] The term "treatment" as used in the present invention includes actions occurring in a subject suffering from a specific disease, disability, or condition, and actions that reduce the severity of the disease, disability, or condition, or delay or slow the progression of the disease, disability, or condition.

[0075] The term "prevention" as used in the present invention means reducing the likelihood (or susceptibility) to at least a disease or condition (i.e., causing at least one clinical symptom of a disease that has not yet occurred in a patient who may have been exposed to or susceptible to the disease but has not yet experienced or exhibited symptoms of the disease).

[0076] As used in the present invention, the term "effective amount" refers to an amount of therapeutic agent, prophylactic agent, and / or diagnostic agent sufficient to treat, alleviate, improve, alleviate symptoms, prevent, delay onset, inhibit progression, reduce severity, and / or reduce incidence of the disease, disorder, and / or condition when applied to a subject who has or is at risk of having a disease, disorder, and / or condition.

[0077] As used in this invention, the term "dosage" refers to an amount capable of improving or delaying the progression of a disease, degenerative, or impaired condition. This may vary depending on the specific disease to be treated, as well as other factors including age, weight, health, severity of symptoms, route of administration, frequency of treatment, and whether other drugs are used concurrently during treatment.

[0078] The term "inflammatory bowel disease" or "IBD" as used in this invention refers to an idiopathic intestinal inflammatory disease affecting the small intestine, rectum, and colon. Clinical symptoms include diarrhea and abdominal pain, and sometimes bloody stools may occur. Although the cause and pathogenesis of this disease are not fully and clearly known, it is known that inflammatory responses resulting from abnormal reactions of the intestinal mucosal immune system play a significant role in the development of IBD, and that it is caused by the interaction of various factors such as environment, genetics, infection, and immune factors.

[0079] The term "ulcerative colitis" as used in the present invention is a chronic, non-specific, non-infectious inflammatory bowel disease affecting the mucosa and submucosa of the large intestine. It is characterized by a continuous and extensive distribution and extends through various pathways starting from the rectum to the cecum as continuous mucosal ulcers of the rectum and colon.

[0080] As used in this invention, the term "Crohn's disease" refers to discontinuous, full-thickness inflammation that can affect the entire digestive tract, most commonly occurring in the terminal small intestine, colon, and perianal area. Symptoms of the disease generally include abdominal pain, diarrhea (bleeding may occur in severe cases), fever, and weight loss.

[0081] The term "indeterminate colitis" used in the present invention refers to a small number of patients for whom it is difficult to determine whether the inflammatory bowel disease they are suffering from is Crohn's disease or ulcerative colitis, and this type is currently defined as indeterminate colitis.

[0082] The solution of the present invention will be described with reference to the following examples. Those skilled in the art will understand that the following examples are used merely to illustrate the invention and should not be construed as limiting the scope of the invention. In the following description, descriptions of known technology are omitted to avoid unnecessary confusion regarding the concept of the invention. Such technology is described in numerous publications, such as *Molecular Cloning: A Laboratory Manual*, Forth Edition (Cold Spring Harbor Laboratory Press).

[0083] If specific techniques or conditions are not specified in the examples, the examples shall be carried out using techniques or conditions described in the literature of the relevant field or according to product specifications. If the manufacturer of the reagents or equipment used is not specified, they are all general products available on the market.

[0084] The inventors [discuss] regulatory T cells (T reg We discovered that a decrease in the level of ) is associated with increased disease severity and can predict disease progression and survival, suggesting that they could be potential therapeutic targets. In addition, T reg The production, activation, and survival of depend entirely on the cytokine Interleukin 2 (IL-2). Based on this, the inventors conducted the following experiments.

[0085] Examples of terms and expressions included in the embodiments are as follows.

[0086] IL-2-HSA fusion protein is an abbreviation for the interleukin 2 fusion protein.

[0087] IL-2 refers to human interleukin 2.

[0088] HSA stands for human serum albumin.

[0089] 125 Ala IL-2 represents an interleukin 2 mutation in which the amino acid at position 125 is alanine. For specifics, refer to the amino acid sequence indicated in SEQ ID NO. 1.

[0090] D0, D2, D4, D8, D 15 means 0 days, 2 days, 4 days, 8 days, and 15 days, respectively.

[0091] DSS stands for sodium dextran sulfate.

[0092] The DSS period refers to the period of drinking water with added sodium dextran sulfate.

[0093] sc stands for subcutaneous injection.

[0094] The commercially available short-acting IL-2 product name is Xinjier, and it is manufactured by Beijing SL Pharm Co., LTD.

[0095] Examples

[0096] Example 1: Establishment of a stably transfected cell line

[0097] One day before transfection, the density of CHO-K1 cells was 0.5×10 6The cell density was adjusted to 1 cell / mL. On the day of transfection, a linearized high-concentration endotoxin-free plasmid was prepared, and the density and viability of CHO-K1 cells were measured to confirm that the cell viability was 97% or higher. After washing the CHO-K1 cells twice with CD CHO medium, an electroporation reaction system was constructed using 700 μL of cell suspension + 40 μg of plasmid; after thorough mixing, the mixture was transferred to a 4 mm electrode cup. The electrode cup was placed in the electroporator, the electroshock parameters were set to 300 V and 1000 μF, and one electroshock was performed. Subsequently, the electroshocked cell suspension was transferred to preheated fresh CD CHO medium and incubated at 37°C for 20 minutes. The cultured cell suspension was uniformly inoculated into a 96-well plate, and 24 hours after transfection, pressure was applied and CD CHO medium containing methionine sulfoxymine (MSX) was added. The cells were cultured statically at a final screening pressure of 25–50 μM MSX, 5% CO2, and 37°C.

[0098] Example 2: Screening of high-expression monoclonal cell lines

[0099] After the monoclones in the 96-well plate grew to a suitable size, monoclonal screening was initiated. All clones were transferred to a new 96-well plate and cultured statically at 37°C under 5% CO2. After the wells were filled with cells, the supernatant from the well plate was taken and subjected to reduction electrophoresis to detect the expression of the fusion protein. The 9 clone lines with the highest expression levels (see Fig. 1) were screened and gradually amplified and cultured in a shaking flask. The 9 clones were fed-batch cultured in a 25 mL shaking flask. The culture supernatant was harvested and identified by non-reduction electrophoresis (see Fig. 2A). The protein expression levels of the mediating cell lines were calculated (see Fig. 2B), and cell line #9 with the best expression was screened. Monoclonal cell line screening was performed on cell line #9 using the limiting dilution method, and 11 high-expression cell lines were screened by seeding them into 96-well plates at a density of 0.3 cells / well. The cells were fed-batch cultured in 25 mL shaking flasks, the supernatant was identified by non-reducing electrophoresis (see Fig. 3A), and the protein expression levels of the mediating cell lines were calculated (see Fig. 3B). Cell line #9-6, which showed the best expression, was screened, and monoclonal cell line screening was performed again on cell line #9-6 using the limiting dilution method. Seven high-expression cell lines were screened, and the cells were fed-batch cultured in 25 mL shaking flasks. The supernatant was identified by non-reducing electrophoresis (see Fig. 4A), and the protein expression levels of the mediating cell lines were calculated (see Fig. 4B). Cell line #9-6-7, which has good stability and high expression levels, was used as the stable and high-expression cell line IL-2-HSA / CHOK1.

[0100] Example 3: 125 mL shaking flask fed-batch culture of a stable cell line

[0101] On the day of initiation of IL-2-HSA / CHO-K1 cell culture and expression, 25 mL of basal medium containing 25–50 μM MSX was added to a 125 mL shaking flask in a 0.3 x 10⁻³ flask. 6Canine cells / mL were inoculated and recorded as D0, and cultured in a shaker at 135 rpm under 5% CO2 at 37°C. Sampling and counting began from inoculation D4, and the cell density was 10×10 6 When the cell count reached 1 cell / mL, the culture temperature was lowered to 33℃. Feed-batch culture was started by supplying supplemental medium starting from D5, and the glucose concentration was adjusted to 3–4 g / L. Culture was terminated on D13, the cell culture supernatant was collected, and the measured expression level of the fusion protein was 4.36 mg / mL.

[0102] The IL-2-HSA / CHO-K1 cell culture kinetics curve is shown in Fig. 5. As can be seen from Fig. 5, in the early stages of culture, the cells are in the logarithmic growth phase, causing a rapid increase in density; in the later stages of culture, the cells enter the protein synthesis phase, and the cell density tends to stabilize. The maximum viable cell density is 19.3 x 10⁻¹⁰. 6 It reached 1 cell / mL.

[0103] The expression kinetic curve of the IL-2-HSA / CHO-K1 cell supernatant is shown in Figure 6. As can be seen from Figure 6, the cell protein expression level showed an increasing trend as the culture period increased from D7 to D13. The expression level at D13 was 4.36 mg / mL.

[0104] The non-reducing electrophoresis analysis of the supernatant for IL-2-HSA / CHO-K1 cell expression is shown in Figure 7. As can be seen from Figure 7, the cell protein expression level showed an increasing trend as the culture period increased from D7 to D13. The band of the target protein was single, and there were no non-specific bands.

[0105] Example 4: 1L shaking flask fed-batch culture of a stable cell line

[0106] 50 mL of IL-2-HSA / CHO-K1 cells in a shaking flask 2 x 10 6Cells were inoculated at a density of 1 cell / mL and cultured in a shaker at 135 rpm under 5% CO2 at 37°C. After sampling and counting daily and observing cell status, basal medium containing 25–50 μM MSX was supplied, and the cell density was increased by 2×10 times daily until the cell culture volume reached 300 mL. 6 After adjusting to cells / mL, the supply of basal medium was stopped and culture continued; this point was recorded as D0. Sampling and counting were performed daily, and 1 mL of culture supernatant was retained. The cell density was 6×10⁻¹⁰ 6 ~7X10 6 When the cell count reached 10 cells / mL, the culture temperature was lowered to 33℃. Feed-batch culture was started by supplying supplemental medium starting from D2, and the glucose concentration was adjusted to 3 g / L. Culture was terminated on D10, and after collecting the supernatant of the cell culture medium, the protein expression levels in the supernatants from D0 to D10 were measured. The fusion protein expression level measured by harvesting the supernatant on D10 was 3.12 mg / mL.

[0107] The IL-2-HSA / CHO-K1 cell culture kinetics curve is shown in Fig. 8. As can be seen from Fig. 8, cell density increases rapidly during the logarithmic growth phase; in the late stages of culture, as the cells enter the protein synthesis phase, cell density tends to stabilize. The maximum viable cell density is 16 x 10⁻¹⁰. 6 Reached cells / mL.

[0108] The kinetic curve of IL-2-HSA / CHO-K1 cell supernatant expression is shown in Figure 9. As can be seen from Figure 9, the cell protein expression level showed a tendency to increase as the number of culture days increased.

[0109] The non-reducing electrophoresis analysis of the supernatant for IL-2-HSA / CHO-K1 cell expression is shown in Figure 10. Cell protein expression levels showed an increasing trend as the culture period increased. The target protein band was single, and there were no non-specific bands.

[0110] Compared to the conventional shaking flask fed-batch culture, the cell density and cell viability of the present invention are very high, and as shown in FIG. 8, the cell density on days 6 to 10 is 14×10 6 ~16X10 6 It reached 1 cell / mL, and the expression level of the cell protein of the present invention was also very high; as shown in Fig. 9, the expression level on day 10 was 3.12 mg / mL. In the present invention, the cell density was 14X10 6 ~16X10 6 Expression levels are high even at cell / mL, indicating that cellular activity remains very good at this cell density.

[0111] Example 5: Detection of the proliferative effect of IL-2-HSA on NK-92 cells

[0112] In this embodiment, NK-92 cells (ATCC® CRL-2407 TM The biological activity of IL-2-HSA was evaluated using an IL-2-dependent NK cell line derived from peripheral blood monocytes of a 50-year-old male patient with malignant non-Hodgkin lymphoma.

[0113] (1) NK-92 cells that had been cryopreserved in a liquid nitrogen tank were removed, revived, and cultured until the logarithmic growth phase.

[0114] (2) A sufficient amount of cells were collected by centrifugation, resuspended in complete medium without IL-2, and starved for 24 hours.

[0115] (3) Centrifuge starved cultured NK-92 cells, resuspend in IL-2-free complete medium and count; cell density 5×10 5 It was adjusted to cells / mL and added to a 96-well plate at a volume of 90 μL per well.

[0116] (4) Preparation of sample solutions: IL-2-HSA samples and rhIL-2 (R&D, Cat. No. 202-IL) were pre-diluted to 50.67 nM in the medium and then diluted to 9 concentrations in a 4-fold gradient. 10 μL / well was added to the corresponding wells of a 96-well plate, 3 duplicate wells were set for each concentration, and 10 μL / well of the medium was added to the negative control and mixed thoroughly.

[0117] (5) After culturing for 72 hours at 37°C and 5% CO2, 20 μL / well of the dissolved and mixed MTS detection reagent was added to the above 96-well plate, shaken and mixed, and then placed in a cell incubator at 37°C and 5% CO2 for 1 to 4 hours.

[0118] (6) After incubation, shake to mix; and measure the absorbance at 490 nm using a microplate reader.

[0119] (7) Analyze the data using GraphPad Prism 8 software, plotting the log value of the drug concentration X on the horizontal axis and OD 490 The drug action curve was fitted using four parameters with as the vertical axis. The obtained EC 50 The values ​​are as shown in Table 1, and the growth curve is as shown in Fig. 11.

[0120] IL-2(R&D) IL-2-HSA EC 50 (nM) 0.0420 0.01047

[0121] As can be seen from Figure 11, IL-2-HSA induced NK-92 cell growth in a dose-dependent manner. Under these experimental conditions, the activity of IL-2-HSA in stimulating NK-92 proliferation was superior (about 4 times) to the activity of equimolar rhIL-2.

[0122] Example 6: Detection of the proliferative effect of IL-2-HSA on CTLL-2 cells

[0123] In this embodiment, CTLL-2 cells (ATCC® TIB214 TM The biological activity of IL-2-HSA was evaluated using a mouse cytotoxic lymphocyte cell line (IL-2 dependent).

[0124] Referring to the method for measuring the biological activity of human interleukin-2 in the Chinese Pharmacopoeia (CTLL-2 cell / MTT colorimetric method), CTLL-2 cells were inoculated into a 96-well plate at a density of 30,000 / well, serially gradient diluted national standard material and IL-2-HSA were added, and the cells were cultured for 18–24 hours at 37°C and 5% CO2 conditions. After adding the MTS detection reagent, the cells were cultured for 1–4 hours, and after shaking, the absorbance at a wavelength of 490 nm was measured using a microplate reader.

[0125] Analyze the data using GraphPad Prism 8 software, plot the log value of dilution X on the horizontal axis, and OD 490 The drug action curve was fitted using four parameters with as the vertical axis. The obtained EC 50 The values ​​are as shown in Table 2, and the growth curve is as shown in Fig. 12.

[0126] The biological activity of IL-2-HSA was calculated according to the following formula:

[0127]

[0128] As a result of the calculation, the inactivity of IL-2-HSA is 8.38X10 6 It is IU / mg, and the inactive IL-2 as specified in the pharmacopoeia (1X10 7 It is similar to (IU / mg or more), but since the difference in molecular weight between the two is about 5 times, the inactivity of IL-2-HS is higher.

[0129] IL-2 (National Standard Material) IL-2-HSA EC 50 (Dilution ratio) 13.90 11.68

[0130] Example 7: Preventive and Therapeutic Effects of IL-2-HSA Fusion Protein on a DSS-Induced Ulcerative Colitis Mouse Model

[0131] 1. Animal selection and grouping, model establishment, and administration regimen:

[0132] 108 male C57BL / 6 wild-type mice (7–8 weeks old) were randomly divided into 7 groups according to body weight: G1 was a healthy control, G2 was a model control, and G3 was a commercially available short-acting IL-2 control (125 Ala IL-2, 3Х10 4 IU / animal / d, sc), G4 is the HSA control group (0.3 mg / kg / 4d, sc), G5 is the IL-2-HSA fusion protein low-dose group (1X10 4 IU / animal / 4d, sc), G6 is the IL-2-HSA fusion protein medium-dose group (3X10 4 IU / animal / 4d, sc), G7 is the high-dose IL-2-HSA fusion protein group (1X10 5 IU / animal / 4d, sc). Of these, the healthy control group consisted of 12 animals, and the other groups each consisted of 16 animals.

[0133] To induce a colitis model, model animals in each group began drinking water supplemented with 2.5% (wt / vol) dextran sodium sulfate (DSS) starting on Day 2 (D2) for 6 days (Days 2–7 were defined as the DSS period). From Day 8, drinking water without DSS was provided. The corresponding drugs or sterile water for injection were administered subcutaneously before and after modeling (IL-2-HSA fusion protein and HSA were administered once every 4 days for a total of 3 times across Days 0, 4, and 8; 125 Ala IL-2 was administered once a day for a total of 9 times from D0 to D8.

[0134] 2. Experimental Index

[0135] 2.1 General Clinical Observations

[0136] During the experimental period, general clinical observations were performed twice daily on the animals that survived in each of the above groups, and the observations included, but were not limited to, physical signs, mental state, behavioral activity, food and water intake, stool characteristics, changes in body weight (main observations), or other abnormal conditions.

[0137] 2.2 weight

[0138] The body weight of each animal in the above group was measured: once before grouping, once a day during the experiment, once before euthanasia, and also when it died unexpectedly.

[0139] 2.3 Characteristics of Stool and Amount of Blood in Stool

[0140] Starting from the day of modeling (D2), the fecal characteristics and blood in the stool of the surviving animals in each of the above groups were scored once a day. The rules were as follows: Fecal characteristics: 0 = Normal; 1 = Slightly soft; 2 = Very soft; 3 = Diarrhea; 4 = Heteroplastic diarrhea; Blood in the stool: 0 = Fecal occult blood negative; 1 = Fecal occult blood positive; 2 = Visible blood in the stool; 3 = Visible rectal bleeding.

[0141] 2.4 Measurement of Colon Length and Weight

[0142] After euthanizing half of the animals in each group at the end of the DSS period (D8) and the remaining animals in each group at the end of the experiment, the large intestines were dissected to measure the length and weight of the colon.

[0143] 2.5 Spleen Graft Measurement and Lymphocyte Subpopulation Analysis

[0144] After euthanizing half of the animals in each group at the end of the DSS period (D8) and the remaining animals in each group at the end of the experiment, the spleens were aseptically removed and weighed, a spleen single-cell suspension was prepared, and CD8 was analyzed through flow cytometry. + T cells, CD4 + T cells and T reg Cell (CD4 + CD25 + Foxp3 + The ratio was measured.

[0145] 3. Statistical Analysis

[0146] Measurement data were described using the mean and standard deviation (Mean±SD), graphs were created using GraphPad Prism 8 software, and statistical analysis was performed using SPSS 25. The procedure was as follows: a homogeneity of variance test was performed on the data; one-way ANOVA was conducted on data with homogeneity of variance (P>0.05); LSD multiple comparison analysis was performed on data with a test of variance (P≤0.05); Kruskal-Wallis nonparametric test was applied to data without an ANOVA (P≤0.05); and Mann-Whitney 2 comparison analysis was performed on data with a chi-square test (P≤0.05), with P≤0.05 considered a significant difference. Differences in detection indicators were primarily investigated between the healthy control group and the model control group, and between the model control group and each drug administration group.

[0147] 4. Experimental Results

[0148] 4.1 Animal Survival

[0149] In the experiment, the recovery period (D8~D 15 A total of 8 animals died during the period, including 4 in the model control group, 125One animal was included in the Ala IL-2 group, two in the HSA control group, and one in the IL-2-HSA medium-dose group. Until the end of the experiment, the animal mortality rates for the healthy control group (G1), model control group (G2), commercially available short-acting IL-2 control group (G3), HSA control group (G4), IL-2-HSA fusion protein low-dose group (G5), IL-2-HSA fusion protein medium-dose group (G6), and IL-2-HSA fusion protein high-dose group (G7) were 0%, 50%, 12.5%, 25%, 0%, 12.5%, and 0%, respectively. The effect of IL-2-HSA fusion protein on the survival rate of the DSS-induced C57BL / 6 mouse colitis model during the recovery period (D8–D15) is shown in Figure 13. As shown in Fig. 13, the IL-2-HSA fusion protein is 1×10 4 IU / animal to 1X10 5 Prophylactic administration of an IU / animal dose once every 4 days (i.e., G5~G7: low, medium, and high doses of IL-2-HSA fusion protein) can reduce the mortality rate of DSS-induced colitis mice.

[0150] 4.2 weight

[0151] Changes in animal body weight for the seven groups are shown in Figure 14. Figure 14 illustrates the effect of IL-2-HSA fusion protein on body weight change in a DSS-induced C57BL / 6 mouse colitis model. As can be seen from Figure 14, the body weight of healthy control animals (G1) showed a variable increasing trend during the experimental period. The body weight of animals in each modeling group (G2–G7) decreased significantly from day 3 (D4) after drinking DSS water until death occurred on D10, after which the body weight of surviving animals showed a slow recovery trend. Among these, body weight loss in the high-dose IL-2-HSA fusion protein group (G7) was significantly inhibited, and the degree of body weight loss was lower compared to the model control group (G2). Two-way ANOVA was performed using SPSS statistical software, and the combined effect of treatment and time was found to be significant (P<0.05).

[0152] According to Figure 14, the IL-2-HSA fusion protein improved body weight loss in a dose-dependent manner in mice with DSS-induced ulcerative colitis. In addition, the IL-2-HSA fusion protein 1×10 5 It has a significant inhibitory effect on body weight loss in DSS-induced colitis mice at an IU / animal dose (G7 IL-2-HSA fusion protein high-dose group).

[0153] 4.3 Characteristics of Stool and Amount of Blood in Stool

[0154] Statistical results regarding the stool characteristics and blood scores in the stool of seven animal groups are shown in Figures 15 and 16. Figure 15 shows the effect of IL-2-HSA fusion protein on the stool characteristics of a DSS-induced C57BL / 6 mouse colitis model. Figure 16 shows the effect of IL-2-HSA fusion protein on the degree of bloody stool in a DSS-induced C57BL / 6 mouse colitis model. As can be seen from the results in Figures 15 and 16, compared to the healthy control group (G1), the animals in each modeling group (G2–G7) showed colitis-related clinical symptoms such as loose stools and bloody stools starting from day 2 (D2) after drinking DSS water, and the clinical scores continued to increase until they gradually recovered after the provision of DSS water was stopped.

[0155] Animals in the low, medium, and high dose groups (G5~G7) of IL-2-HSA fusion protein showed clinical symptoms related to colitis, such as loose stools and bloody stools, starting from day 2 (D2) after drinking DSS water; however, these symptoms were significantly milder compared to the model control group (G2), showing a consistent dose-effect relationship. In particular, the stool characteristic scores were significantly lower in the medium dose group (G6) of IL-2-HSA fusion protein at D6~D7 and in the high dose group (G7) of IL-2-HSA fusion protein at D6~D9, and the difference was statistically significant (P<0.05).

[0156] According to Figures 15 and 16, the IL-2-HSA fusion protein improved diarrhea in mice with DSS-induced ulcerative colitis in a dose-dependent manner. In particular, the IL-2-HSA fusion protein 3×10 4 IU / mari (G6) and 1X10 5 At a dose of IU / mice (G7), the clinical symptoms of DSS-induced ulcerative colitis mice were significantly improved.

[0157] 4.4 Length and Weight of the Large Intestine

[0158] In this experiment, colon tissues were evaluated for half of the animals in each group at the end of the DSS period (D8) and for the remaining animals in each group at the end of the recovery period (D15).

[0159] Colon samples from seven groups of animals are shown in Fig. 17, colon length in Fig. 18, and colon weight in Fig. 17. Fig. 17 is a photograph of a colon sample from C57BL / 6 mice euthanized at D8. Fig. 18 shows the effect of IL-2-HSA fusion protein on colon length in a DSS-induced C57BL / 6 mouse colitis model. Fig. 19 shows the effect of IL-2-HSA fusion protein on colon weight in a DSS-induced C57BL / 6 mouse colitis model.

[0160] As can be seen from Figs. 18 and 19, the colon lengths at D8 for animals in each group—healthy control (G1), model control (G2), commercially available short-acting IL-2 control (G3), HSA control (G4), low-dose IL-2-HSA fusion protein group (G5), medium-dose IL-2-HSA fusion protein group (G6), and high-dose IL-2-HSA fusion protein group (G7)—were 7.43±0.67, 5.29±0.48, 4.76±0.99, 5.36±0.68, 5.39±0.47, 6.04±0.91, and 6.47±1.55 cm, respectively, and the colon weights (including contents) were 0.37±0.05, 0.31±0.07, and 0.21±0.04, respectively. The values ​​were 0.30±0.07, 0.30±0.06, 0.38±0.09, and 0.43±0.16g; the colon length of the model control group (G2) animals was significantly shorter compared to the healthy control group (G1), and the difference was statistically significant (P<0.05); in the low, medium, and high dose groups of IL-2-HSA fusion protein (G5~G7), a dose-dependent increase in colon length was observed compared to the model control group (G2), but no statistically significant difference was observed; 125 Compared to the Ala IL-2 group (G3), the colon weight of animals in the medium-dose IL-2-HSA fusion protein group (G6) significantly increased (P<0.01), and the colon length and colon weight of animals in the high-dose IL-2-HSA fusion protein group (G7) significantly increased (P<0.01). These results were obtained at the same dose (3X10 4 The IL-2-HSA fusion protein (IU / animal) exhibits a more significant therapeutic effect than commercially available short-acting IL-2, demonstrating a statistical difference. Therefore, the IL-2-HSA fusion protein of the present invention [is superior to] the existing 125 Compared to Ala IL-2, it not only extends the drug administration interval but also has a better therapeutic effect.

[0161] According to Figures 18 and 19, the IL-2-HSA fusion protein improved colon shortening in a dose-dependent manner in mice with DSS-induced ulcerative colitis. The IL-2-HSA fusion protein is 3×10 4 IU / mari and 1x10 5 Colon shortening in DSS-induced colitis mice can be prevented at a dose of IU / mice (G6 and G7).

[0162] 4.5 Spleen Weight and Organ Count

[0163] In this experiment, the spleen weight of half of the animals in each group was evaluated at the end of the DSS period (D8) and the remaining animals in each group were evaluated at the end of the recovery period (D15).

[0164] Spleen weight and spleen organ counts of the seven animal groups are shown in Figure 20. Figure 20 illustrates the effects of IL-2-HSA fusion protein on spleen weight and organ counts in a DSS-induced C57BL / 6 mouse colitis model. As can be seen from Figure 20, the spleen weight at D8 in each animal group was significantly increased in the high-dose IL-2-HSA fusion protein group compared to the model control group (0.11±0.01g vs. 0.08±0.01g, P<0.05), while the other groups showed levels similar to the model control group. Compared to the healthy control group (G1), the spleen organ count at D8 in the model control group (G2) animals was significantly increased (0.44±0.06% vs. 0.28±0.01%, P<0.05), but there was no significant difference between the other groups and the model control group.

[0165] 4.6 Analysis of Splenic Lymphocyte Subpopulations

[0166] In this experiment, the splenic lymphocyte subpopulations of half of the animals in each group were analyzed at the end of the DSS period (D8) and the remaining animals in each group were analyzed at the end of the recovery period (D15).

[0167] CD8 of the spleen of 7 groups of animals + T cells, CD4 +T cells and CD4 + T in T cells reg Cell (CD4 + CD25 + Foxp3 + The ratio of ) is shown in Fig. 21. Fig. 21 shows the effect of IL-2-HSA fusion protein on the splenic lymphocyte subpopulation in a DSS-induced C57BL / 6 mouse colitis model. In Fig. 21, the following can be confirmed:

[0168] In experiment D8, CD8 in the spleen of animals in the low, medium, and high dose groups (G5–G7) of IL-2-HSA fusion protein compared to the model control group (G2) + T cells and CD4 + The proportion of T cells did not show a significant change, but CD4 + CD4 in T cells + CD25 + Foxp3 + (Classical T reg All subgroups were significantly induced (P<0.05); 125 T in animals of the Ala IL-2 group reg The proportion of the subgroup also increased to some extent.

[0169] In Experiment D15, CD8 in the spleen of animals in the low, medium, and high dose groups (G5–G7) of IL-2-HSA fusion protein compared to the model control group + T cells and CD4 + The proportion of T cells decreased, but CD4 + T in T cells reg The proportion of the subgroup remained at a high level.

[0170] T for D8 and D15 of the 7 groups reg Representative detection results of the cells are shown in Figures 22 and 23. Figure 22 shows the spleen T in a colitis model mouse. reg (CD4 + CD25 + Foxp3 +This shows the representative flow cytometry detection results of ) cells (D8). Figure 23 shows the spleen T in a colitis model mouse. reg (CD4 + CD25 + Foxp3 + This shows the representative flow cytometry detection results of ) cells (D15).

[0171] According to FIGS. 22 and 23, the IL-2-HSA fusion protein is 1×10 4 IU / animal to 1X10 4 Peripheral T of mice at a dose of IU / animal reg It can elevate cell levels over a long period, and CD8 + It can contribute to reducing T-cell toxicity.

[0172] Example 8: Affinity analysis of IL-2-HSA fusion protein for IL-2R of various species

[0173] Interactions between IL-2-HSA fusion proteins and IL-2 receptors (IL-2R) of various species (human, dog, rat, and mouse) were detected and analyzed using Bio-Layer Interferometry (BLI). IL-2R of other species was captured using ProA or hFc probes, and the affinity between the IL-2-HSA fusion proteins and IL-2R of other species was confirmed by observing the binding and dissociation of the IL-2R attached to the ProA or hFc probes after diluting the IL-2-HSA fusion protein by different concentration gradients. The specific methods are as follows:

[0174] (1) Human IL-2R (hIL2R, BIOSYSTEMS, ILGH5257) and mouse IL-2R (mIL2R, BIOSYSTEMS, ILGM5253) were diluted to 30 nM and adsorbed onto a ProA probe at a speed of 400 rpm / min; rat IL-2R (rIL2R, self-made, 20220111, gene registration number: IL2Rα_P26897, IL2Rβ_P26896, IL2Rγ_AAH79343.1) and dog IL-2R (CaIL2R, self-made, 20220421, gene registration number: IL2Rα_O62802, IL2Rβ_F1PGA6, IL2Rγ_P40321) were diluted to 30 nM and adsorbed onto an hFc probe at a speed of 400 rpm / min.

[0175] (2) IL-2-HSA fusion protein was diluted in five concentration gradients starting from 20 nM, and the program was set so that the diluted IL-2-HSA fusion protein would bind to ProA or hFc attached to IL2R within a set time. After binding was complete, the probe complex was transferred to Q buffer without analytes to dissociate the bound analytes.

[0176] (3) The probe was immersed in the regeneration buffer to remove residual binding analytes.

[0177] (4) The regenerated probe was placed in a 15% sucrose protective solution and stored at room temperature.

[0178] The experimental results of the binding ability of IL-2-HSA fusion protein to various species of IL-2R are shown in Figure 24 and Table 3.

[0179] receptor Sample koff(1 / s) kon(1 / Ms) KD (M) hIL2R IL-2-HSA 0.000169 1.96E+06 0.86E-10 rIL2R IL-2-HSA 0.000549 1.77E+06 3.11E-10 mIL2R IL-2-HSA 0.000887 1.65E+06 5.38E-10 CaIL2R IL-2-HSA 0.000412 3.16E+06 1.30E-10

[0180] The above results show that 1) IL-2-HSA fusion protein binds to IL-2R in humans, rats, mice, and dogs, and that there is cross-species interaction. 2) The ranking of affinity between IL-2-HSA fusion protein and IL-2R in other species is human > dog > rat > mouse.

[0181] Example 9: T from healthy human PBMC reg Effects of IL-2-HSA fusion protein on in vitro cell proliferation

[0182] In this embodiment, using flow cytometry technology, IL-2-HSA fusion protein and commercial rhIL-2 (R&D, Product No.: 202-IL) were used to [treat] T of healthy human peripheral blood mononuclear cells (PBMCs). reg (CD3 + CD4 + CD25 + CD127 low / - The effects of the subgroups on in vitro proliferation were compared.

[0183] 1. Extraction of PBMC from human blood

[0184] Fresh anticoagulant blood from healthy humans was collected, and PBMCs were extracted using lymphocyte isolation solution (Ficoll-Paque PREMIUM, Brand: Cytiva, Product No.: 17-5442-02). 3–5 mL of lymphocyte isolation solution was placed in a 5 mL centrifuge tube, a diluted blood sample (a mixture of 3 mL peripheral blood and 3 mL PBS) was carefully added to the supernatant of the lymphocyte isolation solution, and after centrifugation at 400 g for 30–40 minutes, the pia cells were removed, washed twice with PBS, resuspended in RPMI-1640 medium + 10% FBS, and counted.

[0185] 2. Drug treatment

[0186] 1) Cell density in culture medium: 2×10 6 The concentration was adjusted to 1 / mL, and 1800μL was inoculated per well in a 12-well plate.

[0187] 2) IL-2-HSA fusion protein samples and rhIL-2 were pre-diluted to 10 μM using culture medium, then diluted to 6 different concentrations in a 10-fold gradient, and added to the corresponding wells of a 12-well plate at a rate of 200 μL / well. A blank control group was added at a corresponding rate of 200 μL / well of culture medium.

[0188] 3) Placed in a cell culture vessel at 37℃ and 5% CO2 and cultured continuously for 72 hours.

[0189] 3. Flow cytometry

[0190] 1) Transfer the cell suspension to a new Ep test tube, and the concentration is 2×10 6 It is 100 μL of cells per test tube.

[0191] 2) PB450 anti-human CD3 (BD, Product No.: 558124, 1:200), PC5.5 anti-human CD4 (BD, Product No.: 556924, 1:100), APC anti-human CD25 (BD, Product No.: 555434, 1:50), and PE anti-human CD127 (BD, Product No.: 557938, 1:100) were added in the corresponding proportions and established as a negative control, a single positive control, and an isotype control. Incubated at 4°C in the dark for 30 minutes.

[0192] 3) Centrifuge with 400 g for 5 minutes and wash twice with DPBS.

[0193] 4) It was resuspended in 300μL of DPBS and loaded into a flow cytometer for measurement.

[0194] 4. Experimental Results

[0195] T in human PBMC reg (CD3 + CD4 + CD25 + CD127 low / - The results of the in vitro proliferation effects of IL-2-HSA fusion protein and rhIL-2 on the ) subgroups are shown in Figures 25 and 26. Figure 25 shows T in human PBMCs on day 3. reg (CD3 + CD4 + CD25 + CD127 low / - This compares the in vitro proliferation of IL-2-HSA fusion protein and rhIL-2 in ) subgroups. Figure 26 shows T in human PBMCs reg (CD3+ CD4 + CD25 + CD127 low / - This shows the results of flow cytometry analysis for the subgroup.

[0196] According to Fig. 25, after treatment with IL-2-HSA fusion protein for 72 hours, T in human PBMCs reg The proportion of cells increased in a dose-dependent manner. Furthermore, the IL-2-HSA fusion protein was superior to rhIL-2 in human T reg It has biological activity that promotes cell proliferation.

[0197] Example 10: Pharmacokinetics of IL-2-HSA fusion protein in rats and dogs by single subcutaneous injection

[0198] In this example, the pharmacokinetics (PK) of IL-2-HSA fusion protein were studied in SD rats and beagles.

[0199] (1) Pharmacokinetics of single subcutaneous and intravenous injections in SD rats

[0200] In this experiment, a total of 40 SD rats with no prior history of drug administration were used and randomly divided into four groups of 10 animals each, with half being male and half female. Animals in groups 1–3 were administered a single subcutaneous injection into the nape, and the dosages for the three dose groups (low, medium, and high) were 5 × 10, respectively. 5 IU / kg, 1X10 6 IU / kg, 2Х10 6 The dosage is IU / kg, and animals in Group 4 were administered by tail vein injection; the dosage was 1×10⁻⁶ 6 The dosage was IU / kg, and all were administered at a volume of 2 mL / kg. PK blood samples were collected from experimental animals at the following time points:

[0201] Groups 1–3 (Subcutaneous injection groups): Before administration, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 30 hours, 36 hours, 48 ​​hours, 72 hours after administration;

[0202] Group 4 (Intravenous injection group): Before administration, 2 minutes, 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours after administration;

[0203] The concentration of IL-2-HSA in SD rat serum was quantitatively detected using a validated ELISA method. WinNonlin software (Phoenix TM Pharmacokinetic parameters were calculated by non-compartmental analysis using (version 8.1).

[0204] IL-2-HSA fusion protein was administered to SD rats at various doses (5 x 10 5 , 1X10 6 , 2Х10 6 A single subcutaneous injection of IU / kg and 1X10 6 After a single intravenous injection of IU / kg, the major pharmacokinetic parameters are shown in Table 4 below.

[0205] (2) Pharmacokinetics of single subcutaneous and intravenous injections in Beagles

[0206] In this experiment, a total of 24 beagles with no prior history of drug administration were used and randomly divided into four groups of six animals each, with half being male and half female. Animals in groups 1–3 were administered a single subcutaneous injection into the nape of the neck, and the dosages for the three dose groups (low, medium, and high) were 3 × 10, respectively. 5 IU / kg, 6X10 5 IU / kg, 1.2Х10 6 The dosage was IU / kg, and all were administered at a volume of 0.6 mL / kg. Animals in Group 4 were administered intravenously, and the dosage was 6 x 10 5 It was IU / kg. PK blood samples were collected from experimental animals at the following time points:

[0207] Groups 1–3 (Subcutaneous injection groups): Before administration, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 96 hours, 120 hours after administration;

[0208] Group 4 (Intravenous injection group): Before administration, 2 minutes, 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours after administration;

[0209] The concentration of IL-2-HSA in Beagle serum was quantitatively detected using a validated ELISA method. WinNonlin software (Phoenix TM Pharmacokinetic parameters were calculated by non-compartmental analysis using (version 8.1).

[0210] IL-2-HSA fusion protein administered to beagles at various doses (3 x 10 5 , 6Х10 5 , 1.2Х10 6 A single subcutaneous injection of IU / kg and 6×10 5 After a single intravenous injection of IU / kg, the major pharmacokinetic parameters are shown in Table 5 below.

[0211] bell SD Rat Administration method subcutaneous injection intravenous injection Dosage 5×10 5 IU / kg 1×10 6 IU / kg 2×10 6 IU / kg 1×10 6 IU / kg gender female cock female cock female cock female cock PK parameter (average, n=5) T 1 / 2 (h) 3.21 4.21 3.72 3.78 4.52 3.82 5.24 5.40 T max (h) 12.0 12.8 10.4 16.8 12.0 16.8 - - C max (ng / mL) 180 145 410 269 939 556 5980 6362 AUC last (mg / mL) 3376 2946 8593 5602 18787 13339 41544 47666 Vd (mL / kg) 150 181 138 244 152 184 38.4 33.9 CL (mL / h / kg) 31.9 30.6 25.4 42.0 23.6 32.1 5.12 4.40 MRT last (h) 14.0 14.8 14.5 17.3 15.8 18.3 6.41 6.77

[0212] bell Beagle dog Administration method subcutaneous injection intravenous injection Dosage 3×10 5 IU / kg 6×10 5 IU / kg 1.2 × 10 6 IU / kg 6×10 5 IU / kg gender female cock female cock female cock female cock PK parameter (average, n=3) T 1 / 2 (h) 6.56 6.69 4.05 5.71 5.33 7.69 9.90 8.56 T max (h) 10.7 6.67 8.00 13.3 13.3 24.0 - - C max (ng / mL) 178 142 611 456 1183 971 2240 2073 AUC last (mg / mL) 5040 4334 18022 15440 43473 39360 33646 27584 Vd (mL / kg) 120 157 43.2 70.8 44.6 72.5 54.8 57.8 CL (mL / h / kg) 12.7 15.6 7.36 8.60 6.08 6.48 3.82 4.68 MRT last (h) 19.6 21.1 19.5 21.9 25.1 26.5 13.2 12.5

[0213] Pharmacokinetic studies have revealed that the half-life of IL-2-HSA in rats and dogs is significantly longer than the half-life of natural IL-2 (a few minutes in the human body), which can reduce the frequency of administration, the total amount of drug, and the side effects and treatment costs for patients, as well as improve patient compliance and quality of life in clinical applications.

[0214] Although preferred embodiments of the present invention have been described in detail above, the present invention is not limited to the specific details of the above embodiments, and various simple modifications are possible within the scope of the technical spirit of the present invention. All such simple modifications fall within the scope of protection of the present invention.

[0215] In addition, it should be noted that each of the specific technical features described in the aforementioned specific embodiments can be combined without conflict in any appropriate manner, and to avoid unnecessary repetition, the present invention does not further describe various possible combinations.

[0216] In addition, it is possible to implement various embodiments of the present invention in any combination, and these should also be considered as part of the disclosed content of the present invention as long as they do not infringe upon the spirit of the present invention.

[0217]

[0218] Depository Name: China Comprehensive Microbial Culture Collection Center Trustee Number: CGMCC45173 Date of Deposit: 2022-05-26

Claims

Claim 1 A pharmaceutical composition for use in treating or preventing inflammatory bowel disease (IBD) comprising an interleukin 2 fusion protein, wherein the interleukin 2 fusion protein comprises human interleukin 2 or a variant thereof and human serum albumin or a variant thereof; wherein the human interleukin 2 or a variant thereof comprises an amino acid sequence represented by SEQ ID NO. 1; wherein the human serum albumin or a variant thereof comprises an amino acid sequence represented by SEQ ID NO. 2; and wherein the amino acid at position 125 of the amino acid sequence represented by SEQ ID NO. 1 is alinine. Claim 2 A pharmaceutical composition according to claim 1, characterized in that the human interleukin 2 or its variant is directly linked to the human serum albumin or its variant, or the human interleukin 2 or its variant is linked to the human serum albumin or its variant through a linking peptide. Claim 3 A pharmaceutical composition according to claim 2, characterized in that the general formula of the linked peptide is (GnS)m, wherein n and m are each integers from 1 to 10. Claim 4 A pharmaceutical composition according to claim 3, characterized in that the general formula of the linked peptide is (GnS)m, wherein n is an integer from 1 to 4 and m is an integer from 0 to 3. Claim 5 A pharmaceutical composition according to claim 1, characterized in that the fusion protein has an amino acid sequence represented by SEQ ID NO.

4. Claim 6 A pharmaceutical composition according to claim 1, characterized in that the inflammatory bowel disease (IBD) is selected from ulcerative colitis, Crohn's disease, or indeterminate colitis. Claim 7 A pharmaceutical composition according to claim 1, characterized in that the formulation of the pharmaceutical composition is selected from an injectable, a tablet, or a capsule. Claim 8 A pharmaceutical composition according to claim 7, wherein the formulation is selected from a liquid injectable or a lyophilized powder injectable. Claim 9 A pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Claim 10 A pharmaceutical composition according to claim 9, characterized in that the carrier is one or more selected from the group consisting of excipients, diluents, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and stabilizers. Claim 11 A pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition is administered orally or by injection. Claim 12 A pharmaceutical composition according to claim 11, characterized in that the above pharmaceutical composition is injected subcutaneously or intravenously. Claim 13 In claim 1, based on the fusion protein in the pharmaceutical composition, the dosage of the pharmaceutical composition is 3×10 4 IU / dose to 1×10 6 A pharmaceutical composition characterized by being IU / dose. Claim 14 A pharmaceutical composition according to claim 11, characterized in that the above pharmaceutical composition is administered once every 7 to 28 days. Claim 15 A pharmaceutical composition according to claim 14, characterized in that the above pharmaceutical composition is administered once every 14 to 28 days. Claim 16 A pharmaceutical composition according to claim 1, characterized in that the interleukin 2 fusion protein is expressed from an expression system including CHO cells. Claim 17 A pharmaceutical composition according to claim 16, wherein the expression system is a CHO-K1 cell, and the CHO-K1 cell was deposited with the General Microbiology Center of the Chinese Microbial Species Depository Management Committee on May 26, 2022, under deposit number CGMCC No. 45173.

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