Protein formulations, methods for their preparation and uses

Abstract

The present invention relates to a protein formulation, its preparation method and its use. The formulation is an aqueous solvent and contains (1) a cytokine protein at a concentration of more than 1 mg / ml, (2) polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), and (3) a necessary pH adjuster / buffer. The cytokines include, but are not limited to, IL2, IL4, IL7, IL9, IL10, IL15, IL21, G-CSF, GM-CSF, IFN, EPO, GF and TNF. The formulation is used for the preparation of a medicament or formulation to be administered by subcutaneous injection.

Description

[Technical field]

[0001] The present invention is in the field of biotechnology, specifically, to protein formulations, their preparation methods and their uses. [Background technology]

[0002] Cytokines (CK) are small polypeptides or glycoproteins synthesized and secreted by various tissue cells (mainly immune cells). Cytokines can mediate cell-cell interactions and have various biological functions, such as regulating cell proliferation, differentiation and maturation, maintaining function, regulating immune responses, participating in inflammatory responses, wound healing, and regression of tumor growth. Since the discovery of interferon by Lssac in 1957, more than 200 cytokines have been discovered. Interleukins, interferons, tumor necrosis factors, hematopoietic factors, growth factors, chemokines, etc. are all collectively referred to as cytokines.

[0003] 1.Cytokines are broadly classified according to their functions as follows:

[0004] (1) Interleukins (ILs) are cytokines produced by lymphocytes, monocytes or other nonmononuclear cells that play important roles in regulating cell-cell interactions, immune regulation, hematopoiesis and inflammation.

[0005] (2) Colony-stimulating factors (CSFs) are called G (granulocyte)-CSF, M (macrophage)-CSF, GM (granulocyte-macrophage)-CSF, etc., according to their roles in stimulating hematopoietic stem cells or hematopoietic cells at various differentiation stages. Each CSF can not only stimulate the proliferation and differentiation of hematopoietic stem cells and progenitor cells at various developmental stages, but also promote the functions of mature cells.

[0006] (3) Interferons (IFNs) are divided into IFN-α, IFN-β, and IFN-γ depending on their source and structure, and have antiviral, antitumor, and immunomodulatory effects.

[0007] (4) Tumor necrosis factors (TNFs) are divided into TNF-α and TNF-β. The former is produced by monocyte macrophages, and the latter by activated T cells. These two types of TNFs have similar basic vital activities, such as killing tumor cells, regulating the immune system, and contributing to fever and inflammation.

[0008] (5) Growth factors (GFs) such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), IGF-2, leukemia inhibitory factor (LIF), nerve growth factor (NGF), oncostatin M (OSM), platelet-derived endothelial growth factor (PDECGF), transforming growth factor-α (TGF-α), and vascular endothelial growth factor (VEGF).

[0009] (6) Other cytokines with various biological functions include the transforming growth factor-β family (TGF-β family), the chemokine family, and erythropoietin (EPO).

[0010] 2. Common structure of cytokines

[0011] In terms of molecular structure, cytokines are small polypeptides, most of which are composed of about 100 amino acids. By specifically binding to cytokine receptors on the surface of target cells, cytokines can exert biological effects such as promoting the proliferation and differentiation of target cells, enhancing anti-infection and tumor cell killing effects, promoting or suppressing the synthesis of other cytokines, promoting inflammatory processes, and influencing cell metabolism. The effects of these cytokines have the properties of a network; that is, each cytokine can act on various cells, each cell can be regulated by multiple cytokines, and different cytokines have synergistic or restrictive effects on each other, constituting a complex network of cytokine immunoregulation. This network is not clearly understood.

[0012] 3. Cytokine drugs and the challenges in their development

[0013] Interferon alpha was first used in clinical trials and has shown significant efficacy in treating leukemia and viral infections. In China, interferon alpha passed the new drug review in 1991 and is widely used.

[0014] Internationally approved cytokine drugs include EPO, interferon gamma, GM-CSF, G-CSF, and IL-2.

[0015] Compared with many naturally occurring cytokines, it is obvious that the development of cytokine drugs takes a very long time, and the preparation process of cytokine drugs is extremely difficult. Specifically, the main difficulties in the development of cytokine drugs are as follows: Summary of the Invention [Problem to be solved by the invention]

[0016] (1) Cytokine proteins have unstable structures and are prone to aggregation, which reduces the water solubility and stability of the proteins.

[0017] (2) Cytokine proteins usually have a short half-life, low stability, and are easily degraded in vivo, making it difficult to control the therapeutic concentration range of cytokine drugs.

[0018] (3) Cytokine proteins activate a variety of different receptors or receptor combinations, often resulting in nonspecific pharmacological effects and relatively strong side effects.

[0019] Therefore, new ideas and formulation systems are needed for the development of cytokine drugs.

[0020] 4. Interleukin 2 (IL2)

[0021] Interleukin 2 (IL2), also known as T cell growth factor, was discovered in 1976 and stimulates T cell clonal proliferation in in vitro experiments. IL2 is a glycoprotein composed of four α-helices with a molecular weight of 15.5 Kd, and is secreted primarily by activated CD4 T cells, although small amounts of IL2 can also be produced by other cells, such as activated CD8 cells, NK cells, NKT cells, and ILCs [3, 4]. IL2 functions by binding to autologous receptor cells via autocrine and paracrine pathways and plays an important role in regulating T cells and NK cells. The receptor for IL-2 is composed of three subunits, IL2Rα (CD25), IL2Rβ (CD122) and IL2Rγc (also known as the common cytokine receptor γ chain, γc or CD132), and the affinity between IL2 and the three subunits differs. IL2 binds to the α subunit with low affinity (Kd ~ 10 -8 M) and binds to βγ dimers with intermediate affinity (Kd~10 -9 M), binds to the αβγ trimer with high affinity (Kd ~ 10 -11 In addition, IL2Rγc (CD132) is also the receptor for IL4, IL7, IL9, IL15, and IL21.

[0022] The expression levels of receptors with different affinities vary from cell to cell, as follows: βγ receptors are mainly expressed in resting T cells, CD8 T memory cells and NK cells. α receptors are upregulated after cell activation, and activated T cells express αβγ receptors with high affinity. Tregs consistently express α receptors at high levels and have high affinity for IL2. Due to the difference in receptor expression, low-dose IL2 preferentially activates Tregs expressing high-affinity receptors, and only high-dose IL2 can effectively activate CD8+ T cells and NK cells. However, the clinical use of high-dose IL2 results in greater toxic side effects, limiting the use of IL2.

[0023] To solve this problem, IL2 molecules have been modified by various means to suppress binding to Tregs and enhance binding to effector T cells and NK cells, thereby improving the effectiveness of IL2 molecules in tumor treatment and preventing toxicity due to high doses. In the prior art, the main methods for modifying IL2 include the following:

[0024] (1)PEGylated IL2

[0025] IL2 has a molecular weight of 15.5KD and a half-life of 10-85 minutes in vivo in serum. To obtain a therapeutic effect, IL2 must be administered multiple times. PEGylated IL2 increases the total molecular weight of IL2 and extends the half-life of IL2. In addition, depending on the modification site in PEGylation, IL2 can be made to bind more easily to IL2-Rα or IL2-Rb.

[0026] (2) Complex of IL-2 and anti-IL-2 antibody

[0027] Combining IL2 with anti-IL2 antibodies can extend the half-life of IL2, while anti-IL2 antibodies can bind to various sites on the IL2 molecule, so that the complexes of IL2 and anti-IL2 antibodies show different binding preferences for the receptors CD25 and CD122.

[0028] (3) IL2 mutation modification

[0029] The affinity of IL2 to different receptor subunits can also be altered by changing the receptor binding site on IL-2 through point mutation modification. Point mutations of five amino acids of IL-2, namely L80F, R81D, L85V, I86V and I92F, change the conformation of IL-2, thereby enhancing the binding of IL-2 to CD122 and making downstream signaling independent of CD25. Mutant IL2 tends to proliferate CD8+ T cells and NK cells, has lower toxic side effects and shows better tumor therapeutic effect in mouse tumor-bearing models.

[0030] It remains an important question how to further compare and analyze the differences in the expression of CD25 and CD122 receptors on intratumoral Tregs and effector T cells to further improve the efficacy of IL2.

[0031] However, in the above-mentioned conventional techniques, IL2 modification often requires relatively complicated biological or chemical synthesis processes, which significantly increases the difficulty and uncertainty of pharmaceutical development.

[0032] 5. Granulocyte Colony Stimulating Factor (G-CSF)

[0033] (1) Febrile neutropenia (FN) is a common side effect of chemotherapy for solid tumors.

[0034] In clinical practice, the incidence of febrile neutropenia (FN) due to solid tumor chemotherapy is relatively high, which is mainly related to the type of solid tumor and chemotherapy regimen. The incidence of FN in common myelosuppressive chemotherapy regimens is 13-21%, and the incidence of FN is significantly higher in breast cancer, lung cancer, esophageal cancer and ovarian cancer, reaching 28.1%, 15.6%, 15.6% and 12.5%, respectively. The incidence of FN varies greatly depending on the chemotherapy regimen. In particular, the use of taxanes, platinum and fluorouracil is prone to cause myelosuppression, which is closely related to the incidence of FN.

[0035] (2) Granulocyte colony-stimulating factor (G-CSF) is a factor that promotes neutrophil proliferation and is widely used clinically to treat FN.

[0036] Granulocyte colony-stimulating factor (G-CSF) plays an important role in:

[0037] 1) Maintains survival of granulopoietic hematopoietic stem cells and stimulates their differentiation into mature neutrophils.

[0038] 2) Promotes the release of neutrophils from the bone marrow, enhancing the ability of neutrophils to phagocytose antigens and form reactive oxygen species, making it easier to eliminate bacteria.

[0039] Currently, G-CSF is widely used for treating febrile neutropenia (FN) caused by various causes, and has a very significant therapeutic effect, particularly in radiotherapy and chemotherapy.

[0040] (3) Product defects in commercially available G-CSF preparations

[0041] Similar to interleukin cytokines, there are a variety of different binding modes between G-CSF and the G-CSF receptor, a member of the cytokine receptor superfamily. Initiation of various downstream signaling pathways by G-CSF / G-CSFR binding depends on the structure and function of the intracellular domain of the receptor. G-CSFR signaling helps to activate the STAT1, STAT3, and STAT5 pathways, and the activated form of STAT3 is related to the concentration of G-CSF. Protein / receptor binding at various concentrations can also produce highly diverse biological effects, such as promoting neutrophil proliferation as well as stimulating the proliferation of myeloid-derived immune suppressor cells (MDSCs) and Tregs.

[0042] Despite the importance of G-CSF in the prevention and treatment of FN, it has been largely underutilized in clinical practice, mainly because commercially available preparations still have many deficiencies, in particular those that cause obvious adverse effects (cutaneous vasculitis) and increase the risk of secondary cancers, including:

[0043] 1) The commercially available formulation (formula listed below) has a relatively low pH (4.0), is highly irritating (contains acetic acid and Tween), and is unstable upon dilution.

[0044] 2) Direct exposure of the active ingredient to the injection site activates the release of pro-inflammatory factors (IL-6 and TNF-a).

[0045] 3) Non-selective activation of cells expressing G-CSF receptor. In addition to activating bone marrow cells to improve FN symptoms, G-CSF promotes the development of myeloid-derived suppressor cells (MDSCs) and the proliferation of Tregs, thereby promoting tumor progression. The receptor is also expressed in fibroblasts, endothelial cells, and some tumor cells, which are important sources of clinical side effects.

[0046] Based on the above, the present invention is proposed. [Means for solving the problem]

[0047] The present invention relates first to a cytokine protein pharmaceutical formulation which is a water-soluble formulation. The cytokine protein pharmaceutical formulation comprises: (1) A cytokine protein having a concentration of 40 to 20,000 μg / ml, preferably a concentration of 0.1 to 20 mg / ml, and more preferably a concentration of 1 to 10 mg / ml; (2) polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), and (3) Necessary pH adjusters / buffers wherein the molar ratio of the PEG-DSPE to the cytokine protein is 8 to 100:1, preferably 10 to 80:1, more preferably 15 to 50:1, and most preferably 20 to 40:1; the cytokine protein is a human-derived cytokine having a structure including 3 to 8 α-helices and a molecular weight of 10 to 30 kDa; Preferably, the cytokine protein is a wild-type cytokine protein of human origin having a structure containing only alpha helices; More preferably, the cytokine protein is a recombinantly expressed cytokine protein having a structure containing only 4 to 6 α-helices and a molecular weight of 10 to 20 kDa; Most preferably, the cytokine is IL2, IL4, IL7, IL9, IL10, IL15, IL21, G-CSF, GM-CSF, IFN, EPO, GF or TNF.

[0048] The IL2 protein has the amino acid sequence shown in SEQ ID NO:1, and the G-CSF protein has the amino acid sequence shown in SEQ ID NO:2. TIFF2025504570000002.tif40170 The PEG-DSPE has a purity of 95% or more, and the PEG chains in the PEG-DSPE have a dispersity of 1.1 or less; The length of the PEG chain of the PEG-DSPE molecule is preferably 1000 to 5000 Da, more preferably 1500 to 2500 Da, and most preferably 2000 Da (polyethylene glycol 2000-DSPE).

[0049] The cytokine protein pharmaceutical preparation is preferably adjusted to a pH of 5.5 to 6.0 using a pH adjuster.

[0050] The pH adjusters / buffers include, but are not limited to, hydrochloric acid, acetic acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium dihydrogen carbonate, sodium hydrogen phosphate, sodium dihydrogen phosphate, and amino acid pH adjusters / buffers such as glycine and histidine.

[0051] Optionally, the cytokine protein pharmaceutical formulation may be further lyophilized to provide a lyophilized formulation.

[0052] The lyophilized formulation may further comprise a lyoprotectant, including, but not limited to, glycerin, glycine, sucrose, lactose, albumin, histidine, mannitol, or combinations thereof.

[0053] The present invention further relates to a method for preparing a cytokine protein pharmaceutical formulation, said method comprising the steps of: (1) preparing an aqueous solution having a pH of 3.0 to 4.0, preferably diluting glacial acetic acid with deionized water to a concentration of 10 mM to adjust the pH to 3.0 to 4.0, most preferably diluting glacial acetic acid with deionized water to a concentration of 10 mM to adjust the pH to 3.0 to 3.5; (2) weighing a cytokine protein dry powder, adding the low pH aqueous solution to the cytokine protein dry powder, and thoroughly mixing to obtain a cytokine protein suspension; (3) weighing out an amount of PEG-DSPE dry powder corresponding to the amount of cytokine protein in step (2), adding the PEG-DSPE dry powder to the cytokine protein suspension, and mixing thoroughly; (4) heating in a water bath at 40 to 60°C for 10 to 30 minutes; and (5) allowing the solution obtained in step (4) to reach room temperature, adjusting the pH of the solution to 5.5 to 6.0, and allowing the solution to stand for an additional 10 to 60 minutes to obtain a cytokine protein preparation; The PEG-DSPE has a purity of 95% or more, and the PEG chains in the PEG-DSPE have a dispersity of 1.1 or less, The length of the PEG chain of the PEG-DSPE molecule is preferably 1000 to 5000 Da, more preferably 1500 to 2500 Da, and most preferably 2000 Da (polyethylene glycol 2000-DSPE). The preparation method is optional: (6) diluting the solution of the cytokine protein preparation obtained in step (5) with the same buffer to an appropriate concentration or concentrating the solution to an appropriate concentration by ultrafiltration; and (7) sterilizing the cytokine protein formulation by filtration. Includes.

[0054] The protein recovery rate of the cytokine protein pharmaceutical preparation prepared by the above method is 90% or more, where the recovery rate is the ratio of the protein content in the cytokine protein pharmaceutical preparation to the amount of the added protein.

[0055] The present invention further relates to the use of said cytokine protein pharmaceutical preparation in the manufacture of a medicament, said medicament being preferably administered by subcutaneous injection.

[0056] The present invention further relates to the use of polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) in the manufacture of a formulation for altering the affinity between a protein of interest and its receptor, said protein of interest comprising at least one alpha helix, preferably said protein of interest comprising 4 to 6 alpha helices, more preferably said protein of interest being IL2, IL4, IL7, IL9, IL15, IL21, G-CSF or GM-CSF.

[0057] The aforementioned alteration of the affinity between the protein of interest and its receptor means reducing the binding ability of the protein to a low affinity receptor, preferably the low affinity receptor has a binding affinity of 10 to the protein. -6 Affinity (Kd value) of 10 or more, preferably 10 -7 Affinity (Kd value) of M or more, most preferably 10 -8 It has an affinity (Kd value) of M or higher.

[0058] In the formulation, the ratio of the PEG-DSPE molecules to the protein of interest is: (1) the ratio of the number of PEG-DSPE molecules to the α-helices of the protein of interest is 2 to 25:1, preferably 3 to 20:1, more preferably 4 to 15:1, or (2) The molar ratio of the PEG-DSPE molecule to the target protein is 8 to 100:1, preferably 10 to 80:1, more preferably 15 to 50:1, and most preferably 20 to 30:1.

[0059] The PEG-DSPE has a purity of 95% or more, the PEG chain in the PEG-DSPE has a polydispersity of 1.1 or less, and the length of the PEG chain in the PEG-DSPE molecule is preferably 1000 to 5000 Da, more preferably 1500 to 2500 Da, and most preferably 2000 Da (PEG2000-DSPE).

[0060] The present invention further relates to an IL2 / PP formulation which is a water-soluble formulation, said IL2 / PP formulation comprising: (1) IL2 protein having a concentration of more than 1 mg / ml, preferably 1 to 10 mg / ml; (2) polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), preferably the PEG chain length of the PEG-DSPE molecule is 2000 Da (PEG2000-DSPE), the PEG-DSPE has a purity of 95% or more, and the PEG chain of the PEG-DSPE has a polydispersity of 1.1 or less; and (3) Necessary pH adjusters / buffers The molar ratio of the PEG-DSPE to the IL2 protein is 8 to 100:1, preferably 10 to 80:1, more preferably 15 to 50:1, and most preferably 20 to 30:1. The IL2 protein was expressed in E. coli and purified and has the amino acid sequence shown in SEQ ID NO:1. The IL2 / PP preparation is preferably adjusted to a pH of 5.5 to 6.0 using a pH adjuster.

[0061] Optionally, the IL2 / PP formulation may be further lyophilized to provide an IL2 / PP lyophilized formulation.

[0062] The present invention further relates to a method for preparing an IL2 / PP formulation, said method comprising the steps of: (1) preparing an aqueous solution having a pH of 3.0 to 4.0, preferably by diluting glacial acetic acid with deionized water to a concentration of 10 mM and adjusting the pH to 3.0 to 3.5; (2) weighing the amount of IL2 protein dry powder, adding the aqueous solution having a pH of 3.0 to 3.5 to the IL2 protein dry powder, and thoroughly mixing to obtain an IL2 suspension; (3) weighing out an amount of PEG-DSPE dry powder corresponding to the amount of IL2 protein in step (2), and adding the weighed amount of PEG-DSPE dry powder to the IL2 suspension and mixing thoroughly; (4) heating in a water bath at 40 to 60°C for 10 to 30 minutes; and (5) A step of allowing the solution obtained in step (4) to stand at room temperature, adjusting the pH of the solution to 5.5 to 6.0, and allowing the solution to stand for an additional 10 to 60 minutes to obtain the IL2 / PP formulation. Preferably, the PEG chain length of the PEG-DSPE molecule is 2000 Da (PEG2000-DSPE), the PEG-DSPE has a length of 95% or more, and the PEG chains of the PEG-DSPE have a dispersity of 1.1 or less.

[0063] The method of preparation is optionally (6) diluting the IL2 protein solution obtained in step (5) with the same buffer to an appropriate concentration or concentrating the IL2 protein solution to an appropriate concentration by ultrafiltration; and (7) filtering and sterilizing the preparation Includes.

[0064] The IL2 / PP formulation prepared by the above method has an IL2 protein recovery rate of 90% or more.

[0065] The present invention further comprises: (1) Manufacture of antitumor drugs, (2) The manufacture of a preparation for activating a cellular immune response; or (3) Preparation of an antitumor combination preparation, preferably an antitumor combination preparation further comprising an antibody or antibody construct, a cytotoxic agent, an immunotherapeutic agent or a cellular therapeutic agent. With regard to the use of an IL2 / PP formulation in the treatment of cancer, said agent or formulation has lower toxicity and / or side effects compared to an IL2 formulation in the form of a DLS formulation. The activation of the cellular immune response refers to in vivo or in vitro activation of the cellular immune response. (1) upregulation of CD8+ T cell proliferation, and / or (2) inducing differentiation of T cells into CD8+ T cells; and / or (3) inhibition of T cell differentiation into Tregs, and / or (4) Increase in the proportion of CD8+ T cells or Tregs Preferably, the activation of the cellular immune response refers to The IL2 / PP formulation is compared to an IL2 formulation in the form of a DLS formulation (positive reference formulation) or a formulation in the form of a free protein solution in vivo or in vitro. (1) upregulation of CD8+ T cell proliferation, and / or (2) inducing differentiation of T cells into CD8+ T cells; and / or (3) inhibition of T cell differentiation into Tregs, and / or (4) Increase in the proportion of CD8+ T cells or Tregs It is possible to administer the agent or preparation by subcutaneous injection, preferably.

[0066] The present invention further relates to a G-CSF / PP formulation which is a water-soluble formulation, said G-CSF / PP formulation comprising: (1) G-CSF protein having a concentration of more than 1 mg / ml, preferably 1 to 10 mg / ml; (2) polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), preferably the PEG chain length of the PEG-DSPE molecule is 2000 Da (PEG2000-DSPE), the PEG-DSPE has a purity of 95% or more, and the PEG-DSPE chain has a polydispersity of 1.1 or less; and (3) Necessary pH adjusters / buffers The G-CSF protein was expressed in E. coli and purified and has the amino acid sequence shown in SEQ ID NO:2. The G-CSF / PP formulation is preferably adjusted to a pH of 5.5 to 6.0 using a pH adjuster. Optionally, the G-CSF / PP formulation may be further lyophilized to provide a G-CSF / PP lyophilized formulation.

[0067] The present invention further relates to a method for preparing a G-CSF / PP formulation, said method comprising the steps of: (1) preparing an aqueous solution having a pH of 3.0 to 5.5, preferably by diluting glacial acetic acid with deionized water to a concentration of 10 mM and adjusting the pH to 3.0 to 3.5; (2) weighing the amount of G-CSF protein dry powder, adding the aqueous solution having a pH of 3.0 to 3.5 to the G-CSF protein dry powder, and thoroughly mixing to obtain a G-CSF suspension; (3) weighing out an amount of PEG-DSPE dry powder corresponding to the amount of G-CSF protein in step (2), and adding the weighed amount of PEG-DSPE dry powder to the G-CSF suspension and mixing thoroughly; (4) heating in a water bath at 40 to 60°C for 10 to 30 minutes; and (5) A process of allowing the solution obtained in step (4) to stand at room temperature, adjusting the pH of the solution to 5.5 to 6.0, and allowing the solution to stand for an additional 10 to 60 minutes to obtain a G-CSF / PP formulation. Preferably, the PEG chain length of the PEG-DSPE molecule is 2000 Da (PEG2000-DSPE), the PEG-DSPE has a purity of 95% or more, and the PEG chain of the PEG-DSPE has a polydispersity of 1.1 or less.

[0068] The preparation method is optionally (6) diluting the G-CSF protein from the solution obtained in step (5) with the same buffer to an appropriate concentration or concentrating the G-CSF protein to an appropriate concentration by ultrafiltration; and (7) filtering and sterilizing the preparation. Includes.

[0069] The G-CSF / PP formulation prepared by the above method has a G-CSF protein recovery rate of 90% or more.

[0070] The present invention further relates to the use of a G-CSF / PP preparation in the manufacture of a medicament for treating febrile neutropenia (FN), preferably caused by radiotherapy or chemotherapy of a tumor or organ transplant.

[0071] Preferably, the agent is administered by subcutaneous injection. Effect of the Invention

[0072] The beneficial effects of the present invention are as follows: (1) We developed a novel protein conjugate by utilizing highly specific binding between PEG-PE and cytokine protein molecules, and by remodeling the protein structure and changing its function through the interaction between PEG-PE (PP) and cytokine, we achieved selective binding of cytokines and their receptor de1 and specific activation of downstream cytokines in effector cells. (2) The use of a formulation for subcutaneous administration improves the administration route of cytokine drugs, extends the half-life of the drugs, reduces toxic side effects, and improves their clinical suitability. (3) To provide a new formulation that significantly improves the solubility and stability of cytokine drugs and extends their half-life in vivo. In short, the present invention provides a novel and feasible clinical pharmaceutical solution for cytokine medicines, which overcomes the main drawbacks in clinical use, achieves better therapeutic effects, and greatly expands the scope of clinical application of cytokine medicines. [Brief description of the drawings]

[0073] [Figure 1] Figure 1 is a schematic diagram showing the binding of IL-2 to the low-, intermediate-, and high-affinity IL-2 receptors

[13] . [Diagram 2] FIG. 2(a) is a photograph of the finished IL2 / PP formulation, and FIG. 2(b) is a photograph of the finished G-CSF / PP formulation. [Diagram 3] Figure 3(a) shows the bioactivity of the IL2 / PP formulation and the positive reference DLS as measured by the CTLL-2 / MTT assay, and Figure 3(b) shows the bioactivity of the G-CSF / PP formulation as measured by the NFS-60 cell proliferation assay. [Figure 4]Figure 4 shows the interaction between IL-2 and PEG2000-DSPE and SDS as measured by isothermal titration calorimetry (ITC). Figure 4(a) shows the results of a titration of IL-2 and SDS performed at an experimental temperature of 25°C. Figure 4(b) shows the results of a titration of IL-2 and PEG2000-DSPE performed at an experimental temperature of 25°C. Figure 4(c) shows the results of a titration of IL-2 and PEG2000-DSPE performed at an experimental temperature of 50°C. [Diagram 5] Figure 5 shows the results of evaluating and comparing the inhibition of tumor progression by IL2 / PP formulations and positive reference DLS in a CT26-Balb / C mouse tumor-bearing model (peritoneal + subcutaneous administration). Figure 5(a) shows the change in tumor volume (mean ± SEM) of mice in each group during the entire evaluation period. Figure 5(b) shows the change in body weight (mean ± SEM) of mice in each group from the start of administration to one week after the end of administration. [Figure 6] Figure 6 shows the results of observing and comparing the inhibition of tumor progression by IL2 / PP formulations and positive reference DLS in a CT26-Balb / C mouse tumor-bearing model (tail vein and subcutaneous administration). Figure 6(a) shows the change in tumor volume (mean ± standard error) of mice in each group during the entire evaluation period. Figure 6(b) shows the change in mouse weight (mean ± standard error) of mice in each group during the entire evaluation period. Figure 6(c) shows the difference in survival rate of mice in each group (in addition to mice that actually died, mice with tumor volumes exceeding 2000 mm3 were also considered dead). [Figure 7]Figure 7 shows the comparison of toxic side effects induced by the continuous administration of IL2 / PP formulation and positive reference DLS in CT26-Balb / C mouse tumor-bearing model (tail vein and subcutaneous administration). Figure 7(a) shows that after the continuous subcutaneous administration of positive reference DLS, visible epidermal ulcers appeared at the injection site and the wounds were crusted (pin-sized crusted ulcers). Figure 7(b) shows that after the continuous subcutaneous administration of IL2 / PP formulation, no obvious adverse effects were observed and the injection site was completely normal. Figure 7C(c) shows that after the continuous tail vein administration of positive reference DLS, the mouse's entire tail developed severe ulcers, congestion and swelling. This effect made it difficult to complete the injection and recovery was very slow after the administration was stopped. Figure 7(d) shows that after the continuous tail vein administration of IL2 / PP formulation, no obvious adverse effects were observed and the entire tail was completely normal. Thus, the continuous injection could be completed. FIG. 7(e) shows that the tails of mice suffering from severe toxic side effects in the tail vein administration group of the positive reference DLS gradually became necrotic after hematoma rupture and were eventually severed. [Figure 8] FIG. 8 shows the results of evaluating and comparing the inhibition of tumor progression by the IL2 / PP formulation and the positive reference DLS in a B16F10-C57BL / 6 mouse tumor-bearing model (tail vein and subcutaneous administration). FIG. 8(a) shows the change in tumor volume (mean ± SEM) of four groups of mice receiving subcutaneous injections over the entire evaluation period. FIG. 8(b) shows the change in tumor volume (mean ± SEM) of two groups of mice receiving tail vein injections over the entire evaluation period. FIG. 8(c) shows the change in tumor volume (mean ± SEM) of three groups of mice receiving DLS formulations over the entire evaluation period. FIG. 8(d) shows the change in tumor volume (mean ± SEM) of three groups of mice receiving IL2 / PP formulations over the entire evaluation period. FIG. 8(e) shows the change in body weight (mean ± SEM) of each group of mice over the entire evaluation period. [Figure 9]Figure 9 shows the comparison of toxic side effects induced by continuous administration of IL2 / PP formulation and positive reference DLS in B16F10-C57NL / 6 mouse tumor-bearing model (tail vein and subcutaneous administration). Figure 9(a) shows that after continuous subcutaneous administration of positive reference DLS, visible epidermal ulcers appeared at the injection site, scabs formed on the wound, and the wound gradually healed 4-6 days after administration was stopped. Figure 9(b) shows that after continuous subcutaneous administration of IL2 / PP formulation, no obvious adverse effects appeared and the injection site was completely normal. Figure 9(c) shows that after continuous tail vein administration of positive reference DLS, severe congestion and swelling appeared in the entire tail of the mouse, ulcers appeared at the injection site, this effect made it difficult to complete the injection, and the wound gradually healed about 1 week after administration was stopped. Figure 9(d) shows that after continuous tail vein administration of IL2 / PP formulation, no obvious adverse effects appeared and the entire tail was completely normal. Figure 9(e) shows that the ulceration and scab formation sites of mice suffering from severe toxic side effects in the subcutaneous administration group of the positive reference DLS did not heal completely two weeks after administration was stopped, and Figure 9(f) shows that the tumor formation sites in the tails of mice suffering from severe toxic side effects in the tail vein administration group of the positive reference DLS did not heal two weeks after administration was stopped. [Figure 10] Figure 10 shows the results of evaluating and comparing the selective effects of IL2 / PP formulations and positive reference DLS on different T cell subsets in a CT26-Balb / C mouse tumor-bearing model. Figure 10(a) shows the percentages of activated immunosuppressive Tregs (CD45+CD4+Foxp3+) and CD8+ effector T cells (CD45+CD3+CD8+) in the spleen of tumor-bearing mice induced by IL2 / PP formulations and positive reference DLS, as well as the ratios of the two cells (mean ± standard error) after successive intraperitoneal administration. Figure 10(b) shows the percentages of activated immunosuppressive Tregs (CD45+CD4+Foxp3+) and CD8+ effector T cells (CD45+CD3+CD8+) in the tumor tissue of tumor-bearing mice induced by IL2 / PP formulations and positive reference DLS, as well as the ratios of the two cells (mean ± standard error) after successive intraperitoneal administration. [Figure 11] Figure 11 shows the interaction between G-CSF and PEG2000-DSPE measured by isothermal titration calorimetry (ITC). Figure 11(a) shows the titration results of G-CSF and PEG2000-DSPE carried out at an experimental temperature of 25°C. Figure 11(b) shows the titration results of G-CSF and PEG2000-DSPE carried out at an experimental temperature of 50°C. [Figure 12] Figure 12 shows the results of a pharmacodynamic study of G-CSF / PP in tumor-bearing mice, which shows that G-CSF / PP had a similar leukocytosis effect as bulk G-CSF protein, significantly reduced the number of MDSCs and Tregs, and effectively reduced the immune-related side effects of G-CSF. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0074] material and method

[0075] The IL-2 and G-CSF proteins were expressed in E. coli and purified and have the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. TIFF2025504570000003.tif39170

[0076] PEG 2000 -DSPE (polyethylene glycol 2000-distearoylphosphatidylethanolamine) was purchased from Lipoid.

[0077] Positive Reference Preparation

[0078] As a positive reference formulation, DLS formulation was used, which is an IL2 protein formulation containing SDS (sodium dodecyl sulfate) as a co-solvent, the composition and preparation of which are prior art and well known to those skilled in the art (see, for example, EP 1688146 B1).

[0079] cell line

[0080] CTLL-2 (ATCC No. TIB-214): A cytotoxic T lymphocyte derived from C57BL / 6 mice that grows and proliferates normally in specific concentrations of IL-2 and is commonly used as a reporter cell to assess the biological activity of IL-2 and its analogues.

[0081] Mouse colon cancer cells CT26 (ATCC number CRL-2638): derived from Balb / c mice, these are undifferentiated mouse colon cancer cells induced by N-nitroso-N-methylurethane (NNMU), which show fibroblast-like morphology, grow adherently, and are commonly used in the validation of immunotherapy protocols or in the investigation of host immune responses.

[0082] Mouse skin melanoma cells B16F10 (ATCC number CRL-6475): derived from the skin of C57BL / 6J strain melanoma tumor mice, it is a subline of B16F0, shows fibroblast-like morphology, grows adherently, and is commonly used for constructing mouse melanoma animal models.

[0083] Cell line culture method

[0084] Culture of mouse IL-2-dependent cytotoxic T lymphocytes CTLL-2 in RPMI-1640 medium supplemented with 10% fetal bovine serum, 400-800 IU / ml rhIL-2, and 50 μM β-mercaptoethanol was performed as follows. Frozen cells were removed from the liquid nitrogen storage tank and immediately placed in a 37°C water bath for rapid thawing. The cell suspension was then transferred to a centrifuge tube containing 10 ml of medium and centrifuged at 1500×g for 5 min to remove the supernatant. After resuspension in fresh medium, the cells were transferred to a cell culture flask containing 10-15 ml of medium and cultured in a humidified atmosphere of 5% CO2 in an incubator at 37°C. The cells were observed daily or every other day and passaged by replacing the medium with fresh medium each time.

[0085] The CT26 cell line used in the mouse colon cancer model was cultured in RPMI-1640 medium (containing 10% fetal bovine serum) as follows. Frozen cells were removed from a liquid nitrogen storage tank and immediately placed in a 37°C water bath for rapid thawing. The cell suspension was then transferred to a centrifuge tube containing 10 ml of medium and centrifuged at 700×g for 5 min to remove the supernatant. After resuspension in fresh medium, the cells were transferred to a cell culture flask containing 10–15 ml of medium and cultured in a 37°C incubator in a humidified atmosphere of 5% CO2. The cells were observed daily or every other day and passaged by replacing the medium with fresh medium each time.

[0086] The mouse melanoma B16F10 cell line was cultured in DMEM medium (containing 10% fetal bovine serum) as follows. Frozen cells were removed from a liquid nitrogen storage tank and immediately placed in a 37°C water bath for rapid thawing. The cell suspension was then transferred to a centrifuge tube containing 10 ml of medium and centrifuged at 700×g for 5 min to remove the supernatant. After resuspension in fresh medium, the cells were transferred to a cell culture flask containing 10–15 ml of medium and cultured in a 37°C incubator in a humidified atmosphere of 5% CO2. The cells were observed daily or every other day and passaged by replacing the medium with fresh medium each time.

[0087] Experimental animals

[0088] BALB / c: grade SPF, 6-8 weeks old, female, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. in accordance with the Beijing Laboratory Animal Quality Standards.

[0089] C57BL / 6N: grade SPF, 6-8 weeks old, female, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. in accordance with the Beijing Laboratory Animal Quality Standards. EXAMPLES

[0090] [Example 1] Preparation of IL2 / PP and G-CSF / PP preparations

[0091] 1. Preparation of IL2 / PP formulation

[0092] (1) Glacial acetic acid was diluted with deionized water to a concentration of 10 mM and pH 3.0-3.5, filtered through a 0.22 μm filter membrane, and used as a solvent.

[0093] (2) The lyophilized powder of IL-2 protein was weighed, and 10 mM diluted acetic acid solution was added thereto and thoroughly mixed to obtain an IL-2 suspension.

[0094] (3) The molar ratio of the two is 15 to 30:1 (PEG 2000 - PEG in an amount corresponding to the amount of IL-2 in step (2) so that the total amount of IL-2 is 100%. 2000 -DSPE powder was weighed and added to the IL-2 suspension and mixed thoroughly.

[0095] (4) The mixture was heated in a water bath at 40 to 60° C. for 10 to 30 minutes.

[0096] (5) The sample was allowed to stand to reach room temperature.

[0097] (6) The sample was adjusted to pH 5.5 to 6.0 by adding 1 M NaOH solution, and then allowed to stand for an additional 10 to 60 minutes.

[0098] (7) The obtained sample was diluted to an appropriate concentration with a solvent having the same salt concentration and pH.

[0099] (8) The sample was filtered through a 0.22 μm filter membrane to obtain an IL2 / PP formulation.

[0100] The protein concentration of the filtered product was detected by BCA and stored in a sealed container at 4° C. The appearance of the obtained IL2 / PP formulation was a colorless, transparent solution containing no visible insoluble matter, as shown in Figure 2(a).

[0101] 2. Preparation of G-CSF / PP formulation

[0102] (1) Glacial acetic acid was diluted with deionized water to a concentration of 10 mM and pH 3.0-3.5, filtered through a 0.22 μm filter membrane, and used as a solvent.

[0103] (2) The freeze-dried powder of G-CSF protein was weighed, and 10 mM diluted acetic acid solution was added thereto and mixed thoroughly to obtain a G-CSF suspension.

[0104] (3) The molar ratio of the two is 15 to 30:1 (PEG 2000 According to the amount of G-CSF in step (2), a corresponding amount of PEG2000-DSPE powder was weighed out and added to the G-CSF suspension, and mixed thoroughly to obtain a total weight of 1000 g of PEG2000-DSPE (1:1000:IL-2).

[0105] (4) The mixture was heated in a water bath at 40 to 60° C. for 10 to 30 minutes.

[0106] (5) The sample was allowed to stand at room temperature.

[0107] (6) The pH of the sample was adjusted to 5.5 to 6.0 by adding 1 M NaOH solution, and the sample was allowed to stand for an additional 10 to 60 minutes.

[0108] (7) The obtained sample was diluted to an appropriate concentration with a solvent having the same salt concentration and pH.

[0109] (8) The sample was filtered through a 0.22 μm filter membrane to obtain a G-CSF / PP formulation.

[0110] The protein concentration of the filtered product was detected by BCA and stored in a sealed container at 4° C. The appearance of the obtained G-CSF / PP formulation was a colorless, transparent solution containing no visible insoluble matter, as shown in FIG. 2(b).

[0111] [Example 2] Measurement of biological activity of IL2 / PP and G-CSF / PP preparations by cell assay

[0112] 1. Measurement of biological activity of IL2 / PP preparations by CTLL-2 / MTT cell proliferation assay

[0113] reagent

[0114] (1) Basal medium: RPMI1640 + 10% FBS + 100 U / ml penicillin + 100 U / ml streptomycin

[0115] (2) Complete medium: basal medium supplemented with IL2 at a final concentration of 400-800 IU / ml

[0116] (3) PBS: 1 L contains 8 g of NaCl, 0.20 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4. Sterilized by high-pressure steam at 121°C for 15 minutes.

[0117] (4) Methylthiazolyl tetrazolium solution (MTT): concentration 5 mg / ml, sterilized by filtration through a 0.22 μm filter membrane, stored in the dark at 4°C

[0118] (5) Dissolving solution: 15% sodium dodecyl sulfate solution (SDS)

[0119] Experimental protocol

[0120] (1) Sample preparation: A positive reference DLS with known biological activity was used as the standard, and the IL2 / PP formulation obtained in Example 1 was used as the test sample. The two formulations were diluted with basal medium to an appropriate concentration, for example, 400 to 800 IU / ml.

[0121] (2) Preparation of CTLL-2 cell suspension: CTLL-2 cells cultured in complete medium and effectively proliferated after thawing were washed three times with 1640 medium containing no other components, then resuspended in basal medium to obtain an appropriate concentration, e.g., 2–6 × 10 5 A cell suspension of 1 / ml was prepared.

[0122] (3) Addition of samples: A flat-bottom 96-well plate was used. The test samples and standards were serially diluted with basal medium to obtain samples with at least eight concentrations that differed by more than two-fold. Each concentration of sample was added to 2-3 wells at 50 μl per well.

[0123] (4) Cell seeding: 50 μl of cell suspension was added to each well, mixed thoroughly, and placed in a cell incubator at 37°C with 5% CO2 for 18-24 hours.

[0124] (5) 20 μl of MTT solution was added to each well and incubated in an incubator for 4 to 6 hours.

[0125] (6) 150 μl of 15% SDS solution was added to each well, mixed thoroughly, and incubated in an incubator for 18 to 24 hours.

[0126] (7) The absorbance at 570 nm was measured using a microplate reader, and the IL-2 activity of the test sample was calculated according to the following method.

[0127] Specific activity calculation: Biological activity of test sample (IU / ml) = Pr x (Ds x Es) / (Dr x Er) Pr: standard biological activity, IU / ml Ds: Pre-dilution factor of the test sample Dr: Predilution factor of standard Es: Dilution factor of the test sample corresponding to the median effective dose of the standard Er: Dilution factor of the median standard effective dose

[0128] The experimental results (Figure 3(a)) showed the following results. (1) The biological activity of the IL-2 protein molecule in the IL2 / PP formulation was essentially the same as that of the protein molecule in the positive reference (DLS) and was slightly higher than that of the DLS. (2) Half-maximal inhibitory concentration (IC 50 ) is approximately 0.00723 mM, and the IC 50 This was significantly lower than (0.01147nM).

[0129] 2. Measurement of biological activity of G-CSF / PP formulations using NFS-60 cell proliferation assay

[0130] reagent

[0131] (1) Basal medium: RPMI1640 + 10% FBS + 100 U / ml penicillin + 100 U / ml streptomycin

[0132] (2) Complete medium: basal medium supplemented with 5.5 mM β-mercaptoethanol and 62 ng / mL M-CSF

[0133] (3) PBS: 1 L contains 8 g of NaCl, 0.20 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4. Sterilized by high-pressure steam at 121°C for 15 minutes.

[0134] (4) Methylthiazolyl tetrazolium solution (MTT): concentration 5 mg / ml, sterilized by filtration through a 0.22 μm filter membrane, stored in the dark at 4°C

[0135] (5) Lysis solution: 10% Triton X-100, 2.8% concentrated hydrochloric acid, and 87.2% isopropanol.

[0136] Experimental protocol

[0137] (1) Sample preparation: A positive reference SH protein bulk with known biological activity was used as the standard, and the G-CSF / PP formulation obtained in Example 1 was used as the test sample. The two formulations were diluted with basal medium to an appropriate concentration, for example, 0 to 120 IU / ml.

[0138] (2) Preparation of NFS-60 cell suspension: NFS-60 cells cultured in complete medium and effectively proliferated after thawing were washed three times with basal medium, then resuspended in basal medium to a concentration of 2 × 10 5 A cell suspension of 1 / ml was prepared.

[0139] (3) Cell seeding: 50 μL of cell suspension (1 × 10 4 Cells were seeded into flat-bottom 96-well plates by adding 100 μl of 10 ...

[0140] (4) Sample addition: A flat-bottom 96-well plate was used. Test samples and standards were serially diluted with basal medium to obtain at least 8 concentrations of samples that differed from each other by more than 2-fold. Each concentration of sample was added to 2 wells at 50 μl per well. The cells were incubated in an incubator for 48 hours.

[0141] (5) 20 μl of MTT solution was added to each well and incubated in an incubator for 4 to 6 hours.

[0142] (6) 100μl of the dissolving solution was added to each well and mixed thoroughly. Then, the absorbance at 570nm was measured using a microplate reader with a reference wavelength of 630nm, and the measurement results were recorded. The test data was processed by a computer program or a four-parameter regression calculation method, and the G-CSF activity of the test sample was calculated according to the following method.

[0143] Specific activity calculation: Biological activity of test sample (IU / ml) = Pr x (Ds x Es) / (Dr x Er) Pr: standard biological activity, IU / ml Ds: Pre-dilution factor of the test sample Dr: Predilution factor of standard Es: Dilution factor of the test sample corresponding to the median effective dose of the standard Er: Dilution factor of the median standard effective dose

[0144] The experimental results (Figure 3(a)) showed the following results. (1) The biological activity of the G-CSF protein molecule in the G-CSF / PP formulation was substantially the same as that of the protein molecule in the positive reference (SH). (2) Half-maximal effective concentration (EC 50 ) was approximately 146.0 IU / mL, and the EC 50 , 149.4 IU / mL, which was not significantly different.

[0145] [Example 3] Measuring the affinity of the interaction between a protein of interest and PEG2000-DSPE

[0146] 1. Measurement of the affinity of interaction between IL2 and PEG2000-DSPE and SDS

[0147] (1) Sample preparation A.PEG 2000 -DSPE solution: Dissolved in 10 mM sodium acetate buffer (pH = 5.5), concentration 0.5 mM B. SDS solution: Dissolved in 7.69 mM sodium phosphate buffer (pH = 7.6), concentration 1.73 mM C. IL-2 solution: Dissolved in 10 mM sodium acetate buffer (pH = 5.5), concentration 0.005 mM D. IL-2 solution: Dissolved in 7.69 mM sodium phosphate buffer (pH = 7.0), concentration 0.0164 mM

[0148] (2) Parameter settings and experimental conditions

[0149] A Malvern Panalytical MicroCal ITC 200 isothermal titration calorimeter and its supporting operating software were used, see table below for specific parameter settings. [Table 1]

[0150] (3) To measure the interaction between PEG2000-PE and IL-2, 40 μl of sample A was added to the syringe, and 250 μl of sample C was added to the sample cell. To measure the interaction between SDS and IL-2, 40 μl of sample B was added to the syringe, and 250 μl of sample D was added to the cell. After the corresponding sample concentrations and reaction temperatures were set in the software, the titration was started. After the reaction was completed, the measured heat changes were integrated, and reaction curve fitting (Figure 4) was performed to calculate various parameters of the interaction between the two samples. The K value measured by ITC was M -1The binding constant is Kd, which has units K, and its reciprocal is the dissociation constant Kd of two substances, which has units M. The smaller the Kd value, the stronger the affinity between the two substances.

[0151] (4) As a result, the interaction between IL-2 protein and SDS was very weak (the calculated equilibrium dissociation constant Kd was approximately 3.73 × 10 -5 On the other hand, IL-2 and PEG2000-DSPE spontaneously interacted under these conditions (Kd was approximately 1.17 × 10 -7 M), the affinity between them was about two orders of magnitude higher than that between IL-2 protein and SDS. IL-2 and PEG 2000 The affinity between the α-DSPE and the α-DSPE significantly increased with increasing reaction temperature (from 25°C to 50°C) (Kd was approximately 1.28 × 10 -8 M) From these results, it was found that under the preparation conditions described in Example 1, IL-2 protein and PEG 2000 -DSPE was shown to be much stronger than the interaction between IL-2 protein and the positive reference SDS.

[0152] 2. Measurement of the affinity of interaction between G-CSF and PEG2000-DSPE

[0153] (1) Sample preparation A. PEG2000-DSPE solution: dissolved in 10 mM sodium acetate buffer (pH = 3.5), concentration 1.24 mM BG-CSF solution: dissolved in 10 mM sodium acetate buffer (pH = 3.5), concentration 0.0062 mM

[0154] (2) Parameter settings and experimental conditions

[0155] A Malvern Panalytical MicroCal ITC 200 isothermal titration calorimeter and its supporting operating software were used, see table below for specific parameter settings. [Table 2]

[0156] (3)PEG 2000 To measure the interaction between β-PE and G-CSF, 40 μl of sample A was added to the syringe and 250 μl of sample B was added to the sample cell. The titration was started after the corresponding sample concentrations and reaction temperatures were set in the software. After the reaction was completed, the measured heat changes were integrated and reaction curve fitting (Figure 11) was performed to calculate various parameters of the interaction between the two samples. The K values ​​measured by ITC were expressed as M -1 The binding constant is Kd, which has units K, and its reciprocal is the dissociation constant Kd of two substances, which has units M. The smaller the Kd value, the stronger the affinity between the two substances.

[0157] (4) As a result, G-CSF and PEG2000-DSPE had a very high affinity under these conditions (Kd of approximately 2.78 × 10 -7 The titration curves showed a typical double binding site pattern, indicating the presence of two or more high affinity interaction sites or regions between G-CSF and PEG2000-DSPE. As the reaction temperature increased (from 25°C to 50°C), the 2000 The affinity between DSPE and DSPE was also significantly improved (Kd was approximately 4.72 × 10 -8 M) From these results, it was found that the G-CSF protein and PEG were 2000 It was shown that a very strong interaction can occur spontaneously between -DSPE and .

[0158] [Example 4] Measurement of functional activity of IL2 / PP preparations in CT26-Balb / C mouse tumor-bearing model

[0159] Experimental protocol (1): Intraperitoneal and subcutaneous administration

[0160] (1) CT26 cells thawed from liquid nitrogen were cultured in RPMI-1640 medium (containing 10% fetal bovine serum) for 5-7 days and passaged at least three times before use to ensure the cells were in a normal state. On the day of the experiment, the cells should be grown to 80-90% confluence and in good condition. The cells were digested with 0.05% trypsin, neutralized with 10 volumes of fresh medium, centrifuged at 700 × g to remove the supernatant, resuspended in an appropriate volume of sterile PBS, and counted. The cell suspension was diluted with sterile PBS to a concentration of 5 × 10 6 / ml and placed on ice for later use.

[0161] (2) Using a 1 ml sterile syringe, the CT26 cell suspension was subcutaneously inoculated into the right back of 6-8 week-old female wild-type Balb / C mice. Each mouse was inoculated with 0.1 ml of the CT26 cell suspension, i.e., 5 × 10 5 of CT26 cells were injected.

[0162] (3) Ten days after inoculation, the volume of the mouse at the inoculation site was 120 ± 10 mm 3 The mice were grouped according to tumor volume and shape so that the distribution and mean tumor volume of each group was substantially the same. A total of five experimental groups were set up, with eight mice in each group. A. Blank CTR B. Positive reference DLS, intraperitoneal administration group (DLS ip) C. Positive reference DLS, subcutaneous administration group (DLS sc) D. IL2 / PP formulation, intraperitoneal administration group (IL2 / PP ip) E. IL2 / PP formulation, subcutaneous administration group (IL2 / PP sc)

[0163] (4) Starting from the 11th day after inoculation, for 8 days, 0.1 ml of the corresponding preparation (the actual dose of IL-2 per injection was 10 μg) was injected into each mouse according to the administration route of the group to which the mouse belonged, twice a day for a total of 16 times (the blank group did not receive any treatment).

[0164] (5) The IL2 / PP formulation was prepared by the method described in Example 1. The lyophilized powder of the positive reference DLS was reconstituted with water for injection to prepare the injection. Both were quantified by BCA and adjusted to the appropriate concentration.

[0165] (6) The condition of the mice in each group was continuously observed after grouping. The tumor volume and body weight changes of the tumor-bearing mice were measured three times a week, and deaths were recorded.

[0166] (7) Experimental results (Figure 5) showed that treatment with this dosing regimen resulted in the following: 1 When administered by intraperitoneal injection, both the IL2 / PP and DLS formulations significantly inhibited tumor growth and controlled smaller tumor volumes compared with the blank group, but there was no significant difference between the two groups. 2 When administered by subcutaneous injection, both IL2 / PP and DLS formulations significantly inhibited tumor growth compared with the blank group, and there was also a significant difference between the two groups. That is, the inhibitory effect in the IL2 / PP formulation group was significantly better than that in the DLS formulation group, and reached the same inhibition level as the intraperitoneal administration group. That is, it was suggested that the IL2 / PP formulation had better activity and efficacy than the positive reference DLS. 3 Regarding changes in body weight, after the start of administration, the mice in the intraperitoneal administration group showed a rapid and significant weight loss, as well as a decrease in activity and body temperature, making it difficult for them to survive, but gradually recovered after the end of administration. On the other hand, the weight of the mice in the subcutaneous administration group continued to increase, and the condition of the mice remained stable. These results suggest that although intraperitoneal administration has the advantages of fast absorption and good efficacy, it has strong toxic side effects and seriously affects the survival of the mice, while subcutaneous administration is relatively safe.

[0167] In other words, the IL2 / PP formulation is able to achieve both therapeutic efficacy and safety when administered subcutaneously, and is generally superior to the positive reference DLS of the prior art.

[0168] Experimental protocol (2): Tail vein and subcutaneous administration

[0169] (1) CT26 cells thawed from liquid nitrogen were cultured in RPMI-1640 medium (containing 10% fetal bovine serum) for 5-7 days and passaged at least three times before use to ensure the cells were in a normal state. On the day of the experiment, the cells should be grown to 80-90% confluence and in good condition. The cells were digested with 0.05% trypsin, neutralized with 10 volumes of fresh medium, centrifuged at 700 × g to remove the supernatant, resuspended in an appropriate volume of sterile PBS, and counted. The cell suspension was diluted with sterile PBS to a concentration of 5 × 10 6 / ml and placed on ice for later use.

[0170] (2) Using a 1 ml sterile syringe, the CT26 cell suspension was subcutaneously inoculated into the right back of 6-8 week-old female wild-type Balb / C mice. Each mouse was inoculated with 0.1 ml of the CT26 cell suspension, i.e., 5 × 10 5 of CT26 cells were injected into the wells of the oocyte (the cell suspension must be mixed thoroughly before each pipetting).

[0171] (3) Eight days after inoculation, the volume of the mouse at the inoculation site was 150 ± 10 mm 3 The mice were grouped according to the tumor volume and shape so that the distribution and average tumor volume of each group was substantially the same. A total of five experimental groups were set up, with five mice in each group. A. Blank CTR B. Positive reference DLS, tail vein administration group (DLS iv) C. Positive reference DLS, subcutaneous administration group (DLS sc) D. IL2 / PP formulation, tail vein administration group (IL2 / PP iv) E. IL2 / PP formulation, subcutaneous administration group (IL2 / PP sc)

[0172] (4) Starting from the 9th day after inoculation, each mouse was injected once a day with 0.1 ml of the corresponding preparation (the actual dose of IL-2 was 16 μg) according to the administration route of the group to which the mouse belonged (the blank group did not receive any treatment).

[0173] (5) The IL2 / PP formulation was prepared by the method described in Example 1. The lyophilized powder of the positive reference DLS was reconstituted with water for injection to prepare the injection. Both were quantified by BCA and adjusted to the appropriate concentration.

[0174] (6) The condition of the mice in each group was continuously observed after grouping. The tumor volume and body weight changes of the tumor-bearing mice were measured three times a week, and deaths were recorded. In addition, toxic side effects of the mice in each group were observed during the administration period.

[0175] (7) Experimental results (Figures 6 and 7) showed that treatment with this dosing regimen resulted in the following: 1 When administered by subcutaneous injection, compared with the blank group, the IL2 / PP formulation significantly inhibited tumor growth and development and extended the survival of tumor-bearing mice, whereas the effect of the DLS formulation was much weaker and only slightly inhibited the growth of tumor volume. 2 When administered by tail vein injection, the IL2 / PP and DLS formulations had inhibitory effects, but they were not significant compared with the blank group. 3 When treated with the same dose of IL2 / PP formulation, subcutaneous administration had a significantly better effect than tail vein administration, and there was a significant difference between the two administration routes. 4 When treated with the same dose of DLS formulation, the effects of subcutaneous administration and tail vein administration were essentially the same, but no significant effect was obtained. 5 When administered continuously, either by subcutaneous or tail vein injection, the IL2 / PP formulation was much safer than the DLS formulation and caused few adverse effects, whereas the DLS formulation caused highly significant and severe side effects.

[0176] In summary, when administered by subcutaneous injection, the IL2 / PP formulation was significantly better than the prior art positive reference DLS in terms of enhanced antitumor efficacy, prolonged survival, and improved safety.

[0177] [Example 5] Measurement of functional activity of IL2 / PP preparations in B16F10-C57BL / 6 mouse tumor-bearing model (tail vein and subcutaneous administration)

[0178] (1) B16F10 cells thawed from liquid nitrogen were cultured in DMEM medium (containing 10% fetal bovine serum) for 5-7 days and passaged at least three times before use to ensure the cells were in a normal state. On the day of the experiment, the cells should be grown to 80-90% confluence and in good condition. The cells were digested with 0.05% trypsin, neutralized with 10 volumes of fresh medium, centrifuged at 700 × g to remove the supernatant, resuspended in sterile PBS, and counted. The cell suspension was diluted with sterile PBS to a concentration of 2 × 10 6 / ml and placed on ice for later use.

[0179] (2) Using a 1 ml sterile syringe, the B16F10 cell suspension was subcutaneously inoculated into the right back of 6-8 week-old female wild-type C57BL / 6 mice. Each mouse was inoculated with 0.1 ml of the B16F10 cell suspension, i.e., 2 × 10 5 of B16F10 cells were injected into the wells of the oocyte (the cell suspension must be mixed thoroughly before each pipetting).

[0180] (3) Ten days after inoculation, the volume of the mouse at the inoculation site was 40 ± 3 mm 3 The mice were grouped according to tumor volume and shape so that the distribution and mean tumor volume of each group was substantially the same. A total of eight experimental groups were set up, with five mice in each group. A. Blank CTR B. Positive reference DLS, tail vein administration group (DLS iv 16 μg) C. IL2 / PP formulation, tail vein administration group (IL2 / PP iv 16μg) D. Positive reference DLS, high dose subcutaneous administration group (DLS sc 16 μg) E. Positive reference DLS, low dose subcutaneous administration group (DLS sc 8 μg) F. IL2 / PP formulation, high-dose subcutaneous administration group (IL2 / PP sc 8μg) G. IL2 / PP formulation, low-dose subcutaneous administration group (IL2 / PP sc 4μg)

[0181] (4) Starting from the 8th day after inoculation, 0.1 ml of the formulations with the corresponding concentrations (actual doses of IL-2 were 4, 8, or 16 μg; see above for specific doses for each group) was administered to each mouse once a day for 8 days for the subcutaneous administration group and for 4 days for the tail vein administration group, depending on the administration route and dose for the administration group to which the mouse belonged (the blank group received no treatment).

[0182] (5) The IL2 / PP formulation was prepared according to the method described in Example 1. The lyophilized powder of the positive reference DLS was reconstituted with water for injection to prepare the injection. Both were quantified by BCA and adjusted to the appropriate concentration.

[0183] (6) The condition of the mice was continuously observed after grouping. The tumor volume and body weight changes of the tumor-bearing mice were measured three times a week, deaths were recorded, and toxic side effects in the mice of each group were observed during the treatment.

[0184] (7) Experimental results (Figures 8 and 9) showed that treatment with this dosing regimen resulted in the following: 1 When administered by subcutaneous injection, both the IL2 / PP and DLS formulations significantly inhibited tumor growth in a clear dose-dependent manner, i.e., the higher doses showed better therapeutic effects. The IL2 / PP formulation also showed significantly better tumor inhibition effects than the DLS formulation, showed the same therapeutic effect at a much lower dose than the DLS formulation, and showed better therapeutic effects at only 1 / 4 the dose of the DLS formulation. 2 When administered via tail vein injection, the IL2 / PP formulation showed significantly better tumor-inhibiting effect than the DLS formulation, but the tumor-inhibiting effect was not significant when compared with the blank group. 3 When treated with the IL2 / PP formulation, subcutaneous administration showed significantly better effects than tail vein administration, and treatment required a smaller dose, with significant differences between the two administration routes. 4 When treated with DLS formulation, subcutaneous administration also showed better effects than tail vein administration. 5 The IL2 / PP formulation slightly extended the survival of tumor-bearing mice compared with the blank group. Complete elimination of tumors was observed in mice in each of the two IL2 / PP subcutaneously administered groups, but not in the other group. 6 When administered continuously by either subcutaneous or tail vein injection, the IL2 / PP formulation was much safer than the DLS formulation and showed few adverse effects, whereas the DLS formulation showed highly significant and severe side effects.

[0185] In other words, when administered either by subcutaneous injection or tail vein injection, the IL2 / PP formulation was significantly superior to the prior art positive reference DLS in enhancing antitumor efficacy and improving safety. Especially when administered subcutaneously, the IL2 / PP formulation was able to exert the same therapeutic effect as the DLS formulation at a much lower dose.

[0186] [Example 6] Verification of the selective effect of IL2 / PP preparations on cells with different receptors in the CT26-Balb / C mouse tumor-bearing model

[0187] (1) CT26 cells thawed from liquid nitrogen were cultured in RPMI-1640 medium (containing 10% fetal bovine serum) for 5-7 days and passaged at least three times before use to ensure the cells were in a normal state. On the day of the experiment, the cells should be grown to 80-90% confluence and in good condition. The cells were digested with 0.05% trypsin, neutralized with 10 volumes of fresh medium, centrifuged at 700 × g to remove the supernatant, resuspended in sterile PBS, and counted. The cell suspension was diluted with sterile PBS to a concentration of 5 × 106 / ml and placed on ice for later use.

[0188] (2) Using a 1 ml sterile syringe, the CT26 cell suspension was subcutaneously inoculated into the right back of 6-8 week-old female wild-type Balb / C mice. Each mouse was inoculated with 0.1 ml of the CT26 cell suspension, i.e., 5 × 10 5 of CT26 cells were injected.

[0189] (3) Eight days after inoculation, the mouse had a mass of 80 ± 15 mm at the inoculation site. 3 The mice were grouped according to the tumor volume and shape so that the distribution and average tumor volume of each group was substantially the same. A total of two experimental groups were set up, with three mice in each group. A. Positive reference DLS, intraperitoneal administration group B. IL2 / PP formulation, intraperitoneal administration group

[0190] (4) Starting from the 8th day after inoculation, 0.1 ml of the corresponding preparation (actual dose of IL-2 was 10 μg) was intraperitoneally injected into each mouse once a day for 10 days according to the group to which the mouse belonged.

[0191] (5) The IL2 / PP formulation was prepared according to the method described in Example 1. The lyophilized powder of the positive reference DLS was reconstituted with water for injection to prepare the injection. Both were quantified by BCA and adjusted to the appropriate concentration.

[0192] (6) On day 19 after inoculation, the mice were euthanized by cervical dislocation, and the spleen and tumor tissues were dissected from the mice, and lymphocytes were extracted for detection.

[0193] For spleens, samples were thoroughly macerated on a 70 μm filter mesh, the filtered suspension was centrifuged at 500 × g for 5 min, the pellet was resuspended in ACK reagent to lyse red blood cells, the resulting cell suspension was filtered through a filter mesh and centrifuged again, and the cells were resuspended in FACS buffer, counted, and diluted to the appropriate concentration for fluorescent antibody staining.

[0194] For tumor tissue, the samples were cut into small pieces, 2 mg / ml collagenase IV solution was added, and digested in a thermostatic shaker at 37°C for 1-2 hours. The digested tissue samples were minced and filtered on a 70 μm filter mesh, the filtered suspension was centrifuged at 500 × g for 5 minutes, the pellet was resuspended in ACK reagent to lyse red blood cells, the resulting cell suspension was filtered through a filter mesh and centrifuged again, and the cells were resuspended in FACS buffer, counted, and diluted to the appropriate concentration for fluorescent antibody staining.

[0195] (7) Cell staining: 2×10 6 of cells were used for each sample and the antibody staining protocol was as follows: Regulatory Treg: anti-mouse CD45-BV605, anti-mouse CD4-BV421, and anti-Foxp3-PE CD8+ effector T cells: anti-mouse CD45-BV605, anti-mouse CD3-FITC, and anti-mouse CD8-PE-Cy7 Foxp3 is an intracellular antigen and requires fixation, permeabilization and staining after staining for other surface antigens. After staining, cells were washed, suspended in FACS buffer, detected by a flow cytometer (BD LSR Fortessa) and analyzed by FlowJo X software.

[0196] (8) The experimental results (Figure 10) showed that with this dosing regimen: 1 The IL2 / PP and DLS formulations showed significant differences in their ability to activate different receptor-bearing cell subsets, with the IL2 / PP formulation being able to activate a higher percentage of CD8+ effector T cells while decreasing the percentage of activated immunosuppressive Tregs compared to the positive reference DLS formulation. 2 DLS formulations predominantly activated immunosuppressive Treg cell subsets expressing high-affinity IL2 receptors and had a reduced ability to activate functional CD8+ effector T cells expressing only intermediate-affinity IL2 receptors. 3 IL2 / PP formulation significantly suppressed the activation of Tregs and significantly increased the proportion of activated CD8+ effector T cells, and this trend was very obvious in spleen and tumor tissues. 4 Although the percentages of Treg and CD8+ T cells and the ratio values ​​of the two cells were different in the two different samples, i.e., spleen and tumor tissues, the differences and trends induced by the two preparations were consistent, which means that the selective effects of the two preparations on target cells in different immune environments were essentially the same.

[0197] In other words, compared with the positive reference (DLS formulation), the IL2 / PP formulation had a significant selective effect on the activation of IL2 target cells, did not preferentially activate the Treg subset, and more activated CD8+ effector T cells. This suggests that the IL2 / PP formulation changes the structure of the IL2 protein itself, making it easier to directly activate effector T cells that have a tumor therapeutic effect. This is very important for improving the various limitations of IL2 protein in clinical use.

[0198] [Example 7] Measurement of the effect of G-CSF / PP formulations on the infiltration of spleen- and tumor-derived immune cells

[0199] 1. Cell Culture and Tumor Inoculation Methods

[0200] Culture of the CT26 cell line used in a mouse colon cancer model in RPMI-1640 medium (containing 10% fetal bovine serum). A cryopreservation tube containing CT26 tumor cells was removed from a liquid nitrogen storage tank and rapidly thawed in a 37°C water bath. The cell suspension was then transferred to a centrifuge tube containing 10 ml of culture medium and centrifuged at 350×g for 5 min to remove the supernatant. After resuspension in fresh medium, the cells were transferred to a cell culture flask and 10–15 ml of medium was added to suspend the sedimented cells. After adjusting the cell concentration, the cells were cultured in a humidified atmosphere of 5% CO2 in an incubator at 37°C. During culture, the cells were observed daily and replaced with fresh medium each time.

[0201] Tumor cell inoculation: When the cells grew to 90% confluence, they were digested with 0.05% trypsin and subcultured at a ratio of 1:3. On the day of the experiment, cells in good growth condition and growing to 90% confluence were digested with trypsin, neutralized with fresh medium, centrifuged at 350g to remove the supernatant, resuspended in sterile PBS, and counted. For subsequent use, the concentration of the cell suspension was adjusted to 5 × 10 6 / ml. 5 × 10 5 The cells were inoculated subcutaneously.

[0202] 2. Preparation of G-CSF / PP formulation

[0203] Following the procedure for preparing the G-CSF / PP formulation in Example 1, a stock solution with a concentration of 0.2 mg / mL was prepared for subsequent use.

[0204] 3. Experimental protocol: Detection of infiltration of MDSCs, DCs, CD8+ T cells, and regulatory T cells (Tregs) in the spleen and tumor tissues of tumor-bearing mice

[0205] (1) On day 0, female Balb / c mice were subcutaneously inoculated with CT26 tumor cells. On day 6, all mice developed tumors and were monitored daily for tumor growth. By day 11, the average tumor volume was 80-100 mm. 3 After confirming that the tumor-bearing mice had reached the target tumor size, they were randomly grouped (groups are as follows): a) Control group (tumor-bearing mice group, CTR group) b) Positive control group (protein bulk, used for preparing G-CSF formulation, purchased from Sihuanshengwu Co., Ltd.) c) G-CSF / PP group d) Normal mice group (normal)

[0206] (2) After grouping, the mice were subcutaneously injected with different formulations for 5 consecutive days. The positive control was used in the G-CSF group at a dose of 20 μg / mouse / day. The prepared G-CSF / PP formulation was used in the G-CSF / PP group at the same dose of 20 μg / mouse / day.

[0207] (3) On day 17, spleens and tumors were removed from tumor-bearing mice, weighed, and recorded. Spleens were minced and filtered through a 70 μm sieve mesh to obtain single cell suspensions. Tumor tissues were enzymatically digested, minced, and filtered through a 70 μm sieve mesh to obtain single cell suspensions. Spleen- and tumor-derived MDSCs (CD45 + CD11b + Gr-1 + ), DC(CD45 + CD11c + MHCII + ), total T cells (CD45 + CD3 + ), CD4+ T cells (CD45 + CD3 + CD4 + ) and Tregs (CD45 + CD4 + CD25 + ) were detected by flow cytometry (Figure 12).

[0208] Results analysis

[0209] In the spleen, G-CSF / PP showed similar effects to the positive control on bone marrow-derived cell and MDSC cell numbers, and T cell numbers (CD4 + and Tregs) and showed a similar leukocyte-increasing effect to G-CSF protein bulk. In tumors, G-CSF / PP significantly reduced the numbers of MDSCs and Tregs compared to G-CSF protein bulk. In other words, G-CSF / PP effectively suppressed the immune-related side effects of G-CSF protein.

[0210] Finally, the above embodiments are intended to facilitate understanding of the essence of the present invention for those skilled in the art, and are not intended to limit the protection scope of the present invention.

[0211] This disclosure claims priority based on the specification of China Patent Application Publication No. 202210101468.4, filed with the China Patent Office on January 27, 2022, the disclosure of which is incorporated herein in its entirety.

Claims

1. A cytokine protein pharmaceutical formulation which is a water-soluble formulation, the cytokine protein pharmaceutical formulation comprising: (1) a cytokine protein having a concentration of 40 to 20,000 μg / ml, preferably a concentration of 0.1 to 20 mg / ml, more preferably a concentration of 1 to 10 mg / ml; (2) polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), and (3) Necessary pH adjusters / buffers wherein the molar ratio of said PEG-DSPE to said cytokine protein is 8-100:1, preferably 10-80:1, more preferably 15-50:1, and most preferably 20-40:1; A cytokine protein pharmaceutical preparation, wherein the cytokine protein is a human-derived cytokine having a structure including 3 to 8 α-helices and a molecular weight of 10 to 30 kDa.

2. the cytokine protein is a wild-type cytokine protein derived from a human and having a structure including only α-helices; Preferably, the cytokine protein is a recombinantly expressed cytokine protein having a structure containing only 4-6 alpha helices and a molecular weight of 10-20 kDa; More preferably, the cytokine is IL2, IL4, IL7, IL9, IL10, IL15, IL21, G-CSF, GM-CSF, IFN, EPO, GF or TNF.

3. the PEG-DSPE has a purity of 95% or more, and the PEG chain of the PEG-DSPE has a polydispersity of 1.1 or less; The pharmaceutical formulation according to claim 1 or 2, wherein the length of the PEG chain of the PEG-DSPE molecule is preferably between 1000 and 5000 Da, more preferably between 1500 and 2500 Da, most preferably 2000 Da (PEG2000-DSPE).

4. the pH of the cytokine protein pharmaceutical preparation is adjusted to 5.5 to 6.0 using a pH adjuster; 4. The pharmaceutical formulation of any one of claims 1 to 3, wherein the pH adjusters / buffers include, but are not limited to, hydrochloric acid, acetic acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium dihydrogen carbonate, sodium hydrogen phosphate, sodium dihydrogen phosphate, and amino acid pH adjusters / buffers such as glycine and histidine.

5. The cytokine protein pharmaceutical formulation is further prepared as a lyophilized formulation by lyophilization; 5. The pharmaceutical formulation of claim 1, wherein the lyophilized formulation further comprises a lyoprotectant, including but not limited to glycerin, glycine, sucrose, lactose, albumin, histidine, mannitol, or combinations thereof.

6. A method for preparing a cytokine protein pharmaceutical formulation according to any one of claims 1 to 5, comprising the steps of: (1) preparing an aqueous solution having a pH of 3.0 to 5.5, preferably by diluting glacial acetic acid with deionized water to a concentration of 10 mM to adjust the pH to 3.0 to 3.5; (2) weighing out a cytokine protein dry powder, adding the aqueous solution having a low pH to the cytokine protein dry powder, and thoroughly mixing to obtain a cytokine protein suspension; (3) weighing out a corresponding amount of PEG-DSPE dry powder according to the amount of cytokine protein in step (2), and adding the PEG-DSPE dry powder to the cytokine protein suspension and mixing thoroughly; (4) heating in a water bath at 40 to 60° C. for 10 to 30 minutes; and (5) A step of allowing the solution obtained in step (4) to stand at room temperature, adjusting the pH of the solution to 5.5 to 6.0, and allowing the solution to stand for an additional 10 to 60 minutes to obtain a cytokine protein preparation. wherein the cytokine protein pharmaceutical formulation prepared by said preparation method has a protein recovery rate of 90% or more, said recovery rate being the ratio of the protein content in said cytokine protein pharmaceutical formulation to the amount of protein added.

7. the PEG-DSPE has a purity of 95% or more, and the PEG chain of the PEG-DSPE has a polydispersity of 1.1 or less; The preparation method according to claim 6, wherein the length of the PEG chain of the PEG-DSPE molecule is preferably 1000-5000 Da, more preferably 1500-2500 Da, and most preferably 2000 Da (PEG2000-DSPE).

8. (6) diluting the solution of the cytokine protein preparation obtained in step (5) with the same buffer to an appropriate concentration or concentrating the solution to an appropriate concentration by ultrafiltration; and (7) Sterilizing the cytokine protein formulation by filtration. The method of claim 6 or 7, further comprising:

9. Use of a cytokine protein pharmaceutical formulation according to any one of claims 1 to 5 in the manufacture of a medicament, preferably wherein said medicament is administered by subcutaneous injection.

10. Use of polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) in the manufacture of a formulation for altering the affinity between a protein of interest and its receptor, wherein said protein of interest comprises at least one alpha helix, preferably said protein of interest comprises 4 to 6 alpha helices, more preferably said protein of interest is IL2, IL4, IL7, IL9, IL15, IL21, G-CSF or GM-CSF; The alteration of the affinity between the protein of interest and its receptor means decreasing the binding ability of the protein to a low affinity receptor, the low affinity receptor preferably having a 10 to 20 affinity for the protein. －６ Affinity (Kd value) of 10 or more, more preferably 10 －７ Affinity (Kd value) of 10 or more, most preferably 10 －８ Use of PEG-DSPE with an affinity (Kd value) of M or greater.

11. In the formulation, (1) the ratio of the number of molecules of the PEG-DSPE to the α-helices of the protein of interest is between 2 and 25:1, preferably between 3 and 20:1, more preferably between 4 and 15:1; or (2) The use of PEG-DSPE according to claim 10, wherein the molar ratio of the PEG-DSPE to the protein of interest is 8-100:1, preferably 10-80:1, more preferably 15-50:1, and most preferably 20-30:

1.

12. The use of PEG-DSPE according to claim 10 or 11, wherein the PEG-DSPE has a purity of 95% or more, the PEG chain of the PEG-DSPE has a polydispersity of 1.1 or less, and the length of the PEG chain of the PEG-DSPE molecule is preferably 1000-5000 Da, more preferably 1500-2500 Da, and most preferably 2000 Da (PEG2000-DSPE).

13. An IL2 / PP formulation which is a water-soluble formulation, the IL2 / PP formulation comprising: (1) IL2 protein having a concentration of more than 1 mg / ml, preferably 1 to 10 mg / ml; (2) polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), preferably the PEG chain length of the PEG-DSPE molecule is 2000 Da (PEG2000-DSPE), the PEG-DSPE has a purity of 95% or more, and the PEG chain of the PEG-DSPE has a polydispersity of 1.1 or less; and (3) Necessary pH adjusters / buffers wherein the molar ratio of said PEG-DSPE to said IL2 protein is 8-100:1, preferably 10-80:1, more preferably 15-50:1, and most preferably 20-30:1; The IL2 protein is expressed in E. coli and purified, and has the amino acid sequence shown in SEQ ID NO:

1.

14. The formulation of claim 13, wherein the pH of the IL2 / PP formulation is adjusted to 5.5 to 6.0 using a pH adjusting agent.

15. A method for preparing an IL2 / PP formulation according to claim 13 or 14, comprising the steps of: (1) preparing an aqueous solution having a pH of 3.0 to 5.5, preferably by diluting glacial acetic acid with deionized water to a concentration of 10 mM and adjusting the pH to 3.0 to 3.5; (2) weighing the amount of IL2 protein dry powder, adding the aqueous solution having a pH of 3.0 to 3.5 to the IL2 protein dry powder, and thoroughly mixing to obtain an IL2 suspension; (3) weighing out a corresponding amount of PEG-DSPE dry powder according to the amount of IL2 protein in step (2), adding the PEG-DSPE dry powder to the IL2 suspension, and mixing thoroughly; (4) heating in a water bath at 40 to 60° C. for 10 to 30 minutes; and (5) A step of allowing the solution obtained in step (4) to stand at room temperature, adjusting the pH of the solution to 5.5 to 6.0, and allowing the solution to stand for an additional 10 to 60 minutes to obtain the IL2 / PP formulation. Preferably, the length of the PEG chain of the PEG-DSPE molecule is 2000 Da (PEG2000-DSPE), the PEG-DSPE has a length of 95% or more, and the PEG chain in the PEG-DSPE has a dispersity of 1.1 or less; A preparation method, wherein the IL2 / PP formulation prepared by the preparation method has an IL2 protein recovery rate of 90% or more.

16. Use of an IL2 / PP formulation according to claim 13 or 14, (1) Production of antitumor drugs, (2) The manufacture of a preparation for activating a cellular immune response; or (3) Preparation of an antitumor combination preparation, preferably an antitumor combination preparation further comprising an antibody or antibody construct, a cytotoxic agent, an immunotherapeutic agent or a cellular therapeutic agent. wherein the medicament or preparation has lower toxicity and / or side effects compared to an IL2 preparation in the form of a DLS formulation; The activation of the cellular immune response refers to in vivo or in vitro activation of the cellular immune response. (1) upregulation of CD8+ T cell proliferation, and / or (2) Induction of differentiation of T cells into CD8+ T cells, and / or (3) Inhibition of T cell differentiation into Tregs, and / or (4) Increase in the ratio of CD8+ T cells or Tregs Preferably, the activation of the cellular immune response refers to the activation of the cellular immune response in vivo or in vitro when the IL2 / PP preparation is compared to an IL2 preparation in the form of a DLS preparation (positive reference preparation) or a preparation in the form of a free protein solution. (1) upregulation of CD8+ T cell proliferation, and / or (2) Induction of differentiation of T cells into CD8+ T cells, and / or (3) Inhibition of T cell differentiation into Tregs, and / or (4) Increase in the ratio of CD8+ T cells or Tregs Preferably, the drug or preparation is administered by subcutaneous injection.

17. A G-CSF / PP formulation which is a water-soluble formulation, comprising: (1) G-CSF protein having a concentration of more than 1 mg / ml, preferably 1 to 10 mg / ml; (2) polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), preferably the PEG chain length of the PEG-DSPE molecule is 2000 Da (PEG2000-DSPE), the PEG-DSPE has a purity of 95% or more, and the PEG chain of the PEG-DSPE has a polydispersity of 1.1 or less; and (3) Necessary pH adjusters / buffers wherein the G-CSF protein is expressed in and purified from E. coli and has the amino acid sequence shown in SEQ ID NO:

2.

18. The formulation according to claim 17, wherein the pH of the G-CSF / PP formulation is adjusted to 5.5 to 6.0 using a pH adjusting agent.

19. A method for preparing the G-CSF / PP formulation according to claim 17 or 18, comprising the steps of: (1) preparing an aqueous solution having a pH of 3.0 to 5.5, preferably by diluting glacial acetic acid with deionized water to a concentration of 10 mM and adjusting the pH to 3.0 to 3.5; (2) weighing the amount of G-CSF protein dry powder, adding the aqueous solution having a pH of 3.0 to 3.5 to the G-CSF protein dry powder, and thoroughly mixing to obtain a G-CSF suspension; (3) weighing out a corresponding amount of PEG-DSPE dry powder according to the amount of G-CSF protein in step (2), and adding the PEG-DSPE dry powder to the G-CSF suspension and mixing thoroughly; (4) heating in a water bath at 40 to 60° C. for 10 to 30 minutes; and (5) A step of allowing the solution obtained in step (4) to stand at room temperature, adjusting the pH of the solution to 5.5 to 6.0, and allowing the solution to stand for an additional 10 to 60 minutes to obtain a G-CSF / PP formulation. Preferably, the PEG-DSPE molecule has a PEG chain length of 2000 Da (PEG2000-DSPE), the PEG-DSPE has a purity of 95%, and the PEG chain of the PEG-DSPE has a polydispersity of 1.1 or less; A preparation method, wherein the G-CSF / PP preparation prepared by the above preparation method has a G-CSF protein recovery rate of 90% or more.

20. 19. Use of a formulation according to claim 17 or 18 in the manufacture of a medicament for treating febrile neutropenia (FN), preferably said febrile neutropenia being a condition caused by radiotherapy or chemotherapy of a tumor or organ transplant, preferably said medicament being administered by subcutaneous injection.

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