Methods for achieving controlled and sustained active release of biologically active molecules and their applications in medicines

Abstract

Methods for achieving controlled and sustained active release of biologically active molecules and their applications in medicines. [Solution] Protein or peptide drugs have problems such as short half-life, large fluctuations in blood concentration, and low safety. In particular, direct administration of IL-2 and other drugs has significant toxicity and side effects, limiting their clinical application. Conventional techniques attempt to solve the above problems by PEG modification, but the activity of drugs after PEG modification decreases, and active release and bioavailability are poor. The present invention achieves active release control and sustained release of bioactive molecules using a specific PEG modifier. In particular, the complex of the PEG modifier and IL-2 gradually releases the activity of IL-2 as the PEG is removed, achieving a dynamic balance of excellent bioavailability and stable effective blood concentration, and is expected to have benefits in clinical application such as reduced administration frequency, improved drug bioavailability, patient compliance, and safety.

Description

[Technical field]

[0001] The present invention relates to a method for achieving controlled and sustained active release of biologically active molecules and its pharmaceutical application, in particular to a method for the controlled and sustained active release of highly active cytokines such as interleukin-2. [Background technology]

[0002] Interleukin-2 (IL-2) is a member of the interleukin family. Many studies have shown that IL-2 can promote the differentiation and maturation of T cells, NK cells and B cells, activate the biological activities of these cells, induce the activation of lymphokine-activated killer cells (LAK), and promote the synthesis and release of lymphokines such as interferon and tumor necrosis factor, as well as the production of antibodies. The ability of IL-2 to increase lymphocyte population in the body and enhance the effector functions of these cells contributes to its antitumor effects, and high doses of IL-2 were approved for the treatment of metastatic renal cell carcinoma and malignant melanoma already in the early 1980s.

[0003] However, due to the short half-life and low bioavailability of IL-2 in the body, frequent multiple administrations are often required to maintain efficacy in clinical applications. In addition, when human IL-2 binds to the human IL-2 high affinity receptor (IL-2Rαβγ), a large number of Treg cells are inevitably activated. These two factors combine to cause side effects of different degrees. The most serious is vascular leak syndrome (VLS), which is caused by the accumulation of intravascular fluid in the liver, lungs, and other organs, resulting in pulmonary edema and hepatocellular damage, which forces patients to discontinue treatment, significantly reduces treatment adherence, and limits the further clinical application of related therapies.

[0004] In the prior art, researchers often modify drugs with water-soluble polymers such as polyethylene glycol to extend the physiological half-life of drugs and reduce their immunogenicity and toxicity (Nandini V, Proc. Natl. Acad. Sci, 1987; Chang Yuan, PEGylation of Interleukin-2, 1996). Wang Lifu et al. modified wild-type rIL-2 with monomethoxy polyethylene glycol active ester with a purity of 75% and a molecular weight of 5000, but the resulting randomly modified product only retained 69.7% of its in vitro activity (Wang Lifu et al. Preparation of polyethylene glycol-modified rIL-2 and its anti-hepatoma cell activity in vivo and in vivo, 1997). Furthermore, to achieve sustained release of drugs in the body and improve bioavailability, the traditional modification method of directly binding drugs to polyethylene glycol does not achieve the ideal effect. In the prior art, it has been reported that the purpose of sustained and controlled release of drug activity can be achieved by forming a polymer-drug conjugate by binding a water-soluble polymer to a drug via a linker, and then the water-soluble polymer is detached from the conjugate. This allows the drug to remain at the site of the lesion (such as cancer) for a longer period, reducing the frequency of administration and reducing the inconvenience to the patient. For example, patent document CN200680029849.5 discloses a conjugate containing an aromatic moiety containing an ionizable hydrogen atom such as fluorene, a spacer moiety, and a water-soluble polymer, and patent document CN103517718A further discloses a conjugate formed by the above polymer and rIL-2. This conjugate is actually a CD122 (IL-2Rβ) biased agonist called NKTR-214 developed by Nektar, Inc. in the United States. This NKTR-214 conjugate is composed of IL-2 (the amino acid sequence of which is the same as that of aldesleukin) and six branched PEGs with a molecular weight of 20K and a fluorene ring structure bound thereto. Under mild alkaline conditions, fluorene methoxycarbonyl is eliminated by a β-elimination reaction, which allows NKTR-214 to gradually release the five PEGs on the protein surface under physiological conditions (pH = 7.4, weakly alkaline), preferentially bind to the IL-2Rβ receptor, enhancing T cell activation, and at the same time providing the ability to circulate in the body for a long period of time.The development of the NKTR-214 molecule involved screening PEG reagents and optimizing the coupling reaction to increase site-specific modification of lysine residues (e.g., K31, K34, K42, K47, K48, and K75) that accumulate at the IL-2 / IL-2Rα interface, thereby positioning PEG close to the key hydrophobic binding site where IL-2 / IL-2Rα interacts, thereby achieving the goal of reduced binding to CD25 (IL-2Rα) and preferential activation of CD122 (IL-2Rβ) (Charych DH, Hoch U, Langowski JL, et al. NKTR-214, an engineered cytokine with biased IL2receptor binding, increased tumor exposure, and marked efficacy in mouse tumor models [J]. ClinicalCancerResearch An Official Journal of the American Association for CancerResearch,2016,22(3):680-690.). Nektar and BMS entered into a US$3.6 billion collaboration for NKTR214, but two Phase III clinical trials (PIVOT IO 001 and PIVOT-09) were announced to have failed one after the other in 2022, with clinical treatment data not reaching the primary endpoint (2022 EMSO, European Society of Medical Oncology Annual Meeting, Abstract No. 785O, LBA68).

[0005] The inventors of the present invention screened existing mature polyethylene glycol modifiers through a large amount of experiments and research, and selected a PEG modifier with a certain structure. The polyethylene glycol modifier is used to modify IL-2 or its variants, and has excellent active release control and active sustained release effects, and shows excellent bioavailability compared with similar drugs, reducing the frequency of drug administration, and is expected to significantly improve therapeutic effects and patient compliance. In addition, the present invention is fundamentally different from NKTR-214, a PEG-IL2 drug under development, in terms of molecular design. After administration, NKTR-214 has a very complex and uncontrollable separation process of its PEG molecules in the body, so it is theoretically impossible to always maintain the characteristics of a CD122-biased agonist. This will affect the stable expression of the drug effect. The inventors of the present invention achieved the bias to CD122 and the control of the PEG modification site by site-specific mutation of the sequence of the original protein, and solved the potential shortcomings of the molecular design of the NKTR-214 drug from the three aspects of safety, efficacy, and quality control. Furthermore, these differentiated advantages were confirmed through data on pharmacokinetics, efficacy, and other aspects in animals. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] CN200680029849.5 [Patent Document 2] CN103517718A [Non-patent literature]

[0007] [Non-Patent Document 1] Nandini V, Proc. Natl. Acad. Sci, 1987; Tsunehisa, PEGylation of interleukin-2, 1996 [Non-Patent Document 2] Wang Lifu et al. Preparation of polyethylene glycol modified rIL-2 and its anti-hepatoma cell activity in vivo and in vivo, 1997 [Non-Patent Document 3] Charych DH, HochU, Langowski JL, et al. NKTR‐214, an engineered cytokine with biased IL2receptor binding, increased tumor exposure, and marked efficacy in mouse tumor models [J]. ClinicalCancerResearch An Official Journal of the American Association for CancerResearch, 2016, 22(3):680‐690. [Non-Patent Document 4] 2022EMSO, European Society of Medical Oncology Annual Meeting, Abstract No. 785O, LBA68 Summary of the Invention [Problem to be solved by the invention]

[0008] The technical problem that the present invention aims to solve is to overcome the problem of low availability of biologically active molecules such as IL-2 and the generally reduced activity of their polyethylene glycol modified forms. [Means for solving the problem]

[0009] The present invention first provides a method for achieving controlled and sustained release of biologically active molecules. The inventors of the present invention have realized that a biologically active molecule such as IL-2 is reacted with a polyethylene glycol of a certain type and structure to form a conjugate, and under certain in vitro conditions or in vivo physiological conditions, PEG is gradually detached from the conjugate structure, and the activity of the biologically active molecule is gradually released. This allows the biologically active molecule to maintain a stable effective blood concentration, and allows for controlled and sustained release of the active drug both in and outside the body, achieving a relatively excellent dynamic balance of drug bioavailability. It is also expected to provide benefits in clinical applications such as reduced administration frequency, improved drug bioavailability, improved patient compliance and improved safety. Meanwhile, the present invention provides a new type of IL-2 mutant protein. In this mutant protein, the polyethylene glycol modification site of IL-2 is mutated, making it easier to control PEG modification. In addition, this mutant has a mutation in the receptor binding site of IL-2, and has higher activity than wild-type IL-2.

[0010] One of the objectives of the present invention is to provide a method for achieving controlled and sustained release of a biologically active molecule. The method is a method for modifying the amino group of a biologically active molecule with a polyethylene glycol modifying agent. The modified product obtained by this method gradually releases the activity of the biologically active molecule as PEG is gradually removed. The polyethylene glycol modifying agent is polyethylene glycol succinimidyl succinate.

[0011] The bioactive molecule is a protein or peptide, preferably an interleukin, most preferably IL-2.

[0012] The PEG modifier is preferably linear polyethylene glycol succinimidyl succinate.

[0013] Another object of the present invention is to provide a polyethylene glycol modified human IL-2, which is modified using polyethylene glycol succinimide succinate as a PEG modifier, with an average of 4.5 to 8.5 PEG units bound to each human IL-2.

[0014] Preferably, the PEG modifier is linear polyethylene glycol succinimidyl succinate.

[0015] More preferably, the molecular weight of the PEG modifier is 5-20 kDa. The molecular weight is a nominal value, and the actual molecular weight may be 90%-110% of the nominal value. When the molecular weight of the PEG modifier is 5 kDa, 5.5-7.5 PEGs are bound to each human IL-2 on average. When the molecular weight of the PEG modifier is 10-20 kDa, 6.5-8.5 PEGs are bound to each human IL-2 on average.

[0016] More preferably, the structure of the PEG modifier is as shown below, where n is an integer of 97 to 494. When n is an integer of 97 to 494, the actual molecular weight of the PEG modifier is about 4.5k to 22k, and the corresponding nominal molecular weight is 5 to 20kDa.

[0017] [ka]

[0018] More preferably, the structure of the polyethylene glycol modified human IL-2 is as shown below: In the formula, n is an integer of 97 to 494, and m is 4.5 to 8.5.

[0019] [ka]

[0020] Another object of the present invention is to provide a human IL-2 mutant, which does not contain adjacent lysines and can be modified with PEG to obtain a more uniform modification. The inventors of the present invention have found the following through their research: the amino acids at positions 8, 9, 48, and 49 from the N-terminus of wild-type human IL-2 shown in SEQ ID NO:1 are all lysines, and all of them can be modified with PEG, but adjacent positions are rarely modified with PEG at the same time. In the case of saturation modification, the 8th or 9th position of IL-2 is modified, and the 48th or 49th position is modified. That is, four types of isomers with different modified positions are generated, and the obtained human IL-2 polyethylene glycol modified product is heterogeneous. The inventors of the present invention therefore decided to mutate the amino acids at positions 8 and / or 9, and the amino acids at positions 48 and / or 49. This significantly reduced the generation of isomers with different PEG modification sites, making it easier to control the uniformity of the PEG modified product. Prior to the filing of the present invention, those skilled in the art were not aware that the fact that adjacent amino acids in the IL-2 sequence are both lysine would have a negative effect on PEG modification, and therefore there is no motivation to mutate the corresponding sites when modifying IL-2 with polyethylene glycol.

[0021] The human IL-2 mutants provided by the present invention have amino acid substitutions at positions 8 and / or 9 from the N-terminus based on wild-type human interleukin-2, or have amino acid substitutions at positions 48 and / or 49 from the N-terminus based on wild-type human interleukin-2, the amino acid sequence of which is as shown in SEQ ID NO:1.

[0022] Preferably, the amino acids at positions 8 and / or 9 and 48 and / or 49 from the N-terminus have been substituted based on wild-type human IL-2.

[0023] In the specific examples, the lysine at position 8 is mutated to arginine, the lysine at position 9 is mutated to arginine, the lysine at position 48 is mutated to arginine, tryptophan or tyrosine, and the lysine at position 49 is mutated to aspartic acid. However, these examples are illustrative and do not limit the scope of the present invention. The purpose of mutating the amino acids at positions 8, 9, 48, and 49 is to solve the problem that the presence of adjacent lysines in the wild-type IL-2 sequence results in the generation of various isomers of polyethylene glycol modifications, which affects the uniformity of the PEG-modified product. Those skilled in the art will understand that the above purpose can also be achieved by mutating the above amino acid sites to other non-lysine amino acids other than the amino acids shown in the examples.

[0024] On the other hand, the present invention further optimized the human IL-2 lacking the adjacent lysines to develop a human IL-2 mutant with significantly superior in vitro activity to wild-type IL-2, which has one, two or more amino acid substitutions at positions 1, 8, 9, 18, 19, 48, 49, 72 and / or 81 from the N-terminus of wild-type human IL-2. The amino acid sequence of wild-type human IL-2 is as shown in SEQ ID NO:1.

[0025] Preferably, the mutations at these sites are selected from the following residue substitution schemes: position 1: A1 deletion; position 8: K8R; position 9: K9R; position 18: L18M; position 19: L19S; position 48: K48W, R; position 49: K49R; position 72: L72F; position 81: R81D. More preferably, the mutant comprises one or more of the following mutations: position 8: K8R; position 48: K48W; position 72: L72F; and / or position 81: R81D. The amino acid sequence of wild-type human IL-2 is as shown in SEQ ID NO:1.

[0026] Most preferably, the mutation is selected from any one of the mutation schemes shown in the table below.

[0027] [Table 1]

[0028] As a third aspect, the inventors of the present invention also considered the need to reduce the bias of IL-2 mutants towards IL-2Rα and maintain or enhance the bias towards IL-2Rβ and IL-2Rγ. To this end, they effectively screened for one, two or more amino acid substitutions at positions corresponding to positions 1, 18, 19, 27, 35, 38, 41, 42, 43, 45, 54, 64, 65, 72, 78, 79, 80, 81, 82, 83, 87, 92 and / or 97 from the N-terminus of wild-type human IL-2. G438P8 is one example.

[0029] IL-2 acts through the IL-2 receptor (IL-2R). The IL-2R receptor contains three subunits, IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132), which form three distinct receptor types. High avidity receptors contain all three subunits, IL-2Rα, IL-2Rβ, and IL-2Rγ. Medium avidity receptors include IL-2Rβ and IL-2Rγ. Low avidity receptors are IL-2Rα. IL-2Rα is highly expressed on Treg cells, IL-2Rβ is expressed on all CD8+ T cells, NK cells, and Treg cells, and IL-2Rγ is expressed on all immune cells. IL-2 binds to receptors on various cells and is involved in multiple actions in the immune response. On the one hand, IL-2, as a stimulator of the immune system, stimulates the proliferation and differentiation of T cells, induces the production of cytotoxic T lymphocytes, promotes the proliferation and differentiation of B cells and the synthesis of immunoglobulins, and stimulates the production and activation of NK cells. Based on these functions, IL-2 has been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infections. On the other hand, IL-2 promotes the activation and proliferation of immunosuppressive CD4+CD25+ regulatory T cells (i.e., Treg cells), causing immunosuppression. Many studies have been proposed to reduce the toxicity and side effects of IL-2, which is used as an immune system stimulant, and improve its efficacy by changing the bias of IL-2 to IL-2Rα. Many studies have already been reported on changing the bias of IL-2 receptors by site mutation. Those skilled in the art are not limited to the above specific site-mutated IL-2, and may perform polyethylene glycol modification using IL-2 mutated at other sites.

[0030] Another object of the present invention is to provide a polyethylene glycol modified human IL-2 mutant, which uses polyethylene glycol succinimidyl succinate as a PEG modifier, and an average of 5-8 PEGs are bound to each human IL-2. The human IL-2 mutant is one of the above different mutants.

[0031] Another object of the present invention is to provide an application of polyethylene glycol modified human IL-2 or its mutants in the manufacture of medicaments for treating tumor diseases, including but not limited to squamous cell carcinoma, melanoma, colon cancer, breast cancer, ovarian cancer, prostate cancer, gastric cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, thyroid cancer, kidney cancer, bile duct cancer, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease, and adrenal cortical carcinoma.

[0032] Another object of the present invention is to provide a composition for treating tumor diseases, comprising the polyethylene glycol-modified human IL-2, mutant human IL-2, or polyethylene glycol-modified human IL-2 mutant, and also comprising an HER2 antibody, a PD-1 antibody, a PD-L1 antibody, or a CD26 antibody, etc.

[0033] The HER2 antibodies include Roche's Herceptin, Perjeta, and Kadcyla, among others.

[0034] The PD-1 antibodies include Bristol-Myers Squibb's Opidivo, Merck's Keytruda, Kimil Bio's Toripalimab, Cinda Bio's Sintilimab, Hengrui Pharmaceutical's Camrelizumab, and Baekje Shenju's tislelizumab.

[0035] The PD-L1 antibodies include Roche's Tecentriq, AstraZeneca's Imfinzi, Merck's Bavencio, and the like.

[0036] The CD26 antibody may be an anti-CD26 antibody such as YS110 (prior application CN200680034937.4) or prior application CN202111245489.5.

[0037] Tumor diseases that may be treated by the composition include, but are not limited to, squamous cell carcinoma, melanoma, colon cancer, breast cancer, ovarian cancer, prostate cancer, gastric cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, thyroid cancer, kidney cancer, bile duct cancer, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease, and adrenal cortical carcinoma.

[0038] Through the technical solution of the present invention, the following technical effects are mainly achieved: 1. Polyethylene glycol succinimidyl succinate is used to react with the lysine side chain ε-amino group of IL-2 or its mutants, and polyethylene glycol and IL-2 are linked by an amide bond. Since the receptor binding site is completely covered, the modified PEG-IL-2 molecule has no activity. In addition, unlike other modifiers, polyethylene glycol succinimidyl succinate has one ester bond in the complex after binding with a protein or peptide drug. Due to its inherent instability, the ester bond is prone to hydrolysis and the PEG chain is easily removed. This characteristic has traditionally been considered to lead to instability of the modified molecule because the modifier is easily removed, so the application of this PEG has gradually been replaced by other PEG modifiers (such as polyethylene glycol succinimidyl propionate) that are less likely to be removed. However, in the present invention, this hydrolysis property allows IL-2 to gradually release its drug activity in the body, and extremely excellent drug efficacy can be obtained with a certain molecular weight and number of modifications.

[0039] 2. By mutating the lysine site in IL-2 to eliminate the adjacent lysine, the position of PEG coupling was limited, the generation of isomers with different PEG modification sites was significantly reduced, and the uniformity of the PEG-modified product was easily controlled. Based on this, through the design and screening of further mutation sites, we developed a receptor-binding-biased mutant with significantly reduced affinity for α-receptors and maintained affinity for β-receptors, as well as a mutant with significantly superior CTLL-2 cell proliferation activity compared to wild-type IL-2.

[0040] 3. During actual action, the activity of PEGylated IL-2 continues to be released slowly as PEG is gradually removed. Through optimization and screening of different molecular weight PEG and different PEG modification numbers, the present invention can avoid the low bioavailability of highly modified IL-2, and also avoid the high toxicity and side effects of low modified IL-2. As a result, the PEGylated IL-2 of the present invention can effectively achieve a dynamic balance between bioavailability and safety, while by adjusting the dosage and administration frequency, it can achieve stable blood concentration of the drug and better in vivo utilization, and achieve better efficacy in the body. [Brief description of the drawings]

[0041] [Figure 1a] FIG. 1 is a graph showing the curves of IL-2 mutants stimulating the proliferation of CTLL-2 cells. [Figure 1b] FIG. 1 is a graph showing the curves of IL-2 mutants stimulating the proliferation of CTLL-2 cells. [Figure 1c] FIG. 1 is a graph showing the curves of IL-2 mutants stimulating the proliferation of CTLL-2 cells. [Figure 1d] FIG. 1 is a graph showing the curves of IL-2 mutants stimulating the proliferation of CTLL-2 cells. [Figure 1e] FIG. 1 is a graph showing the curves of IL-2 mutants stimulating the proliferation of CTLL-2 cells. [Figure 2a] FIG. 1 shows the activity values ​​of PEG-SS modified products of different mutant proteins activated under alkaline conditions to stimulate CTLL-2 cell proliferation. [Figure 2b] FIG. 1 shows the activity values ​​of PEG-SS modified products of different mutant proteins activated under alkaline conditions to stimulate CTLL-2 cell proliferation. [Figure 2c] FIG. 1 shows the activity values ​​of PEG-SS modified products of different mutant proteins activated under alkaline conditions to stimulate CTLL-2 cell proliferation. [Diagram 3] This is a curve showing the measurement of CTLL-2 phosphorylated STAT5 levels by the modified products of IL-2 mutants modified with PEG-SS of different structures and molecular weights. [Figure 4] FIG. 13 is a measurement curve of CTLL-2 phosphorylated STAT5 levels by the modified products of different IL-2 mutants modified with PEG-SS. [Diagram 5] FIG. 1 shows a comparison of the in vivo pharmacokinetics of highly modified mPEG-SS-5k-GP8 and the original protein, GP8. [Figure 6a] Figure 6 shows the results of evaluating the efficacy of IL-2 modified with PEG-SS of different structures (linear or branched) in a BALB / c mouse subcutaneously implanted model of CT26.WT mouse-derived colon cancer cells. Figure 6a shows the tumor volume increase curves of animals in each group, Figure 6b shows the tumor weights of animals in each group, and Figure 6c shows the changes in body weights of animals in each group. [Figure 6b] Figure 6 shows the results of evaluating the efficacy of IL-2 modified with PEG-SS of different structures (linear or branched) in a BALB / c mouse subcutaneously implanted model of CT26.WT mouse-derived colon cancer cells. Figure 6a shows the tumor volume increase curves of animals in each group, Figure 6b shows the tumor weights of animals in each group, and Figure 6c shows the changes in body weights of animals in each group. [Figure 6c] Figure 6 shows the results of evaluating the efficacy of IL-2 modified with PEG-SS of different structures (linear or branched) in a BALB / c mouse subcutaneously implanted model of CT26.WT mouse-derived colon cancer cells. Figure 6a shows the tumor volume increase curves of animals in each group, Figure 6b shows the tumor weights of animals in each group, and Figure 6c shows the changes in body weights of animals in each group. [Figure 7a] Figure 7 shows the efficacy evaluation results of different IL-2 mutants modified with linear PEG-SS in a B16-F10 murine melanoma cell C57BL / 6 mouse subcutaneous transplant model, in which Figure 7a shows the tumor volume increase curves of animals in each group, and Figure 7b shows the tumor weights of animals in each group. [Figure 7b] Figure 7 shows the efficacy evaluation results of different IL-2 mutants modified with linear PEG-SS in a B16-F10 murine melanoma cell C57BL / 6 mouse subcutaneous transplant model, in which Figure 7a shows the tumor volume increase curves of animals in each group, and Figure 7b shows the tumor weights of animals in each group. [Figure 8a] Figure 8 shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and degrees of modification in a B16-F10 mouse-derived melanoma tumor model. Figure 8a shows the tumor growth curves of animals in each group, Figure 8b shows the tumor weights of animals in each group on day 17 after cell inoculation, and Figure 8c shows the changes in the weight gain rate of animals. [Figure 8b] Figure 8 shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and degrees of modification in a B16-F10 mouse-derived melanoma tumor model. Figure 8a shows the tumor growth curves of animals in each group, Figure 8b shows the tumor weights of animals in each group on day 17 after cell inoculation, and Figure 8c shows the changes in the weight gain rate of animals. [Figure 8c] Figure 8 shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and degrees of modification in a B16-F10 mouse-derived melanoma tumor model. Figure 8a shows the tumor growth curves of animals in each group, Figure 8b shows the tumor weights of animals in each group on day 17 after cell inoculation, and Figure 8c shows the changes in the weight gain rate of animals. [Figure 9a] Figure 9 shows the efficacy of PEG-SS modified IL-2 mutants with different molecular weights and degrees of modification in a CT26.WT mouse-derived colon cancer tumor model: Figure 9a shows the tumor growth curves of animals in each group, Figure 9b shows the tumor weights of animals in each group on the 22nd day after cell inoculation, and Figure 9c shows the changes in the weight gain rate of animals. [Figure 9b] Figure 9 shows the efficacy of PEG-SS modified IL-2 mutants with different molecular weights and degrees of modification in a CT26.WT mouse-derived colon cancer tumor model: Figure 9a shows the tumor growth curves of animals in each group, Figure 9b shows the tumor weights of animals in each group on the 22nd day after cell inoculation, and Figure 9c shows the changes in the weight gain rate of animals. [Figure 9c] Figure 9 shows the efficacy of PEG-SS modified IL-2 mutants with different molecular weights and degrees of modification in a CT26.WT mouse-derived colon cancer tumor model: Figure 9a shows the tumor growth curves of animals in each group, Figure 9b shows the tumor weights of animals in each group on the 22nd day after cell inoculation, and Figure 9c shows the changes in the weight gain rate of animals. [Figure 10a]Figure 10 shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and degrees of modification in A375 human melanoma model, where Figure 10a shows the animal tumor growth curve, Figure 10b shows the tumor weight of animals in each group on day 45 after cell inoculation, and Figure 10c shows the animal weight gain rate. [Figure 10b] Figure 10 shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and degrees of modification in A375 human melanoma model, where Figure 10a shows the animal tumor growth curve, Figure 10b shows the tumor weight of animals in each group on day 45 after cell inoculation, and Figure 10c shows the animal weight gain rate. [Figure 10c] Figure 10 shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and degrees of modification in A375 human melanoma model, where Figure 10a shows the animal tumor growth curve, Figure 10b shows the tumor weight of animals in each group on day 45 after cell inoculation, and Figure 10c shows the animal weight gain rate. [Figure 11a] Figure 11 shows the efficacy evaluation of different doses of PEG-modified IL-2 mutants in A375 human melanoma model, Figure 11a shows the animal tumor growth curve, Figure 11b shows the tumor weight of each group of animals, and Figure 11c shows the weight gain rate of each group of animals. [Figure 11b] Figure 11 shows the efficacy evaluation of different doses of PEG-modified IL-2 mutants in A375 human melanoma model, Figure 11a shows the animal tumor growth curve, Figure 11b shows the tumor weight of each group of animals, and Figure 11c shows the weight gain rate of each group of animals. [Figure 11c] Figure 11 shows the efficacy evaluation of different doses of PEG-modified IL-2 mutants in A375 human melanoma model, Figure 11a shows the animal tumor growth curve, Figure 11b shows the tumor weight of each group of animals, and Figure 11c shows the weight gain rate of each group of animals. [Figure 12a] Figure 12 shows the efficacy evaluation of different doses of PEG-modified IL-2 mutants in A498 human kidney cancer model, Figure 12a shows the growth curve of the animal tumors in each group, Figure 12b shows the tumor weight of each group, and Figure 12c shows the weight gain rate of each group of animals. [Figure 12b]Figure 12 shows the efficacy evaluation of different doses of PEG-modified IL-2 mutants in A498 human kidney cancer model, Figure 12a shows the growth curve of the animal tumors in each group, Figure 12b shows the tumor weight of each group, and Figure 12c shows the weight gain rate of each group of animals. [Figure 12c] Figure 12 shows the efficacy evaluation of different doses of PEG-modified IL-2 mutants in A498 human kidney cancer model, Figure 12a shows the growth curve of the animal tumors in each group, Figure 12b shows the tumor weight of each group, and Figure 12c shows the weight gain rate of each group of animals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Definition: Interleukin-2 (IL-2) can be obtained by recombinant or non-recombinant methods, and may be wild-type IL-2 or a mutant thereof. IL-2 can be produced by expression in bacteria (e.g., E. coli), mammalian cells (e.g., CHO cells), or yeast (e.g., Pichia coli). IL-2 may be of human or animal origin. IL-2 of human origin is preferred. In a specific embodiment of the present invention, the amino acid sequence of human IL-2 is as shown in SEQ ID NO:1. The mutant is obtained by substituting, inserting, or deleting some amino acids based on human IL-2 shown in SEQ ID NO:1. In a specific embodiment, polyethylene glycol-modified IL-2 is used as IL-2 shown in SEQ ID NO:1 and its mutants, but those skilled in the art will understand that the specific embodiment is merely for illustrative purposes and does not limit the scope of the present invention. The IL-2 of the polyethylene glycol-modified IL-2 can be selected from other human IL-2 whose sequences have been published, or can be selected from mutants obtained by mutating the amino acids at the positions corresponding to SEQ ID NO:1 based on the sequences of other published human IL-2 (mutations to eliminate the adjacent lysine, or mutations to reduce the bias toward IL-2Rα and increase the bias toward IL-2Rβ and IL-2Rγ).

[0043] Polyethylene glycol (PEG) is usually formed by polymerization of ethylene oxide, and is available in branched, linear and multi-armed types. Generally, those with a molecular weight of 20,000 or less are called PEG, and those with a higher molecular weight are called PEO. Ordinary polyethylene glycol has one hydroxyl group at each end, and blocking one end with a methyl group gives methoxypolyethylene glycol (mPEG).

[0044] Polyethylene glycol modifier (PEG modifier) ​​refers to a polyethylene glycol derivative having a functional group, which is an activated polyethylene glycol used for modifying protein and polypeptide drugs. The polyethylene glycol modifier used in the present invention is purchased from Jiangsu Zhonghong Bioengineering Drug Discovery Research Institute Co., Ltd. or Beijing Keykai Technology Co., Ltd. The actual molecular weight of a PEG modifier of a particular molecular weight may be 90%-110% of the listed value. For example, the actual molecular weight of PEG5K may be 4.5kDa-5.5kDa. The actual molecular weight of PEG20K may be 18kDa-22kDa. If the listed value of the molecular weight of a PEG modifier is 5kDa-20kDa, its actual molecular weight is 4.5kDa-22kDa.

[0045] V-PEG-SC-20k used in the examples refers to a branched polyethylene glycol succinimidyl carbonate modifier with a molecular weight of 20 kDa. This is a PEG modifier prepared with reference to patent document CN200680029849.5. According to the patent document, this PEG modifier achieves the purpose of sustained release and controlled release of drug activity by reacting with a drug via a linker to form a complex, and then PEG is detached from the complex. This is consistent with the reported structure of NKTR-214. The applicant took the IL-2 modified with this PEG modifier as a positive reference. The structural formula of the V-PEG-SC-20k modifier is as shown below. In the formula, n is an integer between 199 and 244.

[0046] [ka]

[0047] V-PEG-SS-20k used in the examples refers to a branched polyethylene glycol succinimidyl succinate modifier with a molecular weight of 20 kDa. The structural formula of the V-PEG-SS-20k modifier is as shown below. In the formula, n is an integer of 198 to 244.

[0048] [ka]

[0049] The mPEG-SS-20k / 10k / 5k used in the examples refers to linear polyethylene glycol succinimidyl succinate modifiers with molecular weights of 20kDa, 10kDa, and 5kDa. The structural formulas of the mPEG-SS-20k / 10k / 5k modifiers are shown below. For mPEG-SS-20k, n is an integer between 403 and 494, for mPEG-SS-10k, n is an integer between 199 and 244, and for mPEG-SS-5k, n is an integer between 97 and 119.

[0050] [ka]

[0051] The mPEG-SPA-5k used in the examples refers to a linear polyethylene glycol succinimidyl propionate modifier with a molecular weight of 5 kDa. The structural formula of the mPEG-SPA-5k modifier is as shown below. In the formula, n is an integer of 98 to 120.

[0052] [ka] EXAMPLES

[0053] Example 1: Design and construction of IL-2 mutants 1. Design of IL-2 mutants The IL-2 shown in the table below is wild-type IL-2, and its amino acid sequence is as shown in SEQ ID NO:1. The other mutants are mutants obtained by manipulating the wild-type IL-2 with amino acid substitution, insertion, or deletion. For example, the sequence G438 is obtained by mutating the amino acids at positions 8 and 48 of the sequence shown in SEQ ID NO:1, and K8R refers to the mutation of the lysine at position 8 to arginine.

[0054] [Table 2]

[0055] 2. Protein Production The method is not limited to the method described in the Examples, and any conventional recombinant protein production method may be used. The production will be explained using the IL-2 mutant G438 as an example.

[0056] Step 1: Based on the amino acid sequence of G438 (the sequence of wild-type IL-2 with K8R / K48W mutation as shown in SEQ ID NO:1), obtain the DNA sequence as shown in SEQ ID NO:2, optimized for E. coli. This DNA sequence is cloned into pBV220 vector to form recombinant plasmid pBV220-G438. Then, transform the recombinant plasmid into Top10 E. coli host to construct the expression host Top10-pBV220-G438.

[0057] Step 2: The Top10-pBV220-G438 recombinant expression strain is inoculated into TB medium (500 mL medium, 20% liquid volume) and cultured at 30°C and 220 rpm until the OD600 of the culture reaches 1.0±0.1. After that, the temperature of the culture is raised to 42°C while maintaining the rotation speed of the culture, and the strain is induced to express. The induced expression is carried out for 4 hours. After the induced expression, the culture is centrifuged to collect the bacterial cell precipitate.

[0058] Step 3: The bacterial cell precipitate after expression is resuspended at 100 g / L in 10 mM PBS, pH 7.4, and then ultrasonically disrupted using a probe-type ultrasonic disrupter (working power 250W, 3 seconds operation with 4 seconds interval, total disruption time 30 min). Then, the disruption product is collected by centrifugation to obtain the G438 inclusion body precipitate.

[0059] Step 4: Resuspend the G438 inclusion bodies at 50 g / L in PBS + 1% Triton® X-100, wash 3 times for at least 2 hours with stirring, and centrifuge to collect the crude G438 inclusion bodies.

[0060] Step 5: Resuspend the G438 crude inclusion bodies at 1 g / 100 mL in denaturing solution (20 mM Tris, 8 M urea, 5 mM DTT, pH 10.5), denaturate for at least 2 h, centrifuge and collect the supernatant. The supernatant is purified using Superdex 75.

[0061] Step 6: Dilute the purified G438 with a refolding buffer and refold it (refolding solution: 20 mM Tris, 2 M urea, 3 mM cysteine, 1 mM cystine, 0.01% SDS, pH 8.0). The protein concentration in the refolding system should not exceed 0.1 mg / mL, the refolding time should be 36 hours or more, and the refolding temperature should be 15°C.

[0062] Step 7: The refolded G438 is concentrated by ultrafiltration, and the concentrated sample is dialyzed against PBS (pH 7.4) to remove reagents such as urea to obtain a purified G438 product.

[0063] The amino acid sequence of each mutant is obtained by mutating the sequence shown in SEQ ID NO:1 based on the mutation scheme shown in Table 1. Each mutant is prepared in a similar manner as above.

[0064] Example 2: Determination of the affinity of IL-2 mutants to their receptors 1. Experimental Method The affinity of IL-2 mutants to their receptors is measured by biolayer interferometry (BLI).

[0065] 1. Sample preparation Test solution: Take each protein sample, dilute it to 300 mM with 1× Kinetics buffer, mix it evenly, and set aside for later use. Receptor solution: Take samples of receptor IL-2Rα (CD25), receptor IL-2Rβ (CD122), and receptor IL-2Rβ / γ, dilute them to 15-20 μg / mL with 1× Kinetics buffer, mix them evenly, store them in the dark, and set aside for later use.

[0066] 2. Add sample Add samples to the sample wells according to the protocol design. Add 200 μL of reagent or sample to each well.

[0067] II. Experimental results Run the program and perform data analysis with the software Fortebio DataAnalysis 8.0 to calculate affinity binding values. The results are shown in the table below.

[0068] [Table 3]

[0069] [Table 4]

[0070] 3. Results analysis The results of receptor affinity assay showed that mutants G493, G496, G498, G499, G500, G438P4, and G438P5 had significantly reduced or no binding to the two receptors, indicating that the mutations may have caused significant changes in the protein structure. Mutants such as G495, G438P6, G438P7, G438P8, G438P14, and G438P15 showed a binding bias to the IL-2β receptor. Mutations such as G495, G438P7, G438P8, and G438P14 did not significantly affect the binding of IL-2 to IL-2Rβ / γ. The binding of G438P15 to IL-2Rβ / γ was significantly reduced. To select mutants that retained receptor binding and evaluate their biological activity, further in vitro CTLL-2 cell proliferation activity experiments were performed.

[0071] Example 3: Measurement of the activity of IL-2 and its mutants to stimulate proliferation of CTLL-2 cells 1. Experimental Method CTLL-2 is a mouse cell line, and to evaluate the in vitro biological activity of IL-2 and its mutants and modifications, we used a method to measure the proliferation rate of its cell-dependent line, CTLL-2 cells, at different concentrations. This experimental method was based on the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Principle 3524 "Method for Determination of Biological Activity of Human Interleukin-2" (CTLL-2 / MTT colorimetric method).

[0072] 1. Preparation of test solution RPMI1640 culture medium: Take one bag of RPMI1640 culture medium powder (standard: IL), add water to dissolve, and dilute to 1000 ml. Furthermore, add 2.1 g of sodium bicarbonate, dissolve, mix evenly, filter, sterilize, and store at 4°C. Base culture medium: Measure out 10 ml of newborn bovine serum (FBS) and add 90 ml of RPMI1640 culture medium. Store at 4°C. Complete medium: Aspirate 100ml of basal medium and add human IL-2 to a final concentration of 400-800 IU / ml. Store at 4°C. PBS: Take 100 ml of 10x PBS and dilute to 1000 ml by adding water sterilized at 121°C for 20 minutes. Thiazole blue (MTT) solution: Weigh out 0.1 g of MTT, dissolve in PBS, and dilute to 20 mL. Filter through a 0.22 μm membrane for sterilization. Store at 4°C, protected from light. Lysate solution: 15% sodium lauryl sulfate solution, duration of use should not exceed 12 months.

[0073] 2. Sample Preparation Take a sample with a known protein content and dilute it to an appropriate initial concentration. In a 96-well cell culture plate, perform two-fold serial dilutions to create a total of eight dilutions. Add samples of each dilution to two wells. Leave 50 μl of solution in each well and discard the excess solution in the wells. These operations are performed under sterile conditions.

[0074] 3.Cell culture CTLL-2 cells are cultured in complete medium at 37°C under 5% carbon dioxide until sufficient numbers are reached. CTLL-2 cells are harvested by centrifugation and washed three times with RPMI 1640 medium. They are then resuspended in basal medium at 6.0 × 10 cells per mL. 5 Prepare a cell suspension containing cells. Store the cell suspension at 37°C and 5% carbon dioxide for later use. Add 50 μl of cell suspension to each well of the 96-well cell culture plate containing the wild-type and mutant samples, and incubate at 37°C and 5% carbon dioxide for 18-24 hours. Then, add 20 μl of MTT solution to each well, and incubate at 37°C and 5% carbon dioxide for 4-6 hours. Next, add 150 μl of lysate solution to each well, and incubate at 37°C and 5% carbon dioxide for 18-24 hours. All the above operations are performed under sterile conditions. Mix the liquid in the cell plate evenly, set it in a microplate reader, measure the absorbance at 570 nm with 630 nm as the reference wavelength, and record the results. Using the sample concentration (ng / ml) as the horizontal axis and the average OD570 measurement value as the vertical axis, perform four-term parameter fitting using the software ELISACalc to create a test sample reaction curve. Specific activity of sample (IU / mg) = Biological activity of sample (IU / ml) / 20 (ng / ml) x 10 6

[0075] II. Experimental results The measurement results are shown in FIG. 1 and the table below. [Table 5]

[0076] 3. Results analysis The biological activity of the mutants was measured and showed that the newly constructed IL-2 mutants G438P1, G438P8, G438P12, G438P20, G438P21, and G438P22 showed significantly superior proliferation activity in CTLL-2 cells compared with wild-type IL-2, and have potential for clinical application.

[0077] For the sake of brevity, mutants obtained by further modification based on G438, such as G438P1, G438P8, G438P12, G438P20, G438P21, and G438P22, are abbreviated as GP1, GP8, GP12, GP20, GP21, and GP22, respectively.

[0078] Example 4: Preparation of PEG-modified IL-2 Example 4a: Preparation of positive reference V-PEG-SC-20k-rhIL-2 1. Modification method The compound V-PEG-SC-20k (purchased from Xiamen Sinobang) disclosed in the patent document CN200680029849.5 was used as a modifying agent to prepare PEG-IL-2 with similar sustained release effect as a positive control.

[0079] The purified wild-type IL-2 protein sample is concentrated and replaced with a modification buffer (100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate, pH 8.0) to a concentration of approximately 20 mg / mL. PEG is weighed out so that the mass ratio of protein to PEG modifier is 1:20, and the modification reaction is carried out at room temperature. After reacting for 2 hours, 1 M glycine is added to stop the reaction.

[0080] 2. Purification method Chromatographic conditions: mobile phase A is 20 mM PB + 2M NaCl (pH 6.0), mobile phase B is 20 mM PB (pH 6.0). Sample loading: After diluting the modified sample, the sample is loaded at 5 ml / min and allowed to bind to a hydrophobic chromatography column (GE, Phenyl PH). Equilibration: After sample loading, wash with solution A for 15 to 20 column volumes. Elution: Elute with 0-100% solution B, and collect the main peak V-PEG-SC-20k-rhIL-2 sample at an elution volume of 10 column volumes. The modified sample was purified under the above conditions to prepare the target sample.

[0081] 3. Purity analysis The results of SEC-HPLC analysis of the above PEGylated IL-2 and its variant modifications are shown in the table below.

[0082] [Table 6]

[0083] SEC (size exclusion chromatography) is a chromatography technique that separates molecules based on the size of the sample molecules. The PEG-IL-2 sample prepared in this example was measured by SEC chromatography, and the results showed that the main peak of the sample was uniform and the modification degree was uniform, which met the research needs.

[0084] In the following Examples, unless otherwise specified, V-PEG-SC-20k-rhIL-2 (or abbreviated as PEG-SC-20k-rhIL-2) prepared in the present Example was used as a positive control.

[0085] Example 4b: Preparation of other PEG-modified samples provided by the present invention 1. Modification method Concentrate the purified rhIL-2 wild-type / mutant protein sample and replace it with modification buffer (100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate, pH 7.5) to a concentration of approximately 2-15 mg / mL. Based on the modification reaction ratio shown in the table below, weigh out PEG (types of PEG include, but are not limited to, V-PEG-SS-20k, mPEG-SS-20k, m-PEG-SS-10k, mPEG-SS-5k, and mPEG-SPA-5K) and perform the modification reaction at room temperature. After reacting for 4 hours, add 1M glycine to stop the reaction. The main reaction parameters for each modification are as shown in the table below.

[0086] [Table 7]

[0087] 2. Purification method Chromatographic conditions: mobile phase B is 20 mM NaAc + 1 M NaCl (pH 4.0), mobile phase A is 20 mM NaAc (pH 4.0). Sample loading: After diluting the modified sample, the sample is loaded at 5 ml / min and allowed to bind to a cation exchange chromatography column (GE, HPSP). Equilibration: After sample loading, wash with solution A for 15 to 20 column volumes. Elution: Elute with 0-100% solution B, with an elution volume of 10 column volumes. Collect a sample of the main peak. The modified sample was purified under the above conditions to prepare the target sample.

[0088] 3. Purity analysis The results of SEC-HPLC analysis for each PEGylated IL-2 and its mutant modifications are shown in the table below.

[0089] [Table 8]

[0090] SEC (size exclusion chromatography) is a chromatographic technique that separates molecules based on the size of the sample molecules. Each PEG-IL-2 sample prepared in this example was measured by SEC chromatography, and the results showed that the main peak of the sample was uniform and the degree of modification was uniform, which met the research needs. In addition, PEG-IL-2 samples with different degrees of modification of the same type of PEG (e.g., GP8) showed significant differences in retention time. The sample preparation process established by this invention showed that samples with different degrees of modification can be stably prepared by controlling the process parameters. The specific PEG binding numbers of the samples are shown in Example 5.

[0091] Example 5: Measurement of the number of PEG bonds in PEG-modified IL-2 (hydrolysis method) 1. Experimental Method 1. Preparation of test solution (1) PEG standard gradient solution: Take 5μL, 10μL, 20μL, 30μL, 40μL, and 50μL of PEG solution (2.5mg / mL) of V-PEG-SC-20k, V-PEG-SS-20k, mPEG-SS-20k, mPEG-SS-10k, and mPEG-SS-5k, respectively, add them to water to prepare gradient solution with a final volume of 100μL, mix evenly, and obtain standard gradient solution. Add 25μL of 5× non-reducing loading buffer, mix evenly, and set aside for later use.

[0092] (2) Iodine staining solution: Accurately weigh out 17.5 g of BaCl2, 6 g of KI, and 3.9 g of I2, dissolve in 500 mL of double-distilled water, and store away from light.

[0093] (3) Preparation of 10% perchloric acid solution: Measure out 100 mL of perchloric acid into a measuring cylinder and slowly add it to 900 mL of water and mix evenly to obtain a 10% perchloric acid solution.

[0094] 2. Gel Preparation Preparation of 10% polyacrylamide gel: Aspirate and mix 1.6 mL of double distilled water, 1.8 mL of 30% polyacrylamide solution, 1.3 mL of 1.5 mol / L Tris-HCl pH 8.8, 0.53 mL of 1% SDS solution, 0.033 mL of 10% ammonium persulfate solution, and 0.033 mL of TEMED to prepare a separating gel. After the separating gel solidifies and forms, 2.9 mL of double distilled water, 0.9 mL of 30% polyacrylamide solution, 1.5 mL of 1.5 mol / L Tris-HCl pH 6.8, 0.6 mL of 1% SDS solution, 0.047 mL of 10% ammonium persulfate solution, and 0.047 mL of TEMED are sucked up and mixed uniformly to prepare a concentrated gel.

[0095] 3. Sample Preparation Hydrolysis: Take 100μL of a sample solution with a known protein content, add 200μL of 100mM NaHCO3 pH 9.0 activation buffer, and hydrolyze in a water bath at 37℃ for 24 hours (mPEG-SPA-5k-rhIL-2 does not hydrolyze spontaneously, so the sample is incubated with trypsin after high temperature treatment). After that, take out the sample, add it to 5× non-reducing loading buffer, mix evenly, and set aside for later use.

[0096] 4. Detection by electrophoresis Operating voltage: run at 80 V for 30 min, switch to 120 V when the bromophenol blue indicator migrates to the bottom of the stacking gel, and end when the migration of the bromophenol blue indicator reaches the bottom edge of the resolving gel. Staining with iodine solution: After electrophoresis is completed, the glass plate is opened, a mark is made on the film, and it is placed in the staining box. It is first fixed with 10% perchloric acid solution for 10 minutes, and then the 10% perchloric acid is collected and washed with water three times. Next, the film is covered with iodine staining solution and stained for 2 to 3 minutes. The color develops in about 1 minute. When the color develops, it is immediately decolorized with water.

[0097] 5. Gel Imaging and Data Processing Place the destained and clear gel in the gel imaging apparatus and perform gel imaging. Run the quantitative processing function using the software Quantity One 4.4.0. Perform a linear regression against the PEG content of the standards and the gray scale of the film spots to determine the total amount of free PEG in the samples. Calculate the results using the following formula: PEG conjugation number = PEG moles / protein moles

[0098] II. Experimental results The results of in vitro analysis of the number of PEG bonds for each modification product are shown in the table below.

[0099] [Table 9]

[0100] Example 6: Measurement of in vitro sustained release performance of PEG-modified IL-2 1. Experimental Method 1. Refer to Example 5 for the pretreatment method. The specific procedure of hydrolysis is as follows: Take 100μL of the sample solution with known protein content, add 200μL of 100mM NaHCO3 pH 9.0 activation buffer, and carry out hydrolysis in a water bath at 37℃. At different time points (e.g., 8 hours, 16 hours, and 24 hours after the start of hydrolysis), take 100μL from each hydrolysis sample, dilute to a certain extent, add 5× non-reducing loading buffer, mix uniformly, and set aside for later use. 2. The measurement and calculation methods of the samples are the same as those in Example 5.

[0101] II. Experimental results The results of the in vitro analysis of the PEG sustained release performance of each modified product are shown in the table below.

[0102] [Table 10]

[0103] 3. Results analysis The results shown in this example are in vitro at 37℃ in activation buffer (100mM NaHCO3 pH 9.0). The above IL-2 or its mutants modified with multiple types of PEG-SS with different structures (linear or branched) and different molecular weights (5K, 10K, 20K) all show excellent sustained release performance in vitro. The positive reference also shows ideal in vitro sustained release performance. However, other common PEG types with non-PEG-SS structures, such as SPA-PEG, cannot achieve the technical effect of sustained release unless additional groups (such as the fluorene ring structure of the positive reference) are introduced, since PEG cannot be removed. Although molecules with different molecular weights and different degrees of modification showed different release rates in vitro, further verification by in vivo and in vivo activity evaluation is required to determine whether the released active protein structure still exerts its medicinal effect.

[0104] Example 7: Measurement of the activity of PEG-modified IL-2 to stimulate CTLL-2 cell proliferation 1. Experimental Method Due to the shielding effect of the active center of the protein by PEG modification, the biological activity of the modified protein in vitro is reduced to a certain extent. Therefore, it is necessary to activate the sample in advance. To evaluate the biological activity of the modified product in the activated state in vitro after activation treatment, we used a method to measure the proliferation rate of the cell-dependent line, CTLL-2 cells, at different concentrations.

[0105] 1. Sample Preparation Sample activation: Take 100 μL of a sample solution with a known protein content, add 200 μL of 100 mM NaHCO3 pH 9.0 or 300 μL of 100% human serum to each, and incubate in a water bath at 37°C for a certain period of time. Take 100 μL of each hydrolyzed sample at regular intervals, mix evenly, and set aside for later use. Dilute the samples to the appropriate initial concentration and perform 2-fold serial dilutions in a 96-well cell culture plate to create a total of eight dilutions. Add each dilution to two wells. Leave 50 μL of solution in each well and discard the excess solution. The above operations are performed under sterile conditions. 2. Preparation of test solutions and cell culture were the same as in Example 3.

[0106] II. Experimental results The results of the activity of the activated products of each polyethylene glycol modification activated under 1.100 mM NaHCO3 pH 9.0 conditions to stimulate the proliferation of CTLL-2 cells are shown in Figure 2 and the table below (the activity value of the original protein used for PEG modification is set as 100% for calculation of relative biological activity).

[0107] [Table 11]

[0108] [Table 12]

[0109] 3. Results analysis From Table 9, we can see the following. The PEG-SS modified product, like the reference product V-PEG-SC-20k-rhIL-2, does not show biological activity in the unactivated state (activation 0h) because the receptor binding sites on the surface are completely blocked by PEG. In other words, if the PEG is not shed, there is no biological activity (not detectable). On the other hand, after activation for different times under alkaline conditions in vitro, all of them showed biological activity to significantly promote the proliferation of CTLL-2 cells. Furthermore, the PEG-SS modified product showed a tendency to gradually recover and improve its biological activity as the activation time increased. When the biological activity of mPEG-SS-20k-GP8 and mPEG-SS-20k-GP12 with a certain degree of modification is higher than that of V-PEG-SC-20k-rhIL-2 at the time of sampling and the PEG shed behavior is similar, a better active release effect can be obtained by adjusting the degree of modification.

[0110] In addition, different degrees of modification with PEG of different molecular weights can provide different active release effects. This also provides the basis for the controllable sustained release performance of IL-2 modified with PEG-SS molecules. That is, by adjusting the molecular weight of the modified PEG-SS, controlling the number of modified PEGs, and pursuing the optimal release curve of PEG-IL2 in the body, the bioavailability of the effector molecule in the body can be optimized. This allows the production of candidate molecules with better efficacy than conventional techniques.

[0111] Example 8: Measurement of pSTAT5 activity in CTLL-2 cells by IL-2 modified with PEG having different structures 1. Experimental Method CTLL-2 cells were cultured at 2 × 10 in complete medium (RPMI 1640 medium + 2 mM L-glutamine + 1 mM sodium pyruvate + 10% fetal bovine serum + 10% T-STIM, cultures were supplemented with concanavalin A) at 37°C and 5% CO2. 5 Culture the cells until the cell density reaches 1 × 10 cells / mL. Then, wash once with PBSA (PBS, pH 7.2, 1% BSA) to reduce the cell density to 1 × 10 6Adjust the concentration of cells / mL. Dispense 500 μL / tube into flow cytometry tubes and add different concentrations of PEGylated IL-2 prepared in basal culture medium (RPMI1640 + 2 mM L-glutamine + 1 mM sodium pyruvate + 10% fetal bovine serum). After 20 min incubation at room temperature, immediately add paraformaldehyde to a final concentration of 1.5%, vortex mix uniformly, and incubate at room temperature for 10 min. Then add 1 mL of PBS and centrifuge at 1400 rpm at 4 °C for 5 min to remove paraformaldehyde. Resuspend the cells, add 1 mL of 100% methanol pre-chilled at 4 °C, vortex mix uniformly, and incubate at 4 °C for 20 min. Then wash the cells twice by adding 3 mL of PBSA buffer and centrifuge at 1400 rpm at 4 °C for 5 min. Then, add Anti-Stat5(pY694)-Alexa647 (BD, Cat#612599) and incubate at room temperature for 30 minutes in the dark. Finally, add 3 mL of PBSA to wash twice and measure with BD AccuriTM C6.

[0112] II. Experimental results The measurement results are shown in Figure 3, which shows that: In the in vitro pSTAT5 activation level measurement, compared with V-PEG-SC-20k-rhIL-2 and mPEG-SS-20k-GP8 (referring to the above-mentioned mPEG-SS-20k-GP8-medium modification in this example), mPEG-SS-10k-GP8 showed a significant difference in the increase curve of phosphorylation activation level after activation in serum, and the increase in phosphorylation level was relatively slow and showed a tendency to increase gradually. After 72 hours, the phosphorylation activation levels of the three samples were almost the same. This is highly consistent with the tendency of active protein release due to PEG shedding in vitro.

[0113] 3. Results analysis Regardless of the type of cell on which IL-2 binds, it exerts its biological functions via the JAK-STAT pathway. The JAK1 pathway, which is mediated by IL-2Rβ binding, and the JAK3 pathway, which is mediated by IL-2Rγ binding, induce phosphorylation of key tyrosine residues on the β and γ subunits of IL-2R, respectively, which generate anchoring sites for other signaling molecules. Therefore, the in vitro biological activity of IL-2 and its modified forms can be evaluated by measuring the changes in phosphorylation levels in CTLL-2 cells under the action of different concentrations of PEG-modified IL-2.

[0114] Therefore, the above experimental results indicate that the release rate of PEG-SS in serum directly affects the release rate of pSTAT5 activation and CTLL-2 proliferation-promoting activity. PEG with this structure allows IL-2 mutants to be released gradually in the body, thereby providing the ability to regulate the degree of immune activation.

[0115] Example 9: Measurement of pSTAT5 activity in CTLL-2 cells by different variants of IL-2 modified with PEG 1. Experimental Method Same as Example 8.

[0116] II. Experimental results The measurement results are shown in Figure 4, which shows that: In the in vitro pSTAT5 activation level measurement, mPEG-SS-20k-GP1 and mPEG-SS-20k-GP8 (referring to the above-mentioned mPEG-SS-20k-GP8-medium modification in this example) showed similar trends in phosphorylation activation levels after activation in buffer, exhibiting the characteristics of gradual activation over time, and maintaining persistent activity after reaching a peak.

[0117] 3. Results analysis The above experimental results show that different mutants of the PEG-SS-modified IL-2 of the present invention (eg, GP1, GP8, etc.) tend to have similar phosphorylation activation levels in vivo.

[0118] Example 10: Comparison of the in vivo half-life extension effect of PEG-modified IL-2 mutants and active release of the modified products 1. Experimental Method Experiment 1: 0.5mg / kg, 0.1mg / kg, and 0.04mg / kg mPEG-SS-5k-GP8-highly modified and 1mg / kg mutant GP8 were intravenously injected into SD rats, respectively. Serum was collected before and after administration 0.25 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours, and the content of mPEG-SS-5k-GP8-highly modified and mutant GP8 in each serum was measured by two kinds of ELISA.

[0119] Experiment 2: 1mg / kg mPEG-SS-5k-GP8-highly modified and positive reference V-PEG-SC-20k-rhIL-2 were intravenously injected into SD rats. Serum was collected before and after 0.25, 1, 2, 4, 8, 24, 48, 72, and 96 hours, and activity was measured at each time point by CTLL-2 cell / MTT colorimetric method. The unit is IU / ml.

[0120] II. Experimental results Experiment 1: The test results are shown in Figure 5. The test results show that compared with the original protein of mutant GP8, the mPEG-SS-5k-GP8-high modification can significantly extend the half-life of the drug.

[0121] Experiment 2: Based on the measurement results, the drug concentration-time curve was drawn. The study showed that at the same dose and administration method, mPEG-SS-5k-GP8-high modification showed a higher AUC (5683809h.IU / ml vs 2344730h.IU / ml) than the positive reference V-PEG-SC-20k-rhIL-2 (calculated with GraphPad Prism 7.00). Therefore, it was found that mPEG-SS-5k-GP8-high modification has higher bioavailability than the positive reference (V-PEG-SC-20k-rhIL-2).

[0122] Example 11: Evaluation of the efficacy of IL-2 modified with different structures of PEG-SS (linear or branched) in a CT26.WT mouse-derived colon cancer cell subcutaneously implanted in BALB / c mice 1. Experimental Method CT26.WT cells are cultured in 1640 medium containing 10% fetal bovine serum. CT26.WT cells in the exponential growth phase are harvested and resuspended in PBS to an appropriate concentration. 5 × 10 5 The CT26.WT cell suspension at 0.1 mL / cells was mixed thoroughly and then inoculated subcutaneously into the right back of BALB / c mice. Each mouse was inoculated with 0.1 mL. The tumor volume was approximately 100 mm on average. 3 After reaching this volume, the mice were divided into groups based on the tumor volume. The first administration was started on the day of grouping (in this experiment, one administration was performed on the 9th and 16th days after tumor cell inoculation). The detailed administration protocol and administration route are shown in the table below.

[0123] [Table 13]

[0124] General clinical observations: During the quarantine and experimental periods, observations should be made at least once a day, including observations of tumor growth and the effects of treatment on the normal behavior of the animals, including death or moribundity of the animals (tumor-bearing), mental state, behavioral activity, and other abnormalities. Weight: Measure 2-3 times weekly.

[0125] Tumor volume: Measure 2-3 times weekly. Measure the long and short diameters of the tumor with a caliper.

[0126] Tumor volume (mm 3 ) = major axis * minor axis 2 / 2. Relative tumor inhibition rate: TGI(%)=(1-T / C)×100%. In general, T represents the relative tumor volume at a certain time point in the administration group (the ratio of the tumor volume measured at that time point to the tumor volume at the time of grouping), and C represents the relative tumor volume at a certain time point in the model group (the ratio of the tumor volume measured at that time point to the tumor volume at the time of grouping). However, in the present invention, the groups are divided based on body weight on the day of inoculation in the experiment, so in calculating TGI, T and C represent the tumor volumes of the administration group and the model group actually measured at that time point, respectively.

[0127] Tumor weight: After the final measurement, the experimental animals are euthanized, the tumor masses are excised, washed with saline, and dried by absorbing moisture with filter paper, and the tumor masses are weighed and photographed. Relative tumor inhibition rate TGI(%)=(1-T TW / C TW )×100%, T TW represents the mean tumor weight of the treatment group at the end of the experiment, and C TW represents the average tumor weight of the model group at the end of the experiment.

[0128] II. Experimental results 1. Medicinal properties The mean tumor volume of the animals in the model group 20 days after cell inoculation was 1397.48 ± 289.67 mm 3 It was.

[0129] The mean tumor volumes of the mPEG-SS-20k-GP1 (2 mg / kg) group and the mPEG-SS-20k-GP8 (2 mg / kg) group (referring to the above-mentioned mPEG-SS-20k-GP8-medium modification in this example) on the 20th day after cell inoculation were 282.31 ± 103.02 mm 3 , 133.93±105.69mm 3 There was a significant difference compared to the model group (P<0.01). The relative tumor inhibition rates (TGI) (%) were 79% and 92%, respectively.

[0130] The mean tumor volumes in the V-PEG-SS-20k-GP1 (2 mg / kg) and V-PEG-SS-20k-GP8 (2 mg / kg) groups 20 days after cell inoculation were 942.44 ± 209.66 mm 3 , 1037.67±332.97mm 3 There was no significant difference compared to the model group. The relative tumor inhibition rates (TGI) (%) were 26% and 29%, respectively.

[0131] The mean tumor volume of the positive reference V-PEG-SC-20k-rhIL-2 group (2 mg / kg) on ​​the 20th day after cell inoculation (unless otherwise specified in the following examples, all positive references used V-PEG-SC-20k-rhIL-2, abbreviated as PEG-SC-20k-rhIL-2) was 211.23 ± 86.02 mm 3 There was a significant difference compared to the model group (P<0.01). The relative tumor inhibition rate (TGI) (%) was 84%.

[0132] The analysis results of tumor weight are almost consistent with the analysis results of tumor volume. Specific experimental results are shown in the table below and Figure 6.

[0133] [Table 14]

[0134] [Table 15]

[0135] 2, Safety The animals in the PEG-SS modified group lost less than 10% of their body weight during the administration period, and although their body weight recovered slightly in the later stages of the experiment, there was no significant difference compared to the model group (D13, D16, D18, D20).

[0136] In the positive control group, 1 out of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%) on the second day after the second administration (D18). Also, animals in the positive control group regained some weight in the later stages of the experiment, but the difference was significant compared to the model group.

[0137] [Table 16]

[0138] 3. Results analysis The linear PEG modified products mPEG-SS-20k-GP1 and mPEG-SS-20k-GP8 showed significant tumor growth inhibition at a dose of 2 mg / kg in a CT26.WT mouse-derived colon cancer model, with the inhibition rate being equal to or greater than that of the positive reference product. On the other hand, the branched PEG modified products V-PEG-SS-20k-GP1 and V-PEG-SS-20k-GP8 showed no tumor growth inhibition at a dose of 2 mg / kg in a CT26.WT mouse-derived colon cancer model.

[0139] During this efficacy evaluation period, animals in each group showed good tolerance at a dose of 2 mg / kg. In the groups that showed therapeutic effects, the linear PEG-SS modified product group and the positive control group all experienced weight loss during the middle of the administration period, but after the second administration and during the recovery period, the animals in the linear PEG-SS modified product group showed significantly better condition and weight recovery than the positive control group.

[0140] In this embodiment, the researcher was surprised that the linear mPEG-SS-20k modified product and the branched V-PEG-SS-20k modified product showed a significant difference in in vivo experiments, even though they had the same molecular weight. That is, the in vivo efficacy of the branched PEG-SS modified product was much lower than that of the linear PEG-SS modified product. Therefore, the inventor confirmed the use of linear mPEG-SS in developing IL-2 protein drugs as tumor immune agonists.

[0141] Example 12: Efficacy evaluation of different mutants of IL-2 modified with linear PEG-SS in a B16-F10 murine melanoma cell line subcutaneously implanted in C57BL / 6 mice 1. Experimental Method B16-F10 cells are cultured in DMEM medium containing 10% fetal bovine serum. B16-F10 cells in the exponential growth phase are harvested and resuspended in PBS to an appropriate concentration. 5 × 10 5 The B16-F10 cell suspension at 0.1 mL / cells was mixed thoroughly and then subcutaneously inoculated into the right back of C57BL / 6 mice. Each mouse was inoculated with 0.1 mL. The tumor volume was approximately 100 mm on average. 3 After reaching the tumor volume, the mice were divided into groups based on the tumor volume. The first administration was started on the day of grouping (one administration was given on D7). The detailed administration protocol and administration route are shown in the table below. General clinical observations were the same as in Example 11.

[0142] [Table 17]

[0143] II. Experimental results 1. Medicinal properties The experimental results are shown in the table below and in FIG.

[0144] [Table 18]

[0145] [Table 19]

[0146] 2, Safety The weight change rate of animals in the V-PEG-SC-20k-rhIL-2 group was significantly decreased compared with the model group at D11 (all animals in each group were alive at D11). The weight loss of animals in the mPEG-SS-20k-GP1, mPEG-SS-20k-GP13, mPEG-SS-20k-GP21, and mPEG-SS-20k-GP22 groups, which are different mutant products modified with linear PEG-SS, was not statistically different from the model group at D11, but was statistically different from the V-PEG-SC-20k-rhIL-2 group. This indicates that the different mutant products modified with linear PEG-SS (e.g., mPEG-SS-20k-GP1, mPEG-SS-20k-GP13, mPEG-SS-20k-GP21, mPEG-SS-20k-GP22, etc.) had less effect on the animal body weight than V-PEG-SC-20k-rhIL-2.

[0147] [Table 20]

[0148] 3. Results analysis The linear PEG modified different mutant products mPEG-SS-20k-GP1, mPEG-SS-20k-GP13, mPEG-SS-20k-GP21 and mPEG-SS-20k-GP22 showed significant tumor growth inhibition effect in B16-F10 murine melanoma model at the dose of 1 mg / kg, and the inhibition rate was close to that of the positive reference product.The above test products also had less effect on the body weight of the animals than V-PEG-SC-20k-rhIL-2.

[0149] Example 13: Efficacy evaluation of linear PEG-SS modified IL-2 mutants with different degrees of modification in B16-F10 murine melanoma tumor model 1. Experimental Method The experimental method, general clinical observation, etc. were the same as in Example 11. The detailed administration protocol and administration route are shown in the table below.

[0150] [Table 21]

[0151] II. Experimental results 1. Medicinal properties The mean tumor volume of the animals in the model group on the 17th day after cell inoculation was 2614.97 ± 372.77 mm 3 It was.

[0152] On the 17th day after cell inoculation, the mean tumor volumes of the mPEG-SS-20k-GP8 (high modification), mPEG-SS-20k-GP8 (medium modification), mPEG-SS-5k-GP8 (high modification), mPEG-SS-5k-GP8 (medium modification), and mPEG-SS-5k-GP8 (low modification) groups were 550.25 ± 100.23 mm 3 , 544.90±89.12mm 3 , 574.02±108.15mm 3 , 676.17±128.23mm 3 , 570.45±107.25mm 3 There was a significant difference compared to the model group (P<0.01 or P<0.05). The relative tumor inhibition rates (TGI) (%) were 77%, 80%, 79%, 75%, and 79%, respectively.

[0153] The mean tumor volume of the PEG-SC-20k-rhIL-2 group on the 17th day after cell inoculation was 1215.19 ± 184.70 mm 3 There was a significant difference compared to the model group (P<0.05). The relative tumor inhibition rate (TGI) (%) was 57%.

[0154] The analysis results of tumor weight are almost consistent with the analysis results of tumor volume. Specific experimental results are shown in the table below and Figure 8.

[0155] [Table 22]

[0156] [Table 23]

[0157] 2, Safety In the mPEG-SS-20k-GP8 (highly modified) group, 1 out of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 4th day after the first administration (11th day after cell inoculation). 2 out of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 7th day after the first administration (14th day after cell inoculation), and 3 out of 6 animals died. 1 out of 6 animals still showed moderate weight loss (10% < weight loss ≦ 20%) on the 9th day after the first administration (16th day after cell inoculation), and 1 out of 6 animals died. At the end of the experiment, 2 out of 6 surviving animals showed a tendency to recover their weight. More than half of the animals in this group died, and the cause of death is presumed to be related to the test product.

[0158] In the mPEG-SS-20k-GP8 (moderately modified) group, 2 out of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 4th day after the first administration (11th day after cell inoculation). 4 out of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) and 2 out of 6 animals showed severe weight loss (> 20%) on the 7th day after the first administration (14th day after cell inoculation). 1 out of 6 animals still showed moderate weight loss (10% < weight loss ≦ 20%), 1 out of 6 animals showed severe weight loss (> 20%) and 1 out of 6 animals died on the 9th day after the first administration (16th day after cell inoculation). At the end of the experiment, of the 5 out of 6 surviving animals, 4 animals showed a tendency to regain weight, and 1 animal did not regain weight. Moderate to severe weight loss and one animal death occurred in this group, which was suspected to be test article related.

[0159] In the mPEG-SS-5k-GP8 (highly modified) group, 1 out of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 4th day after the first administration (11th day after cell inoculation). 1 out of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 7th day after the first administration (14th day after cell inoculation). At the end of the experiment, the animals showed a tendency to regain their weight.

[0160] In the mPEG-SS-5k-GP8 (moderate modification) group, there was no significant change in body weight during the administration period.

[0161] In the mPEG-SS-5k-GP8 (low modification) group, 1 out of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 4th day after the first administration (11th day after cell inoculation). At the end of the experiment, the animals showed a tendency to regain their weight.

[0162] In the PEG-SC-20k-rhIL-2 group, 1 out of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%) 4 days after the first dose (11 days after cell inoculation), and the animals showed a tendency to regain weight at the end of the study. 3 out of 6 animals had died 7 days after the first dose (14 days after cell inoculation). Half of the animals in this group died, and the cause of death is presumed to be related to the test product.

[0163] In the model group, 1 out of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 7th day after the first administration (14th day after cell inoculation), and showed a tendency to regain weight at the end of the experiment. The specific results are shown in the table below and Figure 8c.

[0164] [Table 24]

[0165] 3. Results analysis The test samples mPEG-SS-20k-GP8 (high modification), mPEG-SS-20k-GP8 (medium modification), mPEG-SS-5k-GP8 (high modification), mPEG-SS-5k-GP8 (medium modification), and mPEG-SS-5k-GP8 (low modification) all had tumor growth inhibitory effects in the B16-F10 murine melanoma model at a dose of 2 mg / kg, and their effects were superior to those of V-PEG-SC-20k-rhIL-2. During the treatment period, animals in the mPEG-SS-20k-GP8 (highly modified) and mPEG-SS-20k-GP8 (moderately modified) groups did not tolerate the 2 mg / kg dose well, whereas animals in the mPEG-SS-5k-GP8 (highly modified) and mPEG-SS-5k-GP8 (lowly modified) groups tolerated the 2 mg / kg dose well. Animals in the mPEG-SS-5k-GP8 (moderately modified) group tolerated the 2 mg / kg dose well. This example provides preliminary evidence that products with different degrees of modification exhibit different pharmacological effects and safety in the body. This result is consistent with the prediction based on the in vitro experiment in Example 7 that "products with different degrees of modification have different performances." Therefore, the inventors further conducted comparative studies of pharmacological effects in other different models.

[0166] Example 14: Efficacy evaluation of linear PEG-SS modified IL-2 mutants with different degrees of modification in a CT26.WT mouse-derived colon cancer tumor model 1. Experimental Method The experimental method, general clinical observation, etc. were the same as in Example 11. The detailed administration protocol and administration route are shown in the table below.

[0167] [Table 25]

[0168] II. Experimental results 1. Medicinal properties The mean tumor volume of the animals in the model group 22 days after cell inoculation was 1,886.67 ± 341.33 mm 3 It was.

[0169] On the 22nd day after cell inoculation, the mean tumor volumes of the mPEG-SS-20k-GP8 (high modification), mPEG-SS-20k-GP8 (medium modification), mPEG-SS-5k-GP8 (high modification), mPEG-SS-5k-GP8 (medium modification), and mPEG-SS-5k-GP8 (low modification) groups were 268.15 ± 163.54 mm 3 , 132.07±132.07mm 3 , 56.90±36.21mm 3 , 429.12±261.88mm 3 , 87.78±87.78mm 3 There was a significant difference compared to the model group (P<0.01). The relative tumor inhibition rates (TGI) (%) were 88%, 90%, 97%, 80%, and 95%, respectively.

[0170] The mean tumor volume in the PEG-SC-20k-rhIL-2 group 22 days after cell inoculation was 163.13±73.03mm 3 There was a significant difference compared to the model group (P<0.01). The relative tumor inhibition rate (TGI) (%) was 92%.

[0171] The analysis results of tumor weight are almost consistent with the analysis results of tumor volume. Specific experimental results are shown in the table below and Figure 9.

[0172] [Table 26]

[0173] [Table 27]

[0174] 2, Safety In the mPEG-SS-20k-GP8 (highly modified) group, 3 of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 5th day after the first injection, and 1 of 6 animals died. 2 of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 2nd day after the second injection, and showed a tendency to regain weight at the end of the experiment.

[0175] In the mPEG-SS-20k-GP8 (moderately modified) group, 3 of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 5th day after the first dose, and 1 of 6 animals died. 1 of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 7th day after the first dose. 1 of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 2nd day after the second dose, and did not recover by the end of the experiment.

[0176] In the mPEG-SS-5k-GP8 (highly modified) group, 1 out of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) 5 days after the first injection. 1 out of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) 2 days after the second injection, and showed a tendency to regain weight at the end of the experiment.

[0177] In the mPEG-SS-5k-GP8 (moderate modification) group, animals did not lose weight or experienced less than 10% weight loss during the administration period, and animals that lost weight showed weight recovery in the later stages of the experiment.

[0178] In the mPEG-SS-5k-GP8 (low modification) group, 1 out of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 2nd day after the second administration, and showed a tendency to regain weight at the end of the experiment.

[0179] In the PEG-SC-20k-rhIL-2 group, 4 of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%) 5 days after the first dose. 2 of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%) 2 days after the second dose. 1 of 6 animals still showed moderate weight loss (10% < weight loss ≤ 20%) 7 days after the second dose, and the remaining animals showed a tendency to regain weight at the end of the study.

[0180] [Table 28]

[0181] 3. Results analysis The test samples mPEG-SS-20k-GP8 (high modification), mPEG-SS-20k-GP8 (medium modification), mPEG-SS-5k-GP8 (high modification), mPEG-SS-5k-GP8 (medium modification), and mPEG-SS-5k-GP8 (low modification) had tumor growth inhibitory effects in a CT26.WT mouse-derived colon cancer model at a dose of 2 mg / kg.

[0182] During the treatment period, animals in the mPEG-SS-5k-GP8 (highly modified), mPEG-SS-5k-GP8 (moderately modified), and mPEG-SS-5k-GP8 (lowly modified) groups tolerated the 2 mg / kg dose well. Animals in the mPEG-SS-20k-GP8 (highly modified) and mPEG-SS-20k-GP8 (moderately modified) groups generally tolerated the 2 mg / kg dose and regained body weight better than the VPEG-SC-20k-rhIL-2 group.

[0183] Example 15: Efficacy evaluation of linear PEG-SS modified IL-2 mutants with different degrees of modification in A375 human melanoma tumor model A375 cells are cultured in DMEM medium containing 10% fetal bovine serum. A375 cells in the exponential growth phase are harvested and resuspended by adding PBS. hu-PBMCs (human peripheral blood mononuclear cells) are cultured in 1640 medium containing 10% fetal bovine serum, and PBMCs are harvested after stimulation with OKT-3 and IL-2 drugs for 3 days, and resuspended by adding PBS. 5 × 10 5 cells / 0.1mL of A375 cells and 5×10 5 The cells / 0.1mL PBMC cell suspension was thoroughly mixed at a 1:1 ratio (cell inoculation volume) and then subcutaneously inoculated into NOD / SCID mice. Each mouse was inoculated with 0.2mL. On the day after cell inoculation, the mice were divided into groups based on body weight. The first dose was started on the day of grouping. The detailed administration protocol and administration route are shown in the table below.

[0184] [Table 29]

[0185] The experimental method, general clinical observation, and measurements of body weight, tumor volume, and tumor weight were the same as in Example 11.

[0186] II. Experimental results 1. Medicinal properties The mean tumor volume of the animals in the model group 45 days after cell inoculation was 1,970.97 ± 298.65 mm 3 It was.

[0187] On the 45th day after cell inoculation, animals in the mPEG-SS-20k-GP8 (high modification), mPEG-SS-20k-GP8 (medium modification), and mPEG-SS-5k-GP8 (medium modification) groups showed no tumor growth. All were significantly different compared with the model group (P<0.01). The mean tumor volume of animals in the PEG-SC-20k-rhIL-2, mPEG-SS-5k-GP8 (high modification), and mPEG-SS-5k-GP8 (low modification) groups was 136.59±134.84mm, respectively. 3 , 83.34±83.34mm 3 , 96.62±64.92mm 3All were significantly different from the model group (P<0.01).

[0188] The results of tumor weight analysis were close to those of tumor volume analysis.The TGI calculated based on tumor weight in the mPEG-SS-20k-GP8 (high modification) group, the mPEG-SS-20k-GP8 (medium modification) group, the mPEG-SS-5k-GP8 (high modification) group, the mPEG-SS-5k-GP8 (medium modification) group, the mPEG-SS-5k-GP8 (low modification) group, and the PEG-SC-20k-rhIL-2 group were 100%, 100%, 95%, 100%, 95%, and 91%, respectively. Specific experimental results are shown in the table below and FIG.

[0189] [Table 30]

[0190] [Table 31]

[0191] [Table 32]

[0192] [Table 33]

[0193] 2, Safety In the mPEG-SS-20k-GP8 (highly modified) group, 1 of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) on the 3rd day after the 4th injection. This animal continued to show moderate weight loss (10% < weight loss ≦ 20%) on the 6th day after the 4th injection and died on the 1st day after the last injection. Furthermore, 1 of 6 animals showed moderate weight loss (10% < weight loss ≦ 20%) from the 9th day after the last injection to the 13th day after the last injection, and this animal showed a tendency to regain weight at the end of the experiment.

[0194] In the mPEG-SS-20k-GP8 (moderately modified) group, 1 of 6 animals showed severe weight loss (weight loss > 20%) 3 days after the second injection. This animal died 6 days after the second injection. 1 of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%) from 3 days after the last injection until the end of the experiment. Furthermore, 1 of 6 animals died 4 days after the last injection.

[0195] In the mPEG-SS-5k-GP8 (moderately modified) group, one of six animals showed moderate weight loss (10% < weight loss ≦ 20%) from day 6 after the last dose to day 9 after the last dose. At the end of the study, this animal showed a tendency to regain weight.

[0196] In the mPEG-SS-5k-GP8 (high modification) and mPEG-SS-5k-GP8 (low modification) groups, animals did not lose weight or experienced less than 10% weight loss during the administration period, and animals that lost weight showed weight recovery in the later stages of the experiment.

[0197] In the PEG-SC-20k-rhIL-2 group, 1 of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%) 6 days after the last dose. 2 of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%) 9 days after the last dose. 2 of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%) 13 days after the last dose, and 1 of 6 animals died.

[0198] 3. Results analysis The test products mPEG-SS-20k-GP8 (high modification), mPEG-SS-20k-GP8 (medium modification), mPEG-SS-5k-GP8 (high modification), mPEG-SS-5k-GP8 (medium modification), and mPEG-SS-5k-GP8 (low modification) all had the effect of inhibiting tumor growth in the A357 human melanoma model at a dose of 0.5 mg / kg (once per week for 5 weeks). In terms of efficacy, the effects of the test products (mPEG-SS-20k-GP8 (high modification), mPEG-SS-20k-GP8 (medium modification), mPEG-SS-5k-GP8 (high modification), mPEG-SS-5k-GP8 (medium modification), and mPEG-SS-5k-GP8 (low modification)) were slightly better than those of PEG-SC-20k-rhIL-2.

[0199] During the treatment period, animals in the mPEG-SS-20k-GP8 (highly modified), mPEG-SS-20k-GP8 (moderately modified), and V-PEG-SC-20k-rhIL-2 groups did not tolerate a dose of 0.5 mg / kg well, whereas animals in the mPEG-SS-5k-GP8 (highly modified) and mPEG-SS-5k-GP8 (lowly modified) groups tolerated a dose of 0.5 mg / kg (once per week for 5 weeks). Animals in the mPEG-SS-5k-GP8 (moderately modified) group generally tolerated a dose of 0.5 mg / kg (once per week for 5 weeks). From a safety standpoint (body weight, death), the test products mPEG-SS-5k-GP8 (high modification), mPEG-SS-5k-GP8 (medium modification), and mPEG-SS-5k-GP8 (low modification) were better tolerated than PEG-SC-20k-rhIL-2.

[0200] Example 16: Efficacy evaluation of PEG-modified IL-2 mutants in the A375 human melanoma model 1. Experimental Method A375 cells are cultured in DMEM medium containing 10% fetal bovine serum. A375 cells in the exponential growth phase are harvested and resuspended by adding PBS. hu-PBMCs (human peripheral blood mononuclear cells) are cultured in 1640 medium containing 10% fetal bovine serum, and PBMCs are harvested after stimulation with OKT-3 and IL-2 drugs for 3 days, and resuspended by adding PBS. 1×10 61×10 cells / 0.1mL A375 cells 6 The cells / 0.1mL PBMC cell suspension was thoroughly mixed at a 1:1 ratio (cell inoculation volume) and then subcutaneously inoculated into NOD / SCID mice. Each mouse was inoculated with 0.2mL. On the day after cell inoculation, the mice were divided into groups based on body weight. The first dose was started on the day of grouping. The detailed administration protocol and administration route are shown in the table below.

[0201] [Table 34]

[0202] General clinical observations, body weight, tumor volume, and tumor weight were measured in the same manner as in Example 11.

[0203] II. Experimental results 1. Medicinal properties The mean tumor volume in the negative control animals 45 days after cell inoculation was 1,197.64 ± 143.79 mm 3 It was.

[0204] On the 45th day after cell inoculation, the mean tumor volumes of animals in the high, medium, and low dose groups (250 μg / kg, 125 μg / kg, and 62.5 μg / kg) of mPEG-SS-5K-GP8-highly modified mice were 144.87 ± 35.11 mm, respectively. 3 , 296.04±61.10mm 3 , 643.88±153.05mm 3 All were significantly different from the negative control group (P<0.01 or P<0.05).

[0205] The mean tumor volume of animals in the positive control group (Quanqi, 1 million IU / kg) 45 days after cell inoculation was 557.54 ± 104.82 mm 3There was a significant difference compared with the negative control group (P<0.01). The mPEG-SS-5K-GP8-highly modified high-dose group had a significant difference in tumor volume compared with the positive control group (Quanqi) (P<0.01). Although the total dose of the positive control group (Quanqi) was higher than that of the mPEG-SS-5K-GP8-highly modified high-dose group, the experimental results showed that the mPEG-SS-5K-GP8-highly modified had better efficacy.

[0206] The mean tumor volume of animals in the positive reference drug (V-PEG-SC-20k-rhIL-2, mimic NKTR214) group 45 days after cell inoculation was 224.37 ± 33.28 mm 3 There was a significant difference compared with the negative control group (P<0.01). The mPEG-SS-5K-GP8-highly modified medium dose group had no significant difference in tumor volume compared with the positive reference group (P>0.05). The experimental results showed that the efficacy of mPEG-SS-5K-GP8-highly modified was close to that of the positive reference drug (V-PEG-SC-20k-rhIL-2, pseudo-NKTR214) at the same dose.

[0207] The tumor weight analysis results were almost consistent with the tumor volume analysis results. The TGIs of the mPEG-SS-5K-GP8-highly modified high, medium and low dose groups calculated based on tumor weight were 87%, 75% and 46%, respectively. The TGIs of the positive control group (Quanqi) and positive reference group (V-PEG-SC-20k-rhIL-2, mock NKTR214) calculated based on tumor weight were 56% and 81%, respectively. Specific experimental results are shown in the table below and FIG.

[0208] [Table 35]

[0209] [Table 36]

[0210] 2, Safety In the mPEG-SS-5k-GP8-highly modified high dose group (250 μg / kg), 3 out of 8 animals showed moderate weight loss during the intravenous treatment period, and the animals generally tolerated the treatment. In the mPEG-SS-5k-GP8-highly modified medium and low dose groups (125 μg / kg, 62.5 μg / kg), no obvious drug toxicity was observed during the intravenous treatment period, and the treatment was well tolerated.

[0211] In animals in the positive control group (Quanqi, 1 million IU / kg), no obvious drug toxicity was observed during the intravenous administration treatment period, and the drug was well tolerated during the treatment period.On the third day (D32) after the last dose and the 16th day (D45) after the last dose, the body weights of the mPEG-SS-5K-GP8-highly modified high-dose group (250 μg / kg) were not statistically different from those of the positive control group (Quanqi, 1 million IU / kg) (P>0.05).

[0212] Animals in the V-PEG-SC-20k-rhIL-2 group (125 μg / kg) generally tolerated the treatment, although 3 of 8 animals showed moderate weight loss and 1 of 8 animals showed moderate to severe weight loss during the intravenous treatment period. On the third day (D32) and 16 days (D45) after the last dose, the body weights of the mPEG-SS-5K-GP8-highly modified medium dose group (125 μg / kg) were statistically different (P<0.01 or P<0.05) compared with the positive reference group (V-PEG-SC-20k-rhIL-2, 125 μg / kg). As a result, the mPEG-SS-5K-GP8-highly modified group has a better safety profile than the positive reference group (mock NKTR214) at the same dose.

[0213] [Table 37]

[0214] [Table 38]

[0215] 3. Results analysis mPEG-SS-5K-GP8-highly modified showed significant tumor suppression effects on A375 human melanoma model at high (250μg / kg) and medium (125μg / kg) doses. mPEG-SS-5K-GP8-highly modified also showed a tendency of tumor suppression at low dose (62.5μg / kg). mPEG-SS-5K-GP8-highly modified showed a good dose-effect relationship at high, medium and low doses. The positive reference group (V-PEG-SC-20k-rhIL-2, pseudo-NKTR214) showed significant tumor suppression effects on A375 human melanoma model at a dose of 125μg / kg, and the positive control group (Quanqi) showed a tendency of tumor suppression at a dose of 1 million IU / kg.

[0216] Animals in the mPEG-SS-5K-GP8-highly modified high-dose group (250 μg / kg) and V-PEG-SC-20k-rhIL-2 group (125 μg / kg) generally tolerated the treatment, while animals in the mPEG-SS-5K-GP8-highly modified medium and low-dose groups (125 μg / kg, 62.5 μg / kg) and positive control group (Quanqi, 1 million IU / kg) tolerated the treatment well. No deaths were observed in any of the treatment groups.

[0217] The above results indicate that mPEG-SS-5K-GP8-high modification is better tolerated by animals than V-PEG-SC-20k-rhIL-2 at the same tumor suppression effect. Also, at the same safety level, the positive control group (Quanqi) was administered a higher total dose than the mPEG-SS-5K-GP8-high modification high dose group, but the mPEG-SS-5K-GP8-high modification had better efficacy.

[0218] Example 17: Efficacy evaluation of PEG-modified IL-2 mutants in the A498 human kidney cancer model 1. Experimental Method A498 cells are cultured in DMEM medium containing 10% fetal bovine serum. A498 cells in the exponential growth phase are harvested and resuspended by adding PBS. hu-PBMCs (human peripheral blood mononuclear cells) are cultured in 1640 medium containing 10% fetal bovine serum, and PBMCs are harvested after stimulation with OKT-3 and IL-2 drugs for 3 days, and resuspended by adding PBS. 5 × 10 6 cells / 0.1mL of A498 cells and 5×10 6 The cells / 0.1mL PBMC cell suspension was thoroughly mixed at a 1:1 ratio (cell inoculation volume) and then subcutaneously inoculated into NOD / SCID mice. Each mouse was inoculated with 0.2mL. On the day after cell inoculation, the mice were divided into groups based on body weight. The first dose was started on the day of grouping. The detailed administration protocol and administration route are shown in the table below.

[0219] [Table 39]

[0220] General clinical observations, body weight, tumor volume, and tumor weight were measured in the same manner as in Example 11.

[0221] II. Experimental results 1. Medicinal properties The mean tumor volume in the negative control animals 61 days after cell inoculation was 1,960.68 ± 398.28 mm 3 It was.

[0222] On the 61st day after cell inoculation, the mean tumor volume of animals in the medium and low dose groups (125 μg / kg and 62.5 μg / kg) with high mPEG-SS-5K-GP8 modification was 18.99 ± 18.99 mm, respectively. 3 , 248.19±209.86mm 3 There was a significant difference compared with the negative control group (P<0.01).

[0223] The mean tumor volume of animals in the positive control group (Quanqi, 712,500 IU / kg) 61 days after cell inoculation was 290.85 ± 145.96 mm 3There was a significant difference compared with the negative control group (P<0.01). The experimental results showed that although the dose of the mPEG-SS-5K-GP8-highly modified medium dose group was only half that of the positive control group (Quanqi), its efficacy was superior to that of the positive control group (Quanqi).

[0224] The mean tumor volume of animals in the V-PEG-SC-20k-rhIL-2 group 61 days after cell inoculation was 110.22 ± 63.15 mm 3 There was a significant difference compared with the negative control group (P<0.01). The mPEG-SS-5K-GP8-highly modified medium dose group had no significant difference in tumor volume compared with the positive reference group (P>0.05). The experimental results showed that the efficacy of mPEG-SS-5K-GP8-highly modified was superior to that of the positive reference drug (V-PEG-SC-20k-rhIL-2, pseudo-NKTR214) at the same dose.

[0225] The tumor weight analysis results were almost consistent with the tumor volume analysis results. The TGIs of the medium and low dose groups of mPEG-SS-5K-GP8 calculated based on tumor weight were 99% and 85%, respectively, while the TGIs of the positive control group (Quanqi) and positive reference group (V-PEG-SC-20k-rhIL-2, mock NKTR214) calculated based on tumor weight were 83% and 93%, respectively. Specific experimental results are shown in the table below and FIG.

[0226] [Table 40]

[0227] [Table 41]

[0228] 2, Safety In the mPEG-SS-5k-GP8 medium dose group (125 μg / kg), one of eight animals showed moderate weight loss during the intravenous treatment period, and the treatment was generally well tolerated. In the mPEG-SS-5k-GP8 highly modified low dose group (62.5 μg / kg), no obvious drug toxicity was observed during the intravenous treatment period, and the treatment was well tolerated.

[0229] In animals in the positive control group (Quanqi, 712,500 IU / kg), no obvious drug toxicity was observed during the treatment period administered intravenously, and the drug was well tolerated during the treatment period.

[0230] Animals in the V-PEG-SC-20k-rhIL-2 group (125 μg / kg) generally tolerated the treatment, although 3 of 8 animals showed moderate weight loss and 1 of 8 animals showed moderate to severe weight loss during the intravenous treatment period. Four days after the last dose (D33), the body weight of the mPEG-SS-5K-GP8-highly modified medium dose group (125 μg / kg) was statistically different (P<0.01) compared with the V-PEG-SC-20k-rhIL-2 group (125 μg / kg). As a result, the mPEG-SS-5K-GP8-highly modified group has a superior safety profile than the positive reference group (mock NKTR214) at the same dose.

[0231] [Table 42]

[0232] [Table 43]

[0233] 3. Results analysis mPEG-SS-5K-GP8-highly modified showed significant tumor suppression effects in the A498 human renal cancer subcutaneously transplanted tumor model at medium (125μg / kg) and low (62.5μg / kg) doses. mPEG-SS-5K-GP8-highly modified showed a certain dose-effect relationship at medium and low doses. V-PEG-SC-20k-rhIL-2 group showed significant tumor suppression effects at a dose of 125μg / kg and positive control group (Quanqi) showed significant tumor suppression effects at a dose of 712,500 IU / kg in the A498 human renal cancer subcutaneously transplanted tumor model.

[0234] Animals in the mPEG-SS-5K-GP8-highly modified medium dose group (125 μg / kg) and V-PEG-SC-20k-rhIL-2 group (125 μg / kg) generally tolerated the treatment, while animals in the mPEG-SS-5K-GP8-highly modified low dose group (62.5 μg / kg) and positive control group (Quanqi, 712,500 IU / kg) tolerated the treatment well. No deaths were observed in animals in any treatment group.

[0235] The above indicates that mPEG-SS-5K-GP8-highly modified IgG was better tolerated by animals than V-PEG-SC-20k-rhIL-2 with comparable tumor suppression effect.

[0236] Example 18: Effect of PEG-modified IL-2 on the activation of cynomolgus monkey PBMC cells Three cynomolgus monkeys were assigned to each group, and different doses of mPEG-SS-5K-GP8-highly modified (0.01mg / kg group, 0.03mg / kg group) were administered intravenously. The blank control group was administered with the solvent as a control. The administration was performed once a week for a total of five times (D1, D8, D15, D22, D29). Thirty days after the first administration (D1), peripheral blood was collected, and peripheral blood mononuclear cells (PBMC) were isolated and labeled with anti-CD25-APC fluorescent antibody (Miltenyi, cargo number 130-113-842) for FACS measurement.

[0237] [Table 44]

[0238] The above data showed that the expression level of CD25 in the 0.03mg / kg group was significantly different from that in the blank control group. CD25 is a marker of T cell activation in PBMC cells, and the results of this experiment demonstrated that the 0.03mg / kg treatment group effectively stimulated the activation of T cells.

[0239] Example 19: Dose-finding experiment of repeated intravenous administration for 4 weeks in cynomolgus monkeys Cynomolgus monkeys are used as experimental animals, and different doses of mPEG-SS-5K-GP8-highly modified are administered multiple times via intravenous injection to observe possible toxic reactions during the administration period. Four dose groups are set up, including 0.03, 0.1, 0.3, and 0.5 mg / kg. After completing three doses in the animals in the 0.03 mg / kg dose group, the dose is increased to 0.3 mg / kg, forming the 0.3 mg / kg dose group, for a total of six doses.

[0240] I. Group assignment and experimental design Six animals are randomly divided into four groups by sex based on body weight according to the table below.

[0241] [Table 45]

[0242] During the experiment, animals will be clinically observed daily to monitor changes in body weight, food intake, body temperature, and blood pressure, as well as to perform clinical pathological examinations, immune cell phenotyping, cytokine measurements, and macroscopic observations.

[0243] 2. Results No deaths occurred in cynomolgus monkeys in the four dose groups including 0.03, 0.1, 0.3 and 0.5 mg / kg. At a dose of 0.03 mg / kg, cynomolgus monkeys showed no abnormalities and showed a tendency to gain weight. At the 0.1 mg / kg dose, male animals exhibited spleen enlargement, a reduced percentage of CD56+ cells, and a trend toward weight gain. At the 0.3 mg / kg dose, animals consumed less food but had a tendency to gain weight. Gross necropsy revealed splenomegaly, and clinical pathology revealed increased white blood cell counts, decreased red blood cell counts, decreased red blood cell volume ratios, decreased platelet counts, and decreased total protein.

[0244] At a dose of 0.5 mg / kg, animals showed adverse reactions after the first dose, including lethargy, decreased spontaneous activity, large amounts of loose stool, and visible mucosal pallor, and administration was discontinued once and administered four times in total. The animals' food intake and body weight decreased, and gross dissection showed symptoms such as spleen enlargement, thymus shrinkage, abdominal distension, and loose skin over the entire body. Clinical pathology showed increases in white blood cell count, mononuclear cell (absolute value), neutrophil (absolute value), eosinophil (absolute value), basophil (absolute value), urea, creatine kinase, and fibrinogen, and decreases in red blood cell count, red blood cell volume ratio, platelet count, and total protein. There was also a decrease in the percentage of CD56+ cells.

[0245] Preliminary study results showed that under the above experimental conditions, at a dose of 0.5 mg / kg, cynomolgus monkeys showed severe adverse reactions, but no deaths had yet occurred.

[0246] In addition, according to a literature report on NKTA-214 (NKTA-214, an Engineered Cytokine with Biased IL2 Receptor Binding, Increased Tumor Exposure, and Marked Efficacy in Mouse Tumor Models), in a study in which 4 doses were administered at 14-day intervals in cynomolgus monkeys, the maximum tolerated dose (MTD) of NKTA-214 was 0.1 mg / kg. At this dose, CD25+ increased 24-fold and total lymphocytes increased 4-fold. Compared with NKTA-214, mPEG-SS-5K-GP8-highly modified had no severe adverse reactions at shorter dosing intervals (every 7 days), more frequent doses (5 doses), and a higher dose (0.3 mg / kg), suggesting superiority in safety and tolerability.

Claims

1. 1. A method for achieving controlled and sustained release of activity of a bioactive molecule, comprising: The method is characterized in that a physiologically active molecule is amino-modified using a polyethylene glycol modifying agent, and the activity of the physiologically active molecule is gradually released after PEG in the resulting modified product is gradually lost, and the polyethylene glycol modifying agent is polyethylene glycol succinimidyl succinate.

2. The physiologically active molecule is interleukin-2. The method of claim 1.

3. The PEG modifying agent is preferably a linear polyethylene glycol succinimidyl succinate. The method of claim 1.

4. A polyethylene glycol modified human interleukin-2, The polyethylene glycol modification is characterized in that the PEG modifier used is polyethylene glycol succinimidyl succinate, and an average of 4.5 to 8.5 PEGs are coupled to each human interleukin-2.

5. The PEG modifying agent is a linear polyethylene glycol succinimidyl succinate modifying agent. The polyethylene glycol modified product according to claim 4.

6. The molecular weight of the PEG modifier is 5 to 20 kDa. The polyethylene glycol modified product according to claim 4.

7. When the molecular weight of the PEG modifier is 5 kDa, an average of 5.5 to 7.5 PEGs are coupled to each human interleukin-2, and when the molecular weight of the PEG modifier is 10 to 20 kDa, an average of 6.5 to 8.5 PEGs are coupled to each human interleukin-2. The polyethylene glycol modified product according to claim 4.

8. A polyethylene glycol modified human interleukin-2, The structure of the PEG modifier is as shown in the following figure: 【Chemistry 1】 Here, n is an integer of 97 to 494.

9. The structure of the polyethylene glycol modified human IL-2 is as shown below: 【Chemistry 2】 Here, n is an integer of 97 to 494, and m is an integer of 4.5 to 8.

5. The polyethylene glycol modified product according to claim 4.

10. A human interleukin-2 mutant comprising: The human interleukin-2 mutant as described above, characterized in that it does not contain adjacent lysines.

11. The amino acid sequence of wild-type human IL-2 is characterized in that the 8th and / or 9th amino acids from the N-terminus are substituted, or the 48th and / or 49th amino acids from the N-terminus are substituted, as compared to wild-type human IL-2, and the wild-type human IL-2 amino acid sequence is as shown in SEQ ID NO:

1. The mutant of claim 9.

12. A human interleukin-2 mutant comprising: The human interleukin-2 mutant, characterized in that, compared to wild-type IL-2, the mutations are amino acid substitutions at one, two or more positions corresponding to the 1st, 8th, 9th, 18th, 19th, 48th, 49th, 72nd and / or 81st positions from the N-terminus, and the wild-type human interleukin-2 amino acid sequence is as shown in SEQ ID NO:

1.

13. Each mutation is characterized by a substitution residue selected from the following: A1 deletion at position 1, K8R at position 8, K9R at position 9, L18M at position 18, L19S at position 19, K48W, R at position 48, K49R at position 49, L72F at position 72, and R81D at position 81. The mutant of claim 9.

14. The mutations are as follows: 【Table 1】 The mutant according to claim 9, characterized in that it is selected from one of the following mutation schemes:

15. A polyethylene glycol modified human interleukin-2 mutant, comprising: A polyethylene glycol modified human interleukin-2 mutant, characterized in that the functional group capable of reacting with an amino group in the PEG modifier used is succinimidyl succinate, and on average 4.5 to 8.5 PEGs are coupled to each human interleukin-2, and the human interleukin-2 mutant is one of the different mutants according to any one of claims 10 to 14.

16. The use of polyethylene glycol modified human interleukin-2 or polyethylene glycol modified human interleukin-2 mutant in the preparation of a drug for treating tumor diseases.

17. The tumors include squamous cell carcinoma, melanoma, colon cancer, breast cancer, ovarian cancer, prostate cancer, gastric cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, thyroid cancer, kidney cancer, bile duct cancer, brain tumor, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease, and adrenal cortical carcinoma.

17. The application according to claim 16.

18. A composition for treating a tumor disease, comprising: The composition for treating a tumor disease comprises a polyethylene glycol-modified human interleukin-2 according to any one of claims 4 to 8, a human interleukin-2 mutant according to claims 10 to 14, or a polyethylene glycol-modified human interleukin-2 mutant according to claim 15, and further comprises a HER2 antibody, a PD-1 antibody, a PD-L1 antibody, or a CD26 antibody.

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