Methods for controlling the release of bioactive molecules and achieving sustained release of activity, and their application to drugs.

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] This invention relates to a method for achieving controlled release and sustained release of biologically active molecules and its application to drugs, and more particularly to a method for controlled release and sustained release of highly active cytokines such as interleukin-2. [Background technology]

[0002] Interleukin-2 (IL-2) is a member of the interleukin family. Numerous studies have shown that IL-2 promotes the differentiation and maturation of T cells, NK cells, and B cells, activating their biological activity. It can also induce the activation of lymphokine-activated killer cells (LAKs), promoting the synthesis and release of lymphokines such as interferon and tumor necrosis factor, as well as antibody production. The function of IL-2 in increasing lymphocyte populations in the body and enhancing the effector function of these cells contributes to its antitumor effect, and high doses of IL-2 were approved as early as the early 1980s for the treatment of metastatic renal cell carcinoma and malignant melanoma.

[0003] However, due to the short half-life and low bioavailability of IL-2 in the body, frequent administration is often required multiple times to maintain efficacy in clinical applications. Furthermore, 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 varying degrees of side effects. The most serious is vascular leakage syndrome (VLS), in which fluid from blood vessels accumulates in the liver, lungs, and other organs, resulting in pulmonary edema and hepatocyte damage. This forces patients to discontinue treatment, significantly reducing treatment adherence and limiting the further clinical application of related therapies.

[0004] Conventional techniques have often involved researchers modifying drugs using 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; Tsuneto, PEGylation of Interleukin-2, 1996). Wang Lifu et al. modified wild-type rIL-2 with monomethoxypolyethylene glycol active ester with a purity of 75% and a molecular weight of 5000, but the resulting randomly modified product retained only 69.7% of its in vitro activity (Wang Lifu et al., Preparation of Polyethylene Glycol Modified rIL-2 and its Effects on Antihepatic Cancer Cells Intracellularly and Extracellularly, 1997). Furthermore, conventional modification methods that directly bind drugs to polyethylene glycol do not provide the ideal effect for achieving sustained drug release in the body and improving bioavailability. In conventional technology, it has been reported that by linking a water-soluble polymer and a drug via a linker, a polymer-drug conjugate can be formed, and the detachment of the water-soluble polymer from the conjugate can achieve the objectives of sustained release and controlled release of drug activity. As a result, the drug remains at the site of the lesion (such as cancer) for a longer period, reducing the frequency of administration and alleviating inconvenience for the patient. For example, Patent Document CN200680029849.5 discloses a conjugate containing an aromatic moiety with ionizable hydrogen atoms such as fluorene, a spacer moiety, and a water-soluble polymer, and Patent Document CN103517718A further discloses a complex formed with the aforementioned polymer and rIL-2. This complex is actually NKTR-214, a CD122 (IL-2Rβ) biased agonist developed by Nektar Corporation in the United States. In this NKTR-214 complex, six branched PEG molecules with a molecular weight of 20K and a fluorene ring structure are bound to IL-2 (which has the same amino acid sequence as aldesleukin). Under mild alkaline conditions, fluorene methoxycarbonyl is detached via a β-elimination reaction. This allows NKTR-214 to gradually release its five PEG molecules from its protein surface under physiological conditions (pH=7.4, weakly alkaline), preferentially binding to the IL-2Rβ receptor to enhance T cell activation and simultaneously gaining the ability to circulate in the body for extended periods.The development of the NKTR-214 molecule involved screening PEG reagents and optimizing the coupling reaction to increase site-specific modification of lysine residues (K31, K34, K42, K47, K48, K75, etc.) accumulating at the IL-2 / IL-2Rα interface. This positioned PEG near key hydrophobic binding sites where IL-2 / IL-2Rα interact, achieving the objective of reducing binding to CD25 (IL-2Rα) and preferentially activating CD122 (IL-2Rβ) (Charych DH, Hoch U, Langowski JL, et al. NKTR-214, an engineered cytokine with biased IL2 receptor binding, increased tumor exposure, and marked efficacy in mouse tumor models [J]. ClinicalCancerResearch An Official Journal of the American Association for Cancer Research, 2016, 22(3):680-690). Nektar and BMS entered into a US$3.6 billion partnership regarding NKTR214, but two Phase III clinical trials conducted (PIVOT IO 001 and PIVOT-09) were announced to have failed in 2022, as clinical treatment data did not meet the primary endpoint (2022 EMSO, European Society of Medical Oncology Annual Meeting, Abstract No. 785O, LBA68).

[0005] Through a large number of experiments and studies, the inventor of the present invention screened existing mature polyethylene glycol modifiers and selected a PEG modifier with a certain structure. Using the polyethylene glycol modifier to modify IL-2 or its variants, excellent effects of active release control and active sustained release are obtained. Compared with similar drugs, it shows excellent bioavailability, reduces the drug administration frequency, and significant improvement in therapeutic effect and patient compliance is expected. It should be noted that the present invention is fundamentally different from NKTR-214, a PEG-IL2 drug under development, in terms of molecular design. After administration of NKTR-214, the separation process of its PEG molecules in the body is very complex and uncontrollable, so it is theoretically impossible to always maintain the characteristics of a CD122-biased agonist, which will affect the stable expression of drug effects. The inventor of the present invention achieved the control of the bias towards CD122 and the PEG modification site by site-specific mutation of the sequence of the original protein, and solved the potential drawbacks in molecular design of the NKTR-214 drug from three aspects of safety, effectiveness, and quality control. Furthermore, such differentiated advantages were confirmed by data such as drug dynamics and drug efficacy in animals.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0008] The technical problem to be solved by the present invention is to overcome the problems of low utilization rate of bioactive molecules such as IL-2 and generally decreased activity of their polyethylene glycol-modified products.

Means for Solving the Problems

[0009] First, this invention provides a method for achieving controlled release and sustained release of bioactive molecules. The inventors of this invention have achieved the effect of gradually releasing the activity of the bioactive molecule by reacting a bioactive molecule such as IL-2 with a specific type and structure of polyethylene glycol to form a conjugate, thereby causing PEG to gradually detach from the conjugate structure under certain in vitro or in vivo physiological conditions. This allows the bioactive molecule to maintain a stable effective blood concentration, enabling controlled and sustained release of the drug's activity both in and out of the body, and achieving a relatively excellent dynamic balance of drug bioavailability. Furthermore, benefits in clinical applications such as reduced administration frequency, improved drug bioavailability, and improved patient compliance and safety can be expected. On the other hand, this invention provides a novel 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, resulting in higher activity compared to wild-type IL-2.

[0010] One of the objectives of the present invention is to provide a method for controlling the release of activity and achieving sustained release of activity of a bioactive molecule. The method involves modifying a bioactive molecule with an amino group using a polyethylene glycol modifier. In the modified product obtained by this method, the activity of the bioactive molecule is gradually released as the PEG is successively removed. The polyethylene glycol modifier is polyethylene glycol succinimidyl succinate.

[0011] The aforementioned bioactive molecule is a protein or peptide, preferably an interleukin, and 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. The modified product uses polyethylene glycol succinimide succinate as a PEG modifier, and on average, 4.5 to 8.5 PEGs are bound to each human IL-2 molecule.

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

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

[0016] More preferably, the structure of the PEG modifier is as shown in the figure below. In the formula, n is an integer between 97 and 494. When n is an integer between 97 and 494, the actual molecular weight of the PEG modifier is approximately 4.5k to 22k, and the corresponding molecular weight is expressed as 5 to 20kDa.

[0017] [ka]

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

[0019] [ka]

[0020] Another objective of the present invention is to provide a human IL-2 mutant. This mutant does not contain adjacent lysine, and when modified with PEG, a more uniform modified product is obtained. The inventors of the present invention found the following through 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 lysine and can all be modified with PEG, but adjacent sites are rarely modified with PEG simultaneously. In the case of saturated modification, either position 8 or 9 of IL-2 is modified, and either position 48 or 49 is modified. That is, four different isomers are produced with different modification sites, and the resulting 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 positions 48 and / or 49. This significantly reduced the generation of isomers with different PEG modification sites and made it easier to control the uniformity of the PEG modified product. Prior to the filing of this invention, those skilled in the art were unaware that the presence of lysine in adjacent amino acids in the IL-2 sequence would have an undesirable effect on PEG modification. Therefore, when modifying IL-2 with polyethylene glycol, there was no motivation to mutate the corresponding site.

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

[0022] Preferably, wild-type human IL-2 is used, with substitutions at the 8th and / or 9th amino acid from the N-terminus, and at the 48th and / or 49th amino acid.

[0023] Specific examples include the mutation of lysine at position 8 to arginine, lysine at position 9 to arginine, lysine at position 48 to arginine, tryptophan, or tyrosine, and lysine at position 49 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 lysine in the wild-type IL-2 sequence generates various isomers of polyethylene glycol modified products, affecting the uniformity of the PEG modified product. Those skilled in the art will understand that the above objective can also be achieved by mutating the above amino acid sites to other non-lysine amino acids, in addition to the amino acids shown in the examples.

[0024] On the other hand, the present invention further optimized the aforementioned human IL-2 that does not contain adjacent lysine to develop a human IL-2 mutant that exhibits significantly superior in vitro activity compared to wild-type IL-2. The mutant is based on wild-type human IL-2, with one, two, or more amino acids substituted at positions 1, 8, 9, 18, 19, 48, 49, 72, and / or 81 from the N-terminus. The amino acid sequence of the wild-type human IL-2 is shown in SEQ ID NO:1.

[0025] Preferably, mutations at these sites are selected from the following residue substitution scheme: 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 contains 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 the wild-type human IL-2 is as shown in SEQ ID NO: 1.

[0026] Most preferably, the mutation is selected from 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 after making one, two or more amino acid substitutions 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 via the IL-2 receptor (IL-2R). The IL-2R receptor contains three subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). These three subunits form three different receptor configurations. High-binding receptors contain all three subunits: IL-2Rα, IL-2Rβ, and IL-2Rγ. Medium-binding receptors contain IL-2Rβ and IL-2Rγ. Low-binding 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 functions in the immune response. On the one hand, IL-2 acts as an immune system stimulant, stimulating T cell proliferation and differentiation, inducing the generation of cytotoxic T lymphocytes, promoting B cell proliferation and differentiation, and immunoglobulin synthesis, and stimulating the generation and activation of NK cells. Based on these functions, IL-2 is approved as an immunotherapy 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 proposed reducing the toxicity and side effects of IL-2 used as an immune system stimulant and improving its efficacy by altering the bias of IL-2 towards IL-2Rα. Numerous studies have already reported on altering the bias towards the IL-2 receptor through site mutation. Those skilled in the art may perform polyethylene glycol modification using IL-2 mutated at other sites, without being limited to IL-2 mutated at the specific site mentioned above.

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

[0031] Another object of the present invention is to provide the application of polyethylene glycol-modified human IL-2 or its mutants in the manufacture of drugs for treating tumor diseases. The tumors 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 adrenocortical carcinoma.

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

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

[0034] The aforementioned PD-1 antibodies include Opidivo from Bristol-Myers Squibb, Keytruda from Merck, Toripalimab from Kimmitsu Bio, Sintilimab from Shinda Bio, Camrelizumab from Hengrui Pharmaceutical, and tislelizumab from Baekje Shenju.

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

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

[0037] Tumor diseases 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 adrenocortical carcinoma.

[0038] Through the technical solutions of the present invention, the following technical effects were primarily achieved. 1. By reacting polyethylene glycol succinimidyl succinate with the lysine side chain ε-amino group of IL-2 or its mutant, an amide bond is formed between polyethylene glycol and IL-2. Because the receptor binding site is completely covered, the modified PEG-IL-2 molecule is inactive. Furthermore, unlike other modifiers, polyethylene glycol succinimidyl succinate contains one ester bond in the complex after binding to a protein or peptide drug. Due to the inherent instability of the ester bond, the PEG chain is prone to detachment through hydrolysis. Conventionally, this characteristic has been considered to lead to instability of the modified molecule because the modifier is easily detached, and therefore the application of this PEG has gradually been replaced by other PEG modifiers that are less prone to detachment (e.g., polyethylene glycol succinimidyl propionate). However, in this invention, this hydrolytic property allows IL-2 to gradually release its drug activity in the body, and extremely excellent pharmacological effects can be obtained at specific molecular weights and modification numbers.

[0039] 2. By mutating the lysine site in IL-2 and eliminating the adjacent lysine, the PEG coupling site was limited, significantly reducing the generation of isomers with different PEG modification sites and facilitating the control of the uniformity of PEG modification products. Based on this, through further design and screening of mutation sites, we developed receptor-binding biased mutants with significantly reduced affinity to α receptors while maintaining affinity to β receptors, as well as mutants with significantly superior CTLL-2 cell proliferation activity compared to wild-type IL-2.

[0040] 3. When actually acting, the activity of PEGylated IL-2 is slowly released as the PEG is gradually shed. Through optimization and screening of PEGs with different molecular weights and different PEG modification counts, the present invention avoids the low bioavailability of highly modified IL-2, as well as the high toxicity and side effects of low-modified IL-2. As a result, the PEGylated IL-2 of the present invention effectively achieves a dynamic balance between bioavailability and safety, while by adjusting the dosage and frequency of administration, it achieves stable blood concentrations of the drug and better bioavailability, resulting in superior therapeutic effects in the body. [Brief explanation of the drawing]

[0041] [Figure 1a] This is a curve showing how IL-2 mutants stimulate the proliferation of CTLL-2 cells. [Figure 1b] This is a curve showing how IL-2 mutants stimulate the proliferation of CTLL-2 cells. [Figure 1c] This is a curve showing how IL-2 mutants stimulate the proliferation of CTLL-2 cells. [Figure 1d] This is a curve showing how IL-2 mutants stimulate the proliferation of CTLL-2 cells. [Figure 1e] This is a curve showing how IL-2 mutants stimulate the proliferation of CTLL-2 cells. [Figure 2a] This figure shows the activity levels of PEG-SS modified products of different mutant proteins activated under alkaline conditions, which stimulate CTLL-2 cell proliferation. [Figure 2b] This figure shows the activity levels of PEG-SS modified products of different mutant proteins activated under alkaline conditions, which stimulate CTLL-2 cell proliferation. [Figure 2c] This figure shows the activity levels of PEG-SS modified products of different mutant proteins activated under alkaline conditions, which stimulate CTLL-2 cell proliferation. [Figure 3] This is a curve diagram showing the measurement of CTLL-2 phosphorylated STAT5 levels by modified products of IL-2 mutants modified with PEG-SS having different structures and molecular weights. [Figure 4] This is a curve diagram showing the measurement of CTLL-2 phosphorylated STAT5 levels by modified products of different IL-2 mutants modified with PEG-SS. [Figure 5] This figure shows a comparison of the in vivo pharmacokinetics of highly modified mPEG-SS-5k-GP8 and its proto-protein, GP8. [Figure 6a] This figure shows the results of efficacy evaluation of IL-2 modified with different structural PEG-SS (linear or branched) in a BALB / c mouse subcutaneous transplantation model of CT26.WT mouse-derived colon cancer cells. Figure 6a shows the tumor volume increase curve for each group of animals, Figure 6b shows the tumor weight for each group of animals, and Figure 6c shows the change in body weight for each group of animals. [Figure 6b] This figure shows the results of efficacy evaluation of IL-2 modified with different structural PEG-SS (linear or branched) in a BALB / c mouse subcutaneous transplantation model of CT26.WT mouse-derived colon cancer cells. Figure 6a shows the tumor volume increase curve for each group of animals, Figure 6b shows the tumor weight for each group of animals, and Figure 6c shows the change in body weight for each group of animals. [Figure 6c] This figure shows the results of efficacy evaluation of IL-2 modified with different structural PEG-SS (linear or branched) in a BALB / c mouse subcutaneous transplantation model of CT26.WT mouse-derived colon cancer cells. Figure 6a shows the tumor volume increase curve for each group of animals, Figure 6b shows the tumor weight for each group of animals, and Figure 6c shows the change in body weight for each group of animals. [Figure 7a] This figure shows the results of efficacy evaluation of different IL-2 mutants modified with linear PEG-SS in a C57BL / 6 mouse subcutaneous transplantation model of melanoma cells derived from B16-F10 mice. Figure 7a shows the tumor volume increase curves for each group of animals, and Figure 7b shows the tumor weight for each group of animals. [Figure 7b] This figure shows the results of efficacy evaluation of different IL-2 mutants modified with linear PEG-SS in a C57BL / 6 mouse subcutaneous transplantation model of melanoma cells derived from B16-F10 mice. Figure 7a shows the tumor volume increase curves for each group of animals, and Figure 7b shows the tumor weight for each group of animals. [Figure 8a] This figure shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and modification degrees in a B16-F10 mouse-derived melanoma tumor model. Figure 8a shows the tumor growth curves of the animals in each group, Figure 8b shows the tumor weight of the animals in each group 17 days after cell inoculation, and Figure 8c shows the change in the rate of weight gain of the animals. [Figure 8b] This figure shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and modification degrees in a B16-F10 mouse-derived melanoma tumor model. Figure 8a shows the tumor growth curves of the animals in each group, Figure 8b shows the tumor weight of the animals in each group 17 days after cell inoculation, and Figure 8c shows the change in the rate of weight gain of the animals. [Figure 8c] This figure shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and modification degrees in a B16-F10 mouse-derived melanoma tumor model. Figure 8a shows the tumor growth curves of the animals in each group, Figure 8b shows the tumor weight of the animals in each group 17 days after cell inoculation, and Figure 8c shows the change in the rate of weight gain of the animals. [Figure 9a] This figure shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and modification degrees in a CT26.WT mouse-derived colon cancer tumor model. Figure 9a shows the tumor growth curves of the animals in each group, Figure 9b shows the tumor weight of the animals in each group 22 days after cell inoculation, and Figure 9c shows the change in the rate of weight gain of the animals. [Figure 9b] This figure shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and modification degrees in a CT26.WT mouse-derived colon cancer tumor model. Figure 9a shows the tumor growth curves of the animals in each group, Figure 9b shows the tumor weight of the animals in each group 22 days after cell inoculation, and Figure 9c shows the change in the rate of weight gain of the animals. [Figure 9c] This figure shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and modification degrees in a CT26.WT mouse-derived colon cancer tumor model. Figure 9a shows the tumor growth curves of the animals in each group, Figure 9b shows the tumor weight of the animals in each group 22 days after cell inoculation, and Figure 9c shows the change in the rate of weight gain of the animals. [Figure 10a]This figure shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and modification degrees in the A375 human-derived melanoma model. Figure 10a shows the animal tumor growth curve, Figure 10b shows the tumor weight of each group of animals 45 days after cell inoculation, and Figure 10c shows the rate of animal body weight gain. [Figure 10b] This figure shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and modification degrees in the A375 human-derived melanoma model. Figure 10a shows the animal tumor growth curve, Figure 10b shows the tumor weight of each group of animals 45 days after cell inoculation, and Figure 10c shows the rate of animal body weight gain. [Figure 10c] This figure shows the efficacy evaluation of PEG-SS modified IL-2 mutants with different molecular weights and modification degrees in the A375 human-derived melanoma model. Figure 10a shows the animal tumor growth curve, Figure 10b shows the tumor weight of each group of animals 45 days after cell inoculation, and Figure 10c shows the rate of animal body weight gain. [Figure 11a] This figure shows the efficacy evaluation of PEG-modified IL-2 mutants at different doses in the A375 human melanoma model. Figure 11a shows the animal tumor growth curve, Figure 11b shows the tumor weight of the animals in each group, and Figure 11c shows the body weight gain rate of the animals in each group. [Figure 11b] This figure shows the efficacy evaluation of PEG-modified IL-2 mutants at different doses in the A375 human melanoma model. Figure 11a shows the animal tumor growth curve, Figure 11b shows the tumor weight of the animals in each group, and Figure 11c shows the body weight gain rate of the animals in each group. [Figure 11c] This figure shows the efficacy evaluation of PEG-modified IL-2 mutants at different doses in the A375 human melanoma model. Figure 11a shows the animal tumor growth curve, Figure 11b shows the tumor weight of the animals in each group, and Figure 11c shows the body weight gain rate of the animals in each group. [Figure 12a] This figure shows the efficacy evaluation of PEG-modified IL-2 mutants at different dosages in the A498 human renal cancer model. Figure 12a shows the growth curves of animal tumors in each group, Figure 12b shows the tumor weight of the animals in each group, and Figure 12c shows the body weight gain rate of the animals in each group. [Figure 12b]This figure shows the efficacy evaluation of PEG-modified IL-2 mutants at different dosages in the A498 human renal cancer model. Figure 12a shows the growth curves of animal tumors in each group, Figure 12b shows the tumor weight of the animals in each group, and Figure 12c shows the body weight gain rate of the animals in each group. [Figure 12c] This figure shows the efficacy evaluation of PEG-modified IL-2 mutants at different dosages in the A498 human renal cancer model. Figure 12a shows the growth curves of animal tumors in each group, Figure 12b shows the tumor weight of the animals in each group, and Figure 12c shows the body weight gain rate of the animals in each group. [Modes for carrying out the invention]

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

[0043] Polyethylene glycol (PEG) is typically formed by the polymerization of ethylene oxide and exists in branched, linear, and multi-armed forms. Generally, those with a molecular weight of 20,000 or less are called PEG, while those with a larger molecular weight are called PEO. Ordinary polyethylene glycol has one hydroxyl group at each end, and blocking one end with a methyl group yields methoxy polyethylene glycol (mPEG).

[0044] Polyethylene glycol modifiers (PEG modifiers) refer to polyethylene glycol derivatives having functional groups, and are activated polyethylene glycols used for the modification of proteins and polypeptides into drugs. The polyethylene glycol modifiers used in this invention are purchased from Jiangsu Zhonghong Bioengineering Drug Discovery Research Institute Co., Ltd. or Beijing Jiankai Technology Co., Ltd. The actual molecular weight of a PEG modifier of a specific molecular weight may be 90% to 110% of the stated value. For example, the actual molecular weight of PEG5K may be 4.5 kDa to 5.5 kDa. The actual molecular weight of PEG20K may be 18 kDa to 22 kDa. If the stated molecular weight of a PEG modifier is 5 kDa to 20 kDa, its actual molecular weight is 4.5 kDa to 22 kDa.

[0045] The 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 objective of sustained release and controlled release of drug activity by having PEG react with the drug via a linker to form a complex, and then PEG detaches from the complex. This is consistent with the structure of the reported NKTR-214. The applicant used an IL-2 modifier modified with this PEG modifier as a positive reference. The structural formula of the V-PEG-SC-20k modifier is shown below. In the formula, n is an integer between 199 and 244.

[0046] [ka]

[0047] The 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 shown below. In the formula, n is an integer between 198 and 244.

[0048] [ka]

[0049] The mPEG-SS-20k / 10k / 5k used in the examples refer to linear polyethylene glycol succinimidyl succinate modifiers with molecular weights of 20kDa, 10kDa, and 5kDa, respectively. 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 succinimidylpropionate modifier with a molecular weight of 5 kDa. The structural formula of the mPEG-SPA-5k modifier is shown below. In the formula, n is an integer between 98 and 120.

[0052] [ka] [Examples]

[0053] Example 1: Design and fabrication 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 obtained by performing operations such as amino acid substitution, insertion, or deletion based on this wild-type IL-2. For example, the G438 sequence was 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 lysine at position 8 to arginine.

[0054] [Table 2]

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

[0056] Step 1: Based on the amino acid sequence of G438 (a sequence based on wild-type IL-2 shown in SEQ ID NO:1 with a K8R / K48W mutation), the DNA sequence shown in SEQ ID NO:2 is obtained by optimizing it for E. coli. This DNA sequence is cloned into the pBV220 vector to form the recombinant plasmid pBV220-G438. Subsequently, the recombinant plasmid is transformed into a Top10 E. coli host to construct the expression host Top10-pBV220-G438.

[0057] Step 2: Inoculate the Top10-pBV220-G438 recombinant expression strain into TB medium (500 mL medium, 20% liquid volume) and culture with shaking at 30°C and 220 rpm until the OD600 of the culture medium reaches 1.0 ± 0.1. Then, while maintaining the shaking rotation speed, raise the temperature of the culture medium to 42°C to induce expression of the strain. Induction expression is carried out for 4 hours. After induction expression, the bacterial precipitate was collected by centrifugation.

[0058] Step 3: Resuspend the post-expression bacterial cell precipitate in 10 mM PBS, pH 7.4 at a concentration of 100 g / L, and perform ultrasonic disruption using a probe-type ultrasonic disruptor (working power 250 watts, operate for 3 seconds with a 4-second interval, total disruption time 30 mins). Then, collect the disrupted product by centrifugation to obtain the G438 inclusion body precipitate.

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

[0060] Step 5: Resuspend the G438 crude inclusion bodies in a denaturation solution (20 mM Tris, 8 M urea, 5 mM DTT, pH 10.5) at a concentration of 1 g / 100 mL, stir for at least 2 hours to denaturate, and collect the supernatant by centrifugation. Purify the supernatant using Superdex 75.

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

[0062] Step 7: The G438 after refolding is concentrated by ultrafiltration, and the concentrated sample is dialyzed with PBS (pH 7.4) to remove reagents such as urea and 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 according to the mutation scheme shown in Table 1. Each mutant is generated using the same method as described above.

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

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

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

[0067] 2. Experimental Results The program is executed, and data analysis is performed using the Fortebio DataAnalysis 8.0 software to calculate affinity binding values. The results are shown in the table below.

[0068] [Table 3]

[0069] [Table 4]

[0070] 3. Results analysis Receptor affinity measurements showed that mutants G493, G496, G498, G499, G500, G438P4, and G438P5 exhibited significantly reduced or no binding to two types of receptors. This suggests that the mutation may have caused a major change in the protein structure. Mutants such as G495, G438P6, G438P7, G438P8, G438P14, and G438P15 showed a binding bias to the IL-2β receptor. Mutations in G495, G438P7, G438P8, and G438P14 did not significantly affect the binding of IL-2 to IL-2Rβ / γ. Binding of G438P15 to IL-2Rβ / γ was significantly reduced. To select mutants that retain receptor binding and evaluate their biological activity, further in vitro experiments on the proliferation activity of CTLL-2 cells will be conducted.

[0071] Example 3: Measurement of the activity of IL-2 and its mutants in stimulating the proliferation of CTLL-2 cells. 1. Experimental Method CTLL-2 is a mouse-derived cell line. To evaluate the in vitro biological activity of IL-2 and its mutants and modifications, a method was used to measure the proliferation rate of CTLL-2 cells, a cell-dependent cell line, at different concentrations. This experimental method is based on the 2020 edition of the Chinese Pharmacopoeia, Part 4, General Rules 3524, "Method for Measuring the Biological Activity of Human Interleukin-2" (CTLL-2 / MTT colorimetric method).

[0072] 1. Preparation of the reagent RPMI1640 culture medium: Take one packet (spec: IL) of RPMI1640 culture medium powder, dissolve it in water, and dilute it to 1000 ml. Then, add 2.1 g of sodium bicarbonate, dissolve it, mix thoroughly, filter to sterilize, and store at 4°C. Basic culture medium: Measure out 10 ml of newborn bovine serum (FBS) and add 90 ml of RPMI1640 culture medium. Store at 4°C. Complete culture medium: Aspirate 100 ml of basal culture medium and add human IL-2 to achieve a final concentration of 400-800 IU / ml. Store at 4°C. PBS: Aspirate 100 ml of 10x PBS and dilute it to 1000 ml by adding water that has been sterilized at 121°C for 20 minutes. Thiazole blue (MTT) solution: Weigh out 0.1 g of MTT, dissolve it in PBS, and dilute to 20 mL. Filter and sterilize using a 0.22 μm filter membrane. Store in a light-protected place at 4°C. Lysate solution: 15% sodium lauryl sulfate solution; the usage period 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 a 2-fold serial dilution to create a total of eight dilutions. Add the sample from each dilution to two wells. Leave 50 μl of solution in each well and discard the excess solution in the wells. These operations should be performed under sterile conditions.

[0074] 3.Cell culture CTLL-2 cells are cultured in complete culture medium at 37°C under 5% carbon dioxide conditions until sufficient volume is reached. The cells are collected by centrifugation and washed three times with RPMI1640 culture medium. Then, they are resuspended in basal medium at a concentration of 6.0 × 10⁴ cells per mL. 5 Prepare a cell suspension containing 100 cells. Store the cell suspension at 37°C under 5% carbon dioxide conditions for later use. Add 50 μl of the cell suspension to each well of a 96-well cell culture plate containing wild-type and mutant samples, and incubate at 37°C under 5% carbon dioxide conditions for 18-24 hours. Then, add 20 μl of MTT solution to each well and incubate at 37°C under 5% carbon dioxide conditions for 4-6 hours. Next, add 150 μl of lysate to each well and incubate at 37°C under 5% carbon dioxide conditions for 18-24 hours. All of the above operations should be performed under sterile conditions. Mix the liquids in the cell plate uniformly, set it in a microplate reader, measure the absorbance at a wavelength of 570 nm with 630 nm as the reference wavelength, and record the results. Create a test reaction curve by performing quaternary parameter fitting using ELISACalc software, with sample concentration (ng / ml) on the x-axis and the mean of the OD570 measurement value on the y-axis. Specific activity of sample (IU / mg) = Biological activity of sample (IU / ml) / 20 (ng / ml) x 10 6

[0075] 2. Experimental Results The measurement results are shown in Figure 1 and the table below. [Table 5]

[0076] 3. Results analysis The following can be observed from the measurement of the biological activity of the mutants: The newly constructed IL-2 mutants G438P1, G438P8, G438P12, G438P20, G438P21, and G438P22 exhibit significantly superior proliferative activity of CTLL-2 cells compared to wild-type IL-2, and have potential for clinical application.

[0077] For the sake of simplicity, mutants obtained by further modifying G438, such as G438P1, G438P8, G438P12, G438P20, G438P21, and G438P22, will be 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 material V-PEG-SC-20k-rhIL-2 1. Modification method Using the compound V-PEG-SC-20k (purchased from Xiamen Sinobank), published in Patent Document CN200680029849.5, as a modifier, PEG-IL-2, which similarly exhibits a sustained-release effect, was prepared as a positive control.

[0079] The purified wild-type IL-2 protein sample is concentrated and replaced with 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 2 hours of reaction, 1 M glycine is added to stop the reaction.

[0080] 2. Purification method Chromatography conditions: Fluid phase A is 20 mM PB + 2 M NaCl (pH 6.0), and fluid phase B is 20 mM PB (pH 6.0). Sample Loading: After diluting the modified sample as described above, load the sample at 5 ml / min and attach it to a hydrophobic chromatography column (GE, Phenyl PH). Equilibrium: After sample loading is complete, wash 15-20 column volumes with solution A. Elution: Elute with 0-100% Solution B, and collect the V-PEG-SC-20k-rhIL-2 sample with an elution volume equivalent to 10 column volumes, where the main peak is V-PEG-SC-20k-rhIL-2. The modified sample was purified under the above conditions to prepare the target sample.

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

[0082] [Table 6]

[0083] SEC (Size Exclusion Chromatography) is a chromatographic technique that separates molecules based on their size. The PEG-IL-2 sample prepared in this example was measured using SEC chromatography, and the results showed that the main peak of the sample was uniform, indicating a uniform degree of modification, thus meeting the research needs.

[0084] In all subsequent examples, unless otherwise specified, V-PEG-SC-20k-rhIL-2 (or simply PEG-SC-20k-rhIL-2) prepared in this example will be used as the positive control.

[0085] Example 4b: Preparation of other PEG-modified samples provided by the present invention 1. Modification method The purified rhIL-2 wild-type / mutant protein samples are concentrated and replaced 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 proportions shown in the table below, PEG (including, but not limited to, V-PEG-SS-20k, mPEG-SS-20k, m-PEG-SS-10k, mPEG-SS-5k, and mPEG-SPA-5K) is weighed out and the modification reaction is carried out at room temperature. After 4 hours of reaction, 1M glycine is added to stop the reaction. The main reaction parameters for each modifier are shown in the table below.

[0086] [Table 7]

[0087] 2. Purification method Chromatography conditions: Fluid phase B is 20 mM NaAc + 1 M NaCl (pH 4.0), and fluid phase A is 20 mM NaAc (pH 4.0). Sample Loading: After diluting the modified sample as described above, load the sample at 5 ml / min and attach it to a cation exchange chromatography column (GE HPSP). Equilibrium: After sample loading is complete, wash 15-20 column volumes with solution A. Elution: Elute with 0-100% Solution B, and collect the sample with the main peak when the elution volume is equivalent to 10 column volumes. The modified sample was purified under the above conditions to prepare the target sample.

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

[0089] [Table 8]

[0090] SEC (Size Exclusion Chromatography) is a chromatographic technique that separates molecules based on their size. Each PEG-IL-2 sample prepared in this example was measured by SEC chromatography, and the results showed that the main peaks of the samples were uniform, indicating a uniform degree of modification, thus meeting the needs of the research. Furthermore, PEG-IL-2 samples of the same PEG with different degrees of modification (e.g., GP8) showed significant differences in retention time. The sample preparation process established in this invention demonstrates that samples with different degrees of modification can be stably prepared by controlling process parameters. The specific number of PEG bonds in the samples is 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 the reagent (1) PEG standard gradient solutions: Take 5 μL, 10 μL, 20 μL, 30 μL, 40 μL, and 50 μL of PEG solutions (2.5 mg / mL) of V-PEG-SC-20k, V-PEG-SS-20k, mPEG-SS-20k, mPEG-SS-10k, and mPEG-SS-5k, respectively, and add them to water to prepare gradient solutions with a final volume of 100 μL. Mix them uniformly to obtain standard gradient solutions. Add 25 μL of 5 × non-reducing loading buffer to each and mix uniformly, then set aside for later use.

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

[0093] (3) Preparation of 10% perchloric acid solution: Measure 100 mL of perchloric acid using a graduated cylinder, gradually add it to 900 mL of water, and mix thoroughly to obtain a 10% perchloric acid solution.

[0094] 2. Gel preparation Preparation of 10% polyacrylamide gel: Aspirate 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, and mix them uniformly to prepare a separation gel. After the separation gel has solidified and formed, 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 aspirated and uniformly mixed to prepare a concentrated gel.

[0095] 3. Sample preparation Hydrolysis: Take 100 μL of a test sample solution with a known protein content, add 200 μL of 100 mM NaHCO3 pH 9.0 activation buffer, and hydrolyze in a 37°C water bath for 24 hours (during which time, mPEG-SPA-5k-rhIL-2 does not spontaneously hydrolyze, so the sample is incubated with trypsin after high-temperature treatment). Then, remove the sample, add it to 5× non-reducing loading buffer, mix uniformly, and set aside for later use.

[0096] 4. Detection by electrophoresis Operating voltage: Run at 80V for 30 minutes, then switch to 120V when the bromophenol blue indicator moves to the bottom of the concentrated gel, and terminate when the bromophenol blue indicator reaches the bottom edge of the separated gel. Staining with iodine solution: After electrophoresis is complete, pry open the glass plate, mark the film, place it in the staining box, and first fix it in a 10% perchloric acid solution for 10 minutes. Then collect the 10% perchloric acid and wash it three times with water. Next, cover the film with iodine staining solution and stain it for 2-3 minutes. The color will develop in about 1 minute. Once the color has developed, immediately decolorize it with water.

[0097] 5. Gel Imaging and Data Processing The decolorized, clearly defined gel is placed in a gel imaging system, and gel imaging is performed. Quantitative processing is performed using Quantity One 4.4.0 software. Linear regression is performed on the PEG content of the standard and the grayscale of the film spots to determine the total amount of free PEG in the sample. The results are calculated using the following formula. PEG bond count = PEG moles / protein moles

[0098] 2. Experimental Results The results of in vitro PEG binding number analysis for each modified product are shown in the table below.

[0099] [Table 9]

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

[0101] 2. Experimental Results The table below shows the results of the in vitro analysis of the sustained-release performance of each modified product against PEG.

[0102] [Table 10]

[0103] 3. Results analysis The results shown in this example are measurements performed in vitro at 37°C in an activation buffer (100 mM NaHCO3 pH 9.0). IL-2 or its mutants modified with multiple types of PEG-SS with different structures (linear or branched) and molecular weights (5K, 10K, 20K) all exhibited excellent sustained-release performance in vitro. The positive reference product also showed ideal in vitro sustained-release performance. However, other non-PEG-SS structures of conventional PEG types, such as SPA-PEG, cannot achieve sustained-release because PEG shedding cannot be realized unless further matrix elements (e.g., the fluorene ring structure of the positive reference product) are introduced. While molecules with different molecular weights and modification degrees showed different release rates in vitro, further verification through in vitro and in vitro activity evaluation is necessary to determine whether the released active protein structure still exerts its therapeutic effect.

[0104] Example 7: Measurement of the activity of PEG-modified IL-2 in stimulating CTLL-2 cell proliferation. 1. Experimental Method Due to the shielding effect of PEG modification on the protein's active site, the modified protein exhibits a certain degree of reduced in vitro biological activity. Therefore, it is necessary to pre-activate the sample. To evaluate the in vitro biological activity of the activated modified product after activation, we used a method that measured the proliferation rate of the cell-dependent strain, CTLL-2, at different concentrations.

[0105] 1. Sample preparation Sample Activation: Take 100 μL of a test sample solution with 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 37°C water bath for a set period of time. Take 100 μL from each hydrolyzed sample periodically, mix uniformly, and set aside for later use. Dilute the samples to appropriate initial concentrations and perform 2-fold serial dilutions in a 96-well cell culture plate to create a total of 8 dilutions. Add the sample from each dilution to 2 wells. Leave 50 μL of solution in each well and discard the excess solution. Perform the above operations under sterile conditions. 2. The preparation of the test solution and cell culture are the same as in Example 3.

[0106] 2. Experimental Results The results of the activity of the activated products of each polyethylene glycol modifier, activated under 1,100 mM NaHCO3 pH 9.0 conditions, in stimulating the proliferation of CTLL-2 cells are shown in Figure 2 and the table below (relative biological activity is calculated using the activity value of the proto-protein used for PEG modification as the baseline of 100%).

[0107] [Table 11]

[0108] [Table 12]

[0109] 3. Results analysis Table 9 shows the following: Similar to the reference product V-PEG-SC-20k-rhIL-2, the PEG-SS modified compounds exhibit no biological activity in the inactive state (activation 0h) because the receptor binding site on the surface is completely shielded by PEG. In other words, if PEG is not detached, there is no biological activity (undetectable). On the other hand, after activation for different durations under alkaline in vitro conditions, all compounds showed significant biological activity that promoted the proliferation of CTLL-2 cells. Furthermore, the PEG-SS modified compounds showed a tendency for biological activity to gradually recover and improve as the activation time increased. For specific modification levels, mPEG-SS-20k-GP8 and mPEG-SS-20k-GP12 both exhibited higher biological activity than V-PEG-SC-20k-rhIL-2 at the time of sampling, and when the PEG detachment behavior was similar, adjusting the modification level could yield a superior release effect.

[0110] Furthermore, different release effects can be obtained by modifying PEG with different molecular weights and varying degrees of modification. This also forms the basis for the controllable sustained-release performance of IL-2 modified with PEG-SS molecules. In other words, by adjusting the molecular weight of the PEG-SS used for modification, controlling the number of PEG molecules used, 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 makes it possible to obtain candidate molecules with superior pharmacological efficacy compared to conventional technologies.

[0111] Example 8: Measurement of pSTAT5 activity in CTLL-2 cells using IL-2 modified with structurally different PEGs. 1. Experimental Method CTLL-2 cells were cultured in complete medium (RPMI1640 medium + 2 mM L-glutamine + 1 mM sodium pyruvate + 10% fetal bovine serum + 10% T-STIM, with concanavalin A added to the culture) at 37°C under 5% CO2 conditions, 2 × 10⁶ cells. 5 Incubate until the cell density reaches 1 cell / mL. Then, wash once with PBSA (PBS, pH 7.2, 1% BSA) and reduce the cell density to 1 × 10⁶. 6Adjust to cells / mL. Dispense into flow cytometry tubes at a volume of 500 μL / tube and add different concentrations of PEG-modified IL-2 prepared in basal culture medium (RPMI1640 + 2 mM L-glutamine + 1 mM sodium pyruvate + 10% fetal bovine serum). Incubate at room temperature for 20 minutes, then immediately add paraformaldehyde to a final concentration of 1.5%, vortex mix uniformly, and incubate at room temperature for 10 minutes. Then add 1 mL of PBS and centrifuge at 4°C and 1400 rpm for 5 minutes to remove paraformaldehyde. Resuspend the cells, add 1 mL of 100% methanol pre-cooled to 4°C, vortex mix uniformly, and incubate at 4°C for 20 minutes. Next, add 3 mL of PBSA buffer and wash the cells twice by centrifugation at 4°C and 1400 rpm for 5 minutes. Next, Anti-Stat5(pY694)-Alexa647(BD,Cat#612599) is added, and the mixture is incubated at room temperature in the dark for 30 minutes. Finally, 3 mL of PBSA is added, the mixture is washed twice, and the sample is measured using BD Accuri™ C6.

[0112] 2. Experimental Results The measurement results are shown in Figure 3, and the results indicate the following: In the measurement of pSTAT5 activation levels in vitro, compared to V-PEG-SC-20k-rhIL-2 and mPEG-SS-20k-GP8 (in this example, referring to the moderately modified mPEG-SS-20k-GP8), mPEG-SS-10k-GP8 showed a significant difference in the increase curve of phosphorylation activation level after activation in serum. The increase in phosphorylation level was relatively gradual and showed a tendency to increase gradually. After 72 hours, the phosphorylation activation levels of the three samples were almost identical. This is in close agreement with the trend of release of active protein due to PEG shedding in vitro.

[0113] 3. Results analysis Regardless of the cell type, IL-2 exerts its biological function via the JAK-STAT pathway when bound to IL-2R. The JAK1 pathway, triggered by the binding of IL-2Rβ, and the JAK3 pathway, triggered by the binding of IL-2Rγ, respectively, induce phosphorylation of key tyrosine residues on the β and γ subunits of IL-2R, thereby generating anchor sites for other signaling molecules. Therefore, the in vitro biological activity of IL-2 and its modifications can be evaluated by measuring fluctuations in phosphorylation levels in CTLL-2 cells under the action of different concentrations of PEG-modified IL-2.

[0114] Therefore, the experimental results above show 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 gradually releases IL-2 mutants 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 using different mutants of PEG-modified IL-2 1. Experimental Method This is the same as Example 8.

[0116] 2. Experimental Results The measurement results are shown in Figure 4, and the results indicate the following: In the in vitro measurement of pSTAT5 activation levels, mPEG-SS-20k-GP1 and mPEG-SS-20k-GP8 (in this example, referring to the moderately modified mPEG-SS-20k-GP8 mentioned above) showed similar trends in their phosphorylation activation levels after activation in buffer, exhibiting a characteristic of gradual activation over time, and maintaining sustained activity even after reaching a peak.

[0117] 3. Results analysis The experimental results described above indicate that different mutants of the PEG-SS modified IL-2 of the present invention (e.g., GP1, GP8, etc.) exhibit similar phosphorylation activation levels in the body.

[0118] Example 10: Comparison of in vivo half-life extension effect of PEG-modified IL-2 mutants and activity release of modified products 1. Experimental Method Experiment 1: SD rats were intravenously administered 0.5 mg / kg, 0.1 mg / kg, and 0.04 mg / kg of mPEG-SS-5k-GP8-high modification and 1 mg / kg of mutant GP8. Serum was collected before administration and at 0.25 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours after administration. The content of mPEG-SS-5k-GP8-high modification and mutant GP8 in each serum was measured using two different ELISA methods.

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

[0120] 2. Experimental Results Experiment 1: The measurement results are shown in Figure 5. The measurement results indicate that the mPEG-SS-5k-GP8-high-modification significantly extends the drug half-life compared to the original mutant GP8 protein.

[0121] Experiment 2: A drug concentration-time curve was plotted based on the measurement results. The study demonstrated that, with the same dosage and administration method, mPEG-SS-5k-GP8-highly modified showed a higher AUC (5,683,809 h.IU / ml vs 2,344,730 h.IU / ml) than the positive reference product V-PEG-SC-20k-rhIL-2 (calculated using GraphPad Prism 7.00). Therefore, it was found that mPEG-SS-5k-GP8-highly modified has higher bioavailability than the positive reference product (V-PEG-SC-20k-rhIL-2).

[0122] Example 11: Evaluation of the efficacy of IL-2 modified with different structural PEG-SS (linear or branched) in a BALB / c mouse subcutaneous transplantation model of colon cancer cells derived from CT26.WT mice. 1. Experimental Method CT26.WT cells are cultured in 1640 culture medium containing 10% fetal bovine serum. CT26.WT cells in the exponential growth phase are collected and resuspended in PBS to the appropriate concentration. 5 × 10 5 After thoroughly mixing 0.1 mL of CT26.WT cell suspension, inoculate it subcutaneously into the right back of BALB / c mice. Each mouse is inoculated with 0.1 mL. The average tumor volume is approximately 100 mm². 3 After reaching a certain stage, patients are divided into groups based on tumor volume. The first dose is administered on the day of group division (in this experiment, one dose is administered on day 9 and day 16 after tumor cell inoculation). The detailed administration protocol and route are shown in the table below.

[0123] [Table 13]

[0124] General clinical observation: During the quarantine and experimental periods, observations will be made at least once a day, including observation of tumor growth and the effects of treatment on the animals' normal behavior. Specifically, this includes observations of the animals (with tumors), such as death or near-death, mental state, behavior, and other abnormal conditions. Weight: Measure 2-3 times a week.

[0125] Tumor volume: Measure 2-3 times per week. Measure the longest and shortest diameters of the tumor using calipers.

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

[0127] Tumor weight: After the final measurement, the experimental animals are euthanized, the tumor masses are dissected, rinsed with physiological saline, blotted dry with filter paper, the weights of the tumor masses are measured, and photos are taken. Relative tumor inhibition rate TGI(%) = (1 - T TW / C TW ) × 100%, T TW represents the average 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. Drug efficacy The average tumor volume of the animals in the model group on the 20th day after cell inoculation was 1397.48 ± 289.67 mm 3 .

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

[0130] The mean tumor volume 20 days after cell inoculation was 942.44 ± 209.66 mm² in the V-PEG-SS-20k-GP1 (2 mg / kg) group and the V-PEG-SS-20k-GP8 (2 mg / kg) group, respectively. 3 , 1037.67±332.97mm 3 There was no significant difference compared to the model group. The relative tumor suppression rates (TGI) were 26% and 29%, respectively.

[0131] The mean tumor volume of the positive reference group V-PEG-SC-20k-rhIL-2 (2 mg / kg) 20 days after cell inoculation (unless otherwise specified in later examples, all positive references use V-PEG-SC-20k-rhIL-2, and the abbreviation is PEG-SC-20k-rhIL-2) was 211.23 ± 86.02 mm². 3 This showed a significant difference compared to the model group (P<0.01). The relative tumor suppression rate (TGI) was 84%.

[0132] The results of the tumor weight analysis closely match those of the tumor volume analysis. Specific experimental results are shown in the table below and in Figure 6.

[0133] [Table 14]

[0134] [Table 15]

[0135] 2, Safety Animals in the PEG-SS modified group experienced a weight loss of less than 10% during the administration period, and although their 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 reference group, one out of six animals showed moderate weight loss (10% < weight loss ≤ 20%) on day 2 after the second dose (D18). Furthermore, while the animals in the positive reference group showed some weight recovery in the later stages of the experiment, there was a significant difference compared to the model group.

[0137] [Table 16]

[0138] 3. Results analysis mPEG-SS-20k-GP1 and mPEG-SS-20k-GP8, modified products of linear PEG, showed a significant tumor growth inhibitory effect in a CT26.WT mouse-derived colon cancer model at a dose of 2 mg / kg, with the inhibition rate being equivalent to or greater than that of the positive reference product. On the other hand, V-PEG-SS-20k-GP1 and V-PEG-SS-20k-GP8, modified products of branched PEG, did not show a tumor growth inhibitory effect in the CT26.WT mouse-derived colon cancer model at a dose of 2 mg / kg.

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

[0140] In this example, researchers were surprised to find that, despite having the same molecular weight, the linear mPEG-SS-20k modified product and the branched V-PEG-SS-20k modified product showed considerable differences in in vivo experiments. In other words, the in vivo efficacy of the branched PEG-SS modified product was far lower than that of the linear PEG-SS modified product. Therefore, the inventors confirmed the use of linear mPEG-SS when developing IL-2 protein drugs as tumor immune agonists.

[0141] Example 12: Evaluation of the efficacy of different IL-2 mutants modified with linear PEG-SS in a C57BL / 6 mouse subcutaneous transplantation model of melanoma cells derived from B16-F10 mice. 1. Experimental Method B16-F10 cells are cultured in DMEM culture medium containing 10% fetal bovine serum. B16-F10 cells in the exponential growth phase are collected and resuspended in PBS to the appropriate concentration. 5 × 10 5 After thoroughly mixing 0.1 mL of B16-F10 cell suspension, inoculate it subcutaneously into the right back of C57BL / 6 mice. Each mouse is inoculated with 0.1 mL. The average tumor volume is approximately 100 mm². 3 After reaching a certain stage, patients are divided into groups based on tumor volume. The first dose is administered on the day of group division (one dose on D7). The detailed administration protocol and route are shown in the table below. General clinical observations are the same as in Example 11.

[0142] [Table 17]

[0143] 2. Experimental Results 1. Medicinal effects The experimental results are shown in the table and Figure 7 below.

[0144] [Table 18]

[0145] [Table 19]

[0146] 2, Safety The rate of weight change in the V-PEG-SC-20k-rhIL-2 group was significantly lower at D11 (all animals in each group were still alive at D11) compared to the model group. Animals in the mPEG-SS-20k-GP1, mPEG-SS-20k-GP13, mPEG-SS-20k-GP21, and mPEG-SS-20k-GP22 groups, which are products of different mutants modified with linear PEG-SS, did not show a statistically significant difference in weight loss at D11 compared to the model group, but did show a statistically significant difference compared to the V-PEG-SC-20k-rhIL-2 group. This indicates that the products of different mutants modified with linear PEG-SS (e.g., mPEG-SS-20k-GP1, mPEG-SS-20k-GP13, mPEG-SS-20k-GP21, mPEG-SS-20k-GP22, etc.) have a smaller effect on animal body weight than V-PEG-SC-20k-rhIL-2.

[0147] [Table 20]

[0148] 3. Results analysis The products of different mutants modified with linear PEG—mPEG-SS-20k-GP1, mPEG-SS-20k-GP13, mPEG-SS-20k-GP21, and mPEG-SS-20k-GP22—showed significant tumor growth inhibition in a B16-F10 mouse-derived melanoma model at a dose of 1 mg / kg, with inhibition rates close to those of the positive reference product. Furthermore, these test products had a smaller impact on animal body weight than V-PEG-SC-20k-rhIL-2.

[0149] Example 13: Evaluation of the efficacy of products with different modification levels of linear PEG-SS-modified IL-2 mutants in a B16-F10 mouse-derived melanoma tumor model. 1. Experimental Method The experimental methods and general clinical observations are the same as in Example 11. The detailed administration protocol and route are shown in the table below.

[0150] [Table 21]

[0151] 2. Experimental Results 1. Medicinal effects The mean tumor volume of the model animals 17 days after cell inoculation was 2614.97 ± 372.77 mm². 3 That was the case.

[0152] The mean tumor volume 17 days after cell inoculation for 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 was 550.25 ± 100.23 mm², respectively. 3 544.90±89.12mm 3 574.02±108.15mm 3 676.17±128.23mm 3 570.45±107.25mm 3 The results showed a significant difference compared to the model group (P<0.01 or P<0.05). The relative tumor suppression rates (TGI) were 77%, 80%, 79%, 75%, and 79%, respectively.

[0153] The mean tumor volume of the PEG-SC-20k-rhIL-2 group 17 days after cell inoculation was 1215.19 ± 184.70 mm². 3 This showed a significant difference compared to the model group (P<0.05). The relative tumor suppression rate (TGI) was 57%.

[0154] The results of the tumor weight analysis closely match those of the tumor volume analysis. Specific experimental results are shown in the table below and in Figure 8.

[0155] [Table 22]

[0156] [Table 23]

[0157] 2, Safety In the mPEG-SS-20k-GP8 (high-modification) group, one out of six animals showed moderate weight loss (10% < weight loss ≤ 20%) four days after the first dose (11 days after cell inoculation). Seven days after the first dose (14 days after cell inoculation), two out of six animals showed moderate weight loss (10% < weight loss ≤ 20%), and three out of six animals died. Nine days after the first dose (16 days after cell inoculation), one out of six animals still showed moderate weight loss (10% < weight loss ≤ 20%), and one out of six animals died. At the end of the experiment, two out of the six surviving animals showed a tendency towards weight recovery. More than half of the animals in this group died, and it is presumed that the cause of death was 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 day 4 after the first dose (11 days after cell inoculation). On day 7 after the first dose (14 days 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 (weight loss > 20%). On day 9 after the first dose (16 days 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 (weight loss > 20%), and 1 out of 6 animals died. At the end of the experiment, of the 5 surviving animals, 4 showed a tendency towards weight recovery, and 1 animal did not recover its weight. This group experienced moderate to severe weight loss and the death of one animal. This is presumed to be related to the test substance.

[0159] In the mPEG-SS-5k-GP8 (high-modification) group, one out of six animals showed moderate weight loss (10% < weight loss ≤ 20%) on day 4 after the first dose (11 days after cell inoculation). On day 7 after the first dose (14 days after cell inoculation), one out of six animals showed moderate weight loss (10% < weight loss ≤ 20%). At the end of the experiment, the animals showed a tendency towards weight recovery.

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

[0161] In the mPEG-SS-5k-GP8 (low-modification) group, one out of six animals showed moderate weight loss (10% < weight loss ≤ 20%) on day 4 after the first dose (day 11 after cell inoculation). At the end of the experiment, the animals showed a tendency towards weight recovery.

[0162] In the PEG-SC-20k-rhIL-2 group, one out of six animals showed moderate weight loss (10% < weight loss ≤ 20%) on day 4 after the first dose (day 11 after cell inoculation), and the animals showed a tendency towards weight recovery at the end of the experiment. On day 7 after the first dose (day 14 after cell inoculation), three out of six animals died. 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, one out of six animals showed moderate weight loss (10% < weight loss ≤ 20%) on day 7 after the first dose (day 14 after cell inoculation), and a trend toward weight recovery was observed at the end of the experiment. The specific results are shown in the table and Figure 8c below.

[0164] [Table 24]

[0165] 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 showed inhibitory effects on tumor growth in B16-F10 mouse-derived melanoma models at a dose of 2 mg / kg, and their efficacy was superior to that 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. On the other hand, animals in the mPEG-SS-5k-GP8 (highly modified) and mPEG-SS-5k-GP8 (lowly modified) groups were generally well tolerated at the 2 mg / kg dose. Animals in the mPEG-SS-5k-GP8 (moderately modified) group showed good tolerability at the 2 mg / kg dose. This embodiment provides preliminary evidence that products with different levels of modification exhibit different efficacy and safety in the body. This result is consistent with the prediction in Example 7, based on in vitro experiments, that "products with different levels of modification have different performance characteristics." Therefore, the inventors further conducted comparative studies of efficacy in other different models.

[0166] Example 14: Evaluation of the efficacy of products with different modification levels of linear PEG-SS-modified IL-2 mutants in a CT26.WT mouse-derived colon cancer tumor model. 1. Experimental Method The experimental methods and general clinical observations are the same as in Example 11. The detailed administration protocol and route are shown in the table below.

[0167] [Table 25]

[0168] 2. Experimental Results 1. Medicinal effects The mean tumor volume of the model animals 22 days after cell inoculation was 1,886.67 ± 341.33 mm². 3 That was the case.

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

[0170] On the 22nd day after cell inoculation, the average tumor volume of the PEG-SC-20k-rhIL-2 group was 163.13 ± 73.03 mm 3 respectively, and there were significant differences compared with 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 those of tumor volume analysis. The specific experimental results are shown in the following table and Figure 9.

[0172]

Table 26

[0173]

Table 27

[0174] 2. Safety In the mPEG-SS-20k-GP8 (high-modification) group, 3 out of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%) on day 5 after the first dose, and 1 out of 6 animals died. On day 2 after the second dose, 2 out of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%), and a trend toward weight recovery was observed at the end of the experiment.

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

[0176] In the mPEG-SS-5k-GP8 (high-modification) group, one out of six animals showed moderate weight loss (10% < weight loss ≤ 20%) on day 5 after the first dose. Two days after the second dose, one out of six animals showed moderate weight loss (10% < weight loss ≤ 20%), and a trend toward weight recovery was observed at the end of the experiment.

[0177] In the mPEG-SS-5k-GP8 (moderately modified) group, animals did not experience weight loss during the administration period, or their weight loss was less than 10%, and animals that did experience weight loss showed weight recovery in the later stages of the experiment.

[0178] In the mPEG-SS-5k-GP8 (low-modification) group, one out of six animals showed moderate weight loss (10% < weight loss ≤ 20%) two days after the second dose, and a trend towards weight recovery was observed at the end of the experiment.

[0179] In the PEG-SC-20k-rhIL-2 group, 4 out of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%) on day 5 after the first dose. 2 out of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%) on day 2 after the second dose. 7 out of 6 animals still showed moderate weight loss (10% < weight loss ≤ 20%) on day 7 after the second dose, while the remaining animals showed a tendency toward weight recovery at the end of the experiment.

[0180] [Table 28]

[0181] 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) showed an inhibitory effect on tumor growth 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 a dose of 2 mg / kg well. Animals in the mPEG-SS-20k-GP8 (highly modified) and mPEG-SS-20k-GP8 (moderately modified) groups generally tolerated a dose of 2 mg / kg, and their weight recovery was better than that of the V-PEG-SC-20k-rhIL-2 group.

[0183] Example 15: Evaluation of the efficacy of products with different modification levels of linear PEG-SS-modified IL-2 mutants in the A375 human melanoma tumor model. A375 cells are cultured in DMEM culture medium containing 10% fetal bovine serum. A375 cells in the exponential growth phase are collected and resuspended in PBS. hu-PBMCs (human peripheral blood mononuclear cells) are cultured in 1640 culture medium containing 10% fetal bovine serum, stimulated with OKT-3 and IL-2 for 3 days, collected, and deuterated in PBS. 5 × 10 5 A375 cells and 5 × 10 cells / 0.1 mL 5 After thoroughly mixing cells / 0.1 mL of PBMC cell suspension in a 1:1 ratio (cell inoculation volume), inoculate NOD / SCID mice subcutaneously. Each mouse will receive 0.2 mL of the suspension. On the day of cell inoculation, mice will be divided into groups based on body weight. The first dose will be administered on the day of group division. The detailed administration protocol and route are shown in the table below.

[0184] [Table 29]

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

[0186] 2. Experimental Results 1. Medicinal effects The mean tumor volume of the model animals 45 days after cell inoculation was 1,970.97 ± 298.65 mm². 3 That was the case.

[0187] At 45 days after cell inoculation, the 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 groups showed significant differences compared to the model group (P<0.01). The mean tumor volume of the 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 ± 134.84 mm², respectively. 3 83.34±83.34mm 3 96.62±64.92mm 3There were significant differences in all cases compared with the model group (P<0.01).

[0188] The analysis results of tumor weight were similar to those of tumor volume. The TGI values of the mPEG-SS-20k-GP8 (high modification degree) group, mPEG-SS-20k-GP8 (medium modification degree) group, mPEG-SS-5k-GP8 (high modification degree) group, mPEG-SS-5k-GP8 (medium modification degree) group, mPEG-SS-5k-GP8 (low modification degree) group, and PEG-SC-20k-rhIL-2 group calculated based on tumor weight were 100%, 100%, 95%, 100%, 95%, and 91% respectively. The specific experimental results are shown in the following table and Figure 10.

[0189]

Table 30

[0190]

Table 31

[0191]

Table 32

[0192]

Table 33

[0193] 2. Safety In the mPEG-SS-20k-GP8 (high modification degree) group, 1 out of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%) on the 3rd day after the 4th administration. This animal continued to show moderate weight loss (10% < weight loss ≤ 20%) on the 6th day after the 4th administration and died on the 1st day after the final administration. Furthermore, from the 9th day to the 13th day after the final administration, 1 out of 6 animals showed moderate weight loss (10% < weight loss ≤ 20%), and at the end of the experiment, a tendency of weight recovery was observed in this animal.

[0194] In the mPEG-SS-20k-GP8 (moderate modification) group, one out of six animals showed severe weight loss (weight loss >20%) three days after the second dose. This animal died six days after the second dose. From three days after the final dose to the end of the experiment, one out of six animals showed moderate weight loss (10% < weight loss ≤20%). Furthermore, one out of six animals died four days after the final dose.

[0195] In the mPEG-SS-5k-GP8 (moderate modification) group, one out of six animals showed moderate weight loss (10% < weight loss ≤ 20%) between day 6 and day 9 after the final dose. At the end of the experiment, this animal showed a tendency towards weight recovery.

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

[0197] In the PEG-SC-20k-rhIL-2 group, one out of six animals showed moderate weight loss (10% < weight loss ≤ 20%) on day 6 after the final dose. On day 9 after the final dose, two out of six animals showed moderate weight loss (10% < weight loss ≤ 20%). On day 13 after the final dose, two out of six animals showed moderate weight loss (10% < weight loss ≤ 20%), and one out of six 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 showed inhibitory effects on tumor growth in the A357 human melanoma model at a dose of 0.5 mg / kg (once a week for 5 weeks). From the perspective of pharmacological 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 superior to 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. On the other hand, animals in the mPEG-SS-5k-GP8 (highly modified) and mPEG-SS-5k-GP8 (lowly modified) groups showed good tolerance to a dose of 0.5 mg / kg (once a week for 5 weeks). Animals in the mPEG-SS-5k-GP8 (moderately modified) group were generally well tolerated at a dose of 0.5 mg / kg (once a week for 5 weeks). From a safety perspective (body weight, mortality), the tolerability of the test products mPEG-SS-5k-GP8 (high modification), mPEG-SS-5k-GP8 (medium modification), and mPEG-SS-5k-GP8 (low modification) was superior to that of PEG-SC-20k-rhIL-2.

[0200] Example 16: Evaluation of the efficacy of PEG-modified IL-2 mutants in the A375 human melanoma model. 1. Experimental Method A375 cells are cultured in DMEM culture medium containing 10% fetal bovine serum. A375 cells in the exponential growth phase are collected and resuspended in PBS. hu-PBMCs (human peripheral blood mononuclear cells) are cultured in 1640 culture medium containing 10% fetal bovine serum, stimulated with OKT-3 and IL-2 for 3 days, collected, and deuterated in PBS. 1 × 10 6A375 cells and 1 × 10⁶ cells / 0.1 mL 6 After thoroughly mixing cells / 0.1 mL of PBMC cell suspension in a 1:1 ratio (cell inoculation volume), inoculate NOD / SCID mice subcutaneously. Each mouse will receive 0.2 mL of the suspension. On the day of cell inoculation, mice will be divided into groups based on body weight. The first dose will be administered on the day of group division. The detailed administration protocol and route are shown in the table below.

[0201] [Table 34]

[0202] General clinical observations and measurements of body weight, tumor volume, and tumor weight are the same as in Example 11.

[0203] 2. Experimental Results 1. Medicinal effects The mean tumor volume of negative control animals 45 days after cell inoculation was 1,197.64 ± 143.79 mm². 3 That was the case.

[0204] The mean tumor volume of animals 45 days after cell inoculation 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 was 144.87 ± 35.11 mm², respectively. 3 , 296.04±61.10mm 3 643.88±153.05mm 3 All of these findings showed a statistically significant difference compared to the negative control group (P<0.01 or P<0.05).

[0205] The mean tumor volume of animals in the positive control group (Izumi, 1,000,000 IU / kg) 45 days after cell inoculation was 557.54 ± 104.82 mm². 3The results showed a significant difference compared to the negative control group (P<0.01). The mPEG-SS-5K-GP8-high-modification, high-dose group showed a significant difference in tumor volume compared to the positive control group (Izumi) (P<0.01). Although the total dose administered to the positive control group (Izumi) was higher than that of the mPEG-SS-5K-GP8-high-modification, high-dose group, the experimental results showed that the mPEG-SS-5K-GP8-high-modification group had superior efficacy.

[0206] The mean tumor volume of animals in the positive reference drug group (V-PEG-SC-20k-rhIL-2, pseudo-NKTR214) 45 days after cell inoculation was 224.37 ± 33.28 mm². 3 The results showed a significant difference compared to the negative control group (P<0.01). The mPEG-SS-5K-GP8-highly modified medium-dose group did not show a significant difference in tumor volume compared to the positive reference group (P>0.05). The experimental results showed that the efficacy of mPEG-SS-5K-GP8-highly modified was similar to that of the positive reference drug (V-PEG-SC-20k-rhIL-2, pseudo-NKTR214) at the same dose.

[0207] The results of the tumor weight analysis were in close agreement with the results of the tumor volume analysis. Based on tumor weight, the TGI for the mPEG-SS-5K-GP8 high-modification high, medium, and low-dose groups was 87%, 75%, and 46%, respectively. Based on tumor weight, the TGI for the positive control group (Izumi) and positive reference group (V-PEG-SC-20k-rhIL-2, pseudo-NKTR214) was 56% and 81%, respectively. The specific experimental results are shown in the table and Figure 11 below.

[0208] [Table 35]

[0209] [Table 36]

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

[0211] In the positive control group (Izumi, 1 million IU / kg), no obvious drug toxicity was observed during the intravenous administration treatment period, and the treatment was well-tolerated. On day 3 after the final dose (D32) and day 16 after the final dose (D45), there was no statistically significant difference in body weight between the mPEG-SS-5K-GP8-high-modification, high-dose group (250 μg / kg) and the positive control group (Izumi, 1 million IU / kg) (P>0.05).

[0212] In the V-PEG-SC-20k-rhIL-2 group (125 μg / kg), 3 out of 8 animals showed moderate weight loss and 1 out of 8 animals showed moderate to severe weight loss during the intravenous administration period, but the animals were generally well-tolerated. On day 3 after the final dose (D32) and day 16 after the final dose (D45), the body weight of the mPEG-SS-5K-GP8-high-modification medium-dose group (125 μg / kg) was statistically different compared to the positive reference group (V-PEG-SC-20k-rhIL-2, 125 μg / kg) (P<0.01 or P<0.05). As a result, mPEG-SS-5K-GP8-high-modification was safer than the positive reference group (pseudo-NKTR214) at the same dose.

[0213] [Table 37]

[0214] [Table 38]

[0215] 3. Results analysis mPEG-SS-5K-GP8-high modification showed significant tumor suppression effects in the A375 human melanoma model at high doses (250 μg / kg) and medium doses (125 μg / kg). mPEG-SS-5K-GP8-high modification also showed a tendency towards tumor suppression at low doses (62.5 μg / kg). mPEG-SS-5K-GP8-high modification showed a good dose-response relationship at high, medium, and low doses. The positive reference group (V-PEG-SC-20k-rhIL-2, pseudo-NKTR214) showed significant tumor suppression effects in the A375 human melanoma model at a dose of 125 μg / kg, and the positive control group (Izumi) showed a tendency towards tumor suppression at a dose of 1,000,000 IU / kg.

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

[0217] The above indicates that, for equivalent tumor-suppressing effects, animals tolerate mPEG-SS-5K-GP8-highly modified more well than V-PEG-SC-20k-rhIL-2. Furthermore, for equivalent safety, although the positive control group (Izumi) received a higher total dose than the mPEG-SS-5K-GP8-highly modified high-dose group, the mPEG-SS-5K-GP8-highly modified group demonstrated superior efficacy.

[0218] Example 17: Evaluation of the efficacy of PEG-modified IL-2 mutants in the A498 human kidney cancer model. 1. Experimental Method A498 cells are cultured in DMEM culture medium containing 10% fetal bovine serum. A498 cells in the exponential growth phase are collected and resuspended in PBS. hu-PBMCs (human peripheral blood mononuclear cells) are cultured in 1640 culture medium containing 10% fetal bovine serum, stimulated with OKT-3 and IL-2 for 3 days, collected, and deuterated in PBS. 5 × 10 6 A498 cells and 5 × 10⁶ cells / 0.1 mL 6 After thoroughly mixing cells / 0.1 mL of PBMC cell suspension in a 1:1 ratio (cell inoculation volume), inoculate NOD / SCID mice subcutaneously. Each mouse will receive 0.2 mL of the suspension. On the day of cell inoculation, mice will be divided into groups based on body weight. The first dose will be administered on the day of group division. The detailed administration protocol and route are shown in the table below.

[0219] [Table 39]

[0220] General clinical observations and measurements of body weight, tumor volume, and tumor weight are the same as in Example 11.

[0221] 2. Experimental Results 1. Medicinal effects The mean tumor volume of negative control animals 61 days after cell inoculation was 1,960.68 ± 398.28 mm². 3 That was the case.

[0222] The mean tumor volume of animals in the moderate- and low-dose groups (125 μg / kg and 62.5 μg / kg) of mPEG-SS-5K-GP8-highly modified cells 61 days after inoculation was 18.99 ± 18.99 mm², respectively. 3 , 248.19±209.86mm 3 This showed a significant difference compared to the negative control group (P<0.01).

[0223] The mean tumor volume of animals in the positive control group (Izumi, 712,500 IU / kg) 61 days after cell inoculation was 290.85 ± 145.96 mm². 3The results showed a significant difference compared to the negative control group (P<0.01). Although the dose in the mPEG-SS-5K-GP8 high-modification medium-dose group was only half that of the positive control group (Izumi), the experimental results showed that its efficacy was superior to that of the positive control group (Izumi).

[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 mm². 3 The results showed a significant difference compared to the negative control group (P<0.01). The mPEG-SS-5K-GP8-highly modified medium-dose group did not show a significant difference in tumor volume compared to 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 results of the tumor weight analysis were in close agreement with the results of the tumor volume analysis. Based on tumor weight, the TGI for the intermediate and low-dose mPEG-SS-5K-GP8 groups was 99% and 85%, respectively. Based on tumor weight, the TGI for the positive control group (Izumi) and positive reference group (V-PEG-SC-20k-rhIL-2, pseudo-NKTR214) was 83% and 93%, respectively. The specific experimental results are shown in the table and Figure 12 below.

[0226] [Table 40]

[0227] [Table 41]

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

[0229] In the positive control group (Izumi, 712,500 IU / kg), no obvious drug toxicity was observed during the treatment period via intravenous injection, and the drug was well-tolerated during the treatment period.

[0230] In the V-PEG-SC-20k-rhIL-2 group (125 μg / kg), 3 out of 8 animals showed moderate weight loss and 1 out of 8 animals showed moderate to severe weight loss during the intravenous administration period, but the animals were generally well-tolerated. On day 4 after the final dose (D33), the body weight of the mPEG-SS-5K-GP8-highly modified medium-dose group (125 μg / kg) was statistically different from that of the V-PEG-SC-20k-rhIL-2 group (125 μg / kg) (P<0.01). As a result, mPEG-SS-5K-GP8-highly modified was safer than the positive reference group (pseudo-NKTR214) at the same dose.

[0231] [Table 42]

[0232] [Table 43]

[0233] 3. Results analysis mPEG-SS-5K-GP8-high modification showed significant tumor suppression effects in the A498 human kidney cancer subcutaneous transplant tumor model at moderate (125 μg / kg) and low (62.5 μg / kg) doses. mPEG-SS-5K-GP8-high modification showed a consistent dose-response relationship at moderate and low doses. The V-PEG-SC-20k-rhIL-2 group showed significant tumor suppression effects in the A498 human kidney cancer subcutaneous transplant tumor model at a dose of 125 μg / kg, while the positive control group (Izumi) showed significant tumor suppression effects at a dose of 712,500 IU / kg.

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

[0235] The above indicates that, for equivalent tumor-suppressing effects, animals tolerate mPEG-SS-5K-GP8-high-modification better than V-PEG-SC-20k-rhIL-2.

[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 amounts of mPEG-SS-5K-GP8-highly modified (0.01 mg / kg group, 0.03 mg / kg group) were administered intravenously. A solvent was administered to the blank control group as a control. The drug was administered once a week for a total of five doses (D1, D8, D15, D22, D29). Thirty days after the first dose (D1), peripheral blood was collected, peripheral blood mononuclear cells (PBMCs) were separated, labeled with anti-CD25-APC fluorescent antibody (Miltenyi, cargo number 130-113-842), and measured by FACS.

[0237] [Table 44]

[0238] The data above shows that CD25 expression levels in the 0.03 mg / kg group were significantly different from those in the blank control group. CD25 is a marker of T cell activation in PBMC cells, and the results of this experiment demonstrate that the 0.03 mg / kg dose group effectively stimulated T cell activation.

[0239] Example 19: Dose-finding experiment of repeated intravenous injection in cynomolgus monkeys over 4 weeks. Cynomolgus monkeys will be used as experimental animals, and different amounts of mPEG-SS-5K-GP8-high-modification will be administered intravenously multiple times. Possible toxic reactions during the administration period will be observed. Four dose groups will be established: 0.03, 0.1, 0.3, and 0.5 mg / kg. After completing three administrations in the 0.03 mg / kg dose group, the dose will be increased to 0.3 mg / kg, creating the 0.3 mg / kg dose group, and a total of six administrations will be performed.

[0240] 1. Grouping and Experimental Design According to the table below, the six animals will be randomly divided into four groups based on their weight and sex.

[0241] [Table 45]

[0242] During the experiment, animals will be clinically observed daily, monitoring changes in body weight, food intake, body temperature, and blood pressure. Clinical pathological examinations, immunocytophenotypic analysis, cytokine measurements, and macroscopic observations will also be performed.

[0243] 2. Results No deaths occurred in cynomolgus monkeys in the four dose groups: 0.03, 0.1, 0.3, and 0.5 mg / kg. At a dose of 0.03 mg / kg, cynomolgus monkeys showed no abnormal conditions and tended to gain weight. At a dose of 0.1 mg / kg, male animals showed splenomegaly, a decrease in the percentage of CD56+ cells, and a tendency toward weight gain. At a dose of 0.3 mg / kg, the animals' food intake decreased, but their body weight tended to increase. Gross anatomical examination revealed spleen enlargement, and clinical pathological examination confirmed an increase in white blood cell count, a decrease in red blood cell count, a decrease in red blood cell volume ratio, a decrease in platelet count, and a decrease in total protein.

[0244] At a dose of 0.5 mg / kg, the animals showed adverse reactions after the first dose, including fatigue, decreased spontaneous activity, massive loose stools, and pallor of the visible mucous membranes. Administration was discontinued once, and a total of four doses were administered. The animals' food intake and body weight decreased, and gross anatomical examination revealed symptoms such as splenomegaly, thymus reduction, abdominal distension, and generalized skin laxity. Clinical pathological examination showed increases in white blood cell count, mononuclear cells (absolute value), neutrophils (absolute value), eosinophils (absolute value), basophils (absolute value), urea, creatine kinase, and fibrinogen, while decreases in red blood cell count, red blood cell volume ratio, platelet count, and total protein. In addition, the percentage of CD56+ cells decreased.

[0245] Preliminary studies have shown that, under the experimental conditions described above, a dose of 0.5 mg / kg resulted in severe adverse reactions in cynomolgus monkeys, but no deaths have yet occurred.

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

Claims

1. A human interleukin-2 mutant characterized by not containing adjacent lysine, wherein the mutant is as follows: Table 1 A human interleukin-2 mutant characterized by having a mutation selected from the mutation scheme, wherein the mutation site is based on wild-type human IL-2.

2. The aforementioned mutations are shown in the following table: Table 2 Characterized by being selected from the mutation scheme, The human interleukin-2 mutant according to claim 1.

3. A polyethylene glycol modified form of human interleukin-2, A polyethylene glycol modified human interleukin-2, characterized in that polyethylene glycol succinimidyl succinate is used as the PEG modifier, an average of 4.5 to 8.5 PEG molecules are bound to each human interleukin-2 molecule, and the human interleukin-2 is the human interleukin-2 mutant described in claim 1.

4. The PEG modifier is characterized by being a linear polyethylene glycol succinimidyl succinate modifier. The polyethylene glycol modified product according to claim 3.

5. The PEG modifier is characterized by having a molecular weight of 5 to 20 kDa. The polyethylene glycol modified product according to claim 3.

6. When the molecular weight of the PEG modifier is 5 kDa, an average of 5.5 to 7.5 PEG molecules are bound to each human interleukin-2 molecule, and when the molecular weight of the PEG modifier is 10 to 20 kDa, an average of 6.5 to 8.5 PEG molecules are bound to each human interleukin-2 molecule. The polyethylene glycol modified product according to claim 3.

7. The structure of the PEG modifier is as shown in the following figure: 【Chemistry 1】 The polyethylene glycol modified product according to claim 3, characterized in that n is an integer between 97 and 494 in the formula.

8. The structure of the polyethylene glycol modified human interleukin-2 is as shown below. 【Chemistry 2】 In the formula, n is an integer between 97 and 494, and m is between 4.5 and 8.

5. The polyethylene glycol modified product according to claim 3.

9. Use of the polyethylene glycol modified human interleukin-2 according to claim 3 in the manufacture of a drug for treating tumor diseases.

10. The tumors are characterized by including squamous cell carcinoma, melanoma, colon cancer, breast cancer, ovarian cancer, prostate cancer, stomach 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 adrenocortical carcinoma. The use described in claim 9.

11. A composition for treating tumor diseases, The composition comprises a human interleukin-2 mutant according to claim 1, or a polyethylene glycol modified human interleukin-2 according to claim 3, and further comprises a HER2 antibody, a PD-1 antibody, a PD-L1 antibody, or a CD26 antibody, for the treatment of tumor diseases.

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