IL-2 mutant and application thereof

Abstract

The invention provides an IL-2 mutant and an application thereof. The IL-2 mutant comprises amino acid mutation at the 125th site of a protein with an amino acid sequence as shown in SEQ ID NO: 1 and amino acid mutation at at least one of the following sites: F42 or R38, the protein with the amino acid sequence as shown in SEQ ID NO: 1 is of an IL-2 wild type, the activation capacity of the IL-2 mutant on IL-2R alpha beta gamma is lower than that of the IL-2 wild type on IL-2R alpha beta gamma, and the IL-2 mutant can be used for preparing the IL-2 mutant. The activation capability of the IL-2 mutant on IL-2R beta gamma is not obviously different from the activation capability of the IL-2 wild type on IL-2R beta gamma. According to the IL-2 mutant, the cell bias is improved, CD8 + T effector cells and NK cells can be selectively activated, immunosuppression caused by activation of Treg cells is avoided, and the problem that in the prior art, the cell bias is low is effectively solved.

Description

IL-2 mutants and their applications

[0001] This application is based on the Chinese application with CN application number 202211442050.6 and application date November 17, 2022, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of genetic engineering, and in particular to an IL-2 mutant and applications thereof. Background Art

[0003] In recent years, with the 2018 Nobel Prize in Physiology or Medicine awarded to the research on PD-L1 / PD-1 and CTLA-4 targets, immunotherapy has become one of the hot topics in current drug research. In November 1984, a female melanoma patient experienced the disappearance of her whole body tumors after receiving high-dose IL-2 treatment for only a few months [Rosenberg SA et al. N Engl J Med 1985; 313(23)1485-92]. Based on the significant efficacy of IL-2 in treating cancer, the FDA approved high-dose IL-2 for the treatment of advanced renal cancer and malignant melanoma in 1992 and 1998, respectively, with an efficacy of 15%-20%. Although the higher the dose of IL-2, the better the anti-tumor effect, high-dose IL-2 can cause severe vascular permeability syndrome (VLS), leading to water accumulation in human organs, pulmonary edema and liver cell damage [DF McDermott, Oncoimmunology. 2016 Jun; 5(6): e1163462.]. Recent studies have found that low-dose IL-2 preferentially activates Tregs, suppresses immune responses, and promotes tumor escape. Therefore, low-dose IL-2 cannot be used for tumor treatment. This limited dosage window significantly restricts the further clinical application of IL-2-based immunotherapies.

[0004] The IL-2 receptor is mainly composed of three subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). Among them, CD25 can bind to IL-2 with low affinity (Kd≈10nM), and CD25 is not required for signal transduction [Ye CX, et al. Siganl Transduct Target Ther 2018;3:2]. T cells in the resting state express very low levels of CD25. Once T cells are activated, they will induce high expression of CD25 on their surface. CD122 and CD132 constitute a heterodimeric receptor with medium affinity (Kd≈1nM) for IL-2, which is crucial for downstream proliferation signals [Math Med Biol 2018;35(1):79-119]. When IL-2 binds to CD25, the conformation of IL-2 undergoes a subtle repositioning, which greatly enhances its interaction with the CD122 and CD132 heterodimeric receptors and forms a high-affinity tertiary complex (Kd≈10-50pM) [Science 2005; 310(5751):1159-1163; Nature 2012; 484(7395):529-533].

[0005] IL-2 interacts with receptors on different cells expressing different subunits, producing different biological activities. For example, regulatory T cells constitutively express the high-affinity trimeric receptor IL-2Rαβγ and are more sensitive to low concentrations of IL-2. Therefore, low-dose IL-2 can activate and promote the proliferation of Treg cells, inhibit overactivation of the immune system, and thus regulate immune homeostasis. Currently, many clinical results have shown that low-dose IL-2 is effective for a variety of autoimmune diseases, such as vasculitis, inflammatory myopathy, and systemic lupus erythematosus (SLE). Effector T cells and natural killer cells express IL-2Rβγ receptors with moderate affinity and require higher concentrations of IL-2 to be effectively stimulated. Therefore, when using IL-2 for immunotherapy, high doses must be used to activate Teff and NK cells as much as possible [Nat Rev Immunol 2018; 18(10): 648-659]. However, in addition to Tregs, CD31+ pulmonary endothelial cells express low to moderate levels of IL-2Rαβγ trimer receptors, so high doses of IL-2 will inevitably interact with them and induce severe VLS [Proc Natl Acad Sci USA 2010; 107(26): 11906-11911]. The development of IL-2 with cell-selective ability to stimulate lymphocyte proliferation is the key to solving its toxic side effects.

[0006] Existing modification schemes generally start from two complementary approaches: (1) reducing the affinity of IL-2 for the trimeric receptor or reducing the interaction between IL-2 and its receptor CD25, making it a less toxic molecule, allowing for higher dosages; (2) enhancing the affinity of IL-2 for the dimeric receptor, making it easier to activate effector T cells or NK cells, allowing for lower dosages. For example, NKTR-214 developed by Nektar Therapeutics modified IL-2 with six PEGs through non-site-specific conjugation to make it a prodrug. It is assumed that through gradual hydrolysis in the body, it will eventually degrade to an active form with only one PEG or no PEG. Since PEG conjugation hinders the interaction between IL-2 and CD25, it retains its ability to stimulate CD122, making it more inclined to activate CD8 T and NK cells to kill tumors [Clin Cancer Res 2016; 22(3): 680-690; Plos One 2017; 12(7): e0179431]. The non-site conjugation poses great challenges to the preparation and quality control of NKTR-214.

[0007] Secondly, the protein is only active when it is degraded to a PEG state, which leads to a decrease in the effective concentration of the protein, seriously affecting its anti-tumor activity. Based on this, Synthorx's THOR-707 uses non-natural amino acid technology to insert a lysine derivative with an azide group into the proline at position 65 of IL-2, and then uses click chemistry to site-couple a 30k long polyethylene glycol polymer [Nature Communication 2021; 12(1): 1-14]. The site-coupled PEG not only significantly prolongs the half-life of IL-2, but also effectively blocks the interaction between IL-2 and CD25, making it tend to promote the expansion of CD8 T cells and increase the tumor infiltration of CD8+T, thus playing a strong anti-tumor effect. In terms of safety, even 1000ug / kg of THOR 707 will not cause VLS [WO2019028419A1].

[0008] However, the introduction of unnatural amino acids will affect protein production and increase overall costs. At the same time, compared with wild-type IL-2, THOR707 has a lower proliferative activity on CTLL2 cells than wild-type IL-2. 50 The shift is about 800-fold, and the selected coupling site may not be the optimal site. In addition, through directed evolution, Levin et al. screened out the IL-2 super mutant H9, which has a significantly improved affinity for CD122 compared to wild-type IL-2 [Nature 2012; 484(7395): 529-533].

[0009] Therefore, constructing an IL-2 mutant with stronger cell bias has become a hot topic in the research field.

[0010] Summary of the Invention

[0011] The main purpose of the present invention is to provide an IL-2 mutant and its application to solve the problem of low cell tropism of modified IL-2 in the prior art.

[0012] To achieve the above-mentioned object, according to a first aspect of the present invention, an interleukin-2 (IL-2) mutant is provided, comprising: an amino acid mutation at position 125 of a protein having an amino acid sequence as shown in SEQ ID NO: 1, and an amino acid mutation at at least one of the following sites: F42 or R38, wherein the protein having the amino acid sequence as shown in SEQ ID NO: 1 is a wild-type IL-2, the IL-2 mutant has a lower ability to activate IL-2Rαβγ than the wild-type IL-2, and there is no significant difference between the ability of the IL-2 mutant to activate IL-2Rβγ and that of the wild-type IL-2.

[0013] Furthermore, the mutations of the IL-2 mutant are each independently selected from the following: F42X+C125J, R38X+C125J or F42X+R38X+C125J, wherein the letter before the number represents the original amino acid, the letter after the number represents the mutant amino acid, the amino acid represented by X is any amino acid with a sulfhydryl group, and J represents any of the following amino acids: G, A, S, T or V; preferably, the IL-2 mutant is F42C+R38C+C125S; preferably, the IL- The IL-2 mutant is an IL-2 mutant in which C125 is mutated to S and F42X and R38X directly form an intramolecular disulfide bond; preferably, the IL-2 mutant is an IL-2 mutant in which C125 is mutated to S and the sulfhydryl groups on F42X and R38X are modified; preferably, the IL-2 mutant is an IL-2 mutant in which C125 is mutated to S and the sulfhydryl groups on F42X and R38X are modified with DCA; preferably, the structure in which the sulfhydryl groups on F42X and R38X are modified with DCA is as shown in Formula I:

[0014] Furthermore, the activation ability of wild-type IL-2 on the IL-2Rαβγ complex was recorded as the first EC 50 The activation ability of the above IL-2 mutants on the IL-2Rαβγ complex was recorded as the second EC 50 Value, second EC 50 Value and first EC 50 The ratio of the values ​​is recorded as n, where n≥74, preferably n≥256.

[0015] In order to achieve the above-mentioned object, according to the second aspect of the present invention, an IL-2 mutant conjugate is provided, which is an IL-2 protein-polyethylene glycol (PEG) conjugate obtained by PEG conjugation based on the above-mentioned IL-2 mutant protein.

[0016] Furthermore, the PEG in the above-mentioned IL-2 mutant conjugate is PEG modified with a chemical modifier, the IL-2 protein is an IL-2 mutant having F42X and / or R38X mutation sites and C125J, and the thiol group of F42X and / or R38X is coupled to PEG through a chemical coupling agent in PEG, wherein the letter before the number represents the original amino acid, the letter after the number represents the mutant amino acid, the amino acid represented by X is any amino acid with a thiol group, and J represents any of the following amino acids: G, A, S, T or V; preferably, the chemical coupling agent is a compound with a hydroxylamino group or a hydrazide group; preferably, the compound with a hydroxylamino group is selected from any one of the following: maleimide, succinimide; preferably, the substituent with a hydrazide group is selected from an alkyl group, an aryl group or a heteroaryl group, the number of carbon atoms of the alkyl group is selected from 1 to 8, and the number of carbon atoms of the aryl group or the heteroaryl group is selected from 5 to 10.

[0017] In order to achieve the above object, according to the third aspect of the present invention, a DNA molecule is provided, which encodes the above IL-2 mutant.

[0018] In order to achieve the above object, according to a fourth aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above DNA molecule.

[0019] In order to achieve the above object, according to a fifth aspect of the present invention, a host cell is provided, into which the above recombinant plasmid is transformed.

[0020] In order to achieve the above object, according to the sixth aspect of the present invention, there is provided a use of the above IL-2 mutant or IL-2 mutant conjugate in the preparation of a drug or preparation for treating cancer.

[0021] Furthermore, the above-mentioned cancer is selected from any one of the following: renal cancer, melanoma, pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, mesothelioma, leukemia, multiple myeloma, lymphoma, liver cancer, sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary central nervous system lymphoma, primitive neuroectodermal tumor, bladder cancer, esophageal cancer, uterine cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer and gastric cancer.

[0022] By applying the technical solution of the present invention, site-directed mutagenesis is performed on the basis of the existing IL-2 to obtain a double cys mutant (FRC). The resulting FRC has a higher cell bias and has a reduced ability to activate CD25 while substantially retaining the ability to activate the IL-2Rβγ complex. Since its interaction with CD25 is greatly reduced and the ability to activate the IL-2Rβγ complex is retained, the activation ability of FRC on the IL-2Rαβγ trimer is greatly weakened. This means that the IL-2 mutant of the present invention can avoid the immunosuppression caused by the activation of Tregs by low-dose use of natural IL-2, selectively activate CD8+T cells and / or NK cells, and can be used in high doses in the clinic to achieve the effect of tumor treatment, providing a positive impact on the treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0024] FIG1 shows a schematic structural diagram of the F42C+R38C+C125S mutation predicted by Alphafold in Example 1 of the present invention.

[0025] Figure 2 shows a schematic diagram of the preparation process of FRC-DCA in Example 1 of the present invention, wherein "STAPLE" means staple.

[0026] FIG3 is a schematic diagram showing mass spectrum data of FRC in Example 1 of the present invention.

[0027] FIG4 is a schematic diagram showing mass spectrum data of FRC-DCA in Example 1 of the present invention.

[0028] FIG5 shows a schematic diagram of the identification of the FRC and FRC-PEG proteins purified in Example 1 of the present invention.

[0029] Figure 6 shows a schematic diagram of the activation effects of IL-2, FRC, and FRC-DCA on different cells in Example 2 of the present invention, wherein "Ratio" in Figure (d) means ratio.

[0030] FIG7 shows a schematic diagram of SDS-PAGE of FRC and FRC-2PEG in Example 3 of the present invention.

[0031] FIG8 shows a schematic diagram of size exclusion chromatography of FRC in Example 3 of the present invention.

[0032] FIG9 shows a schematic diagram of size exclusion chromatography of FRC-2PEG in Example 3 of the present invention.

[0033] FIG10 shows the SPR detection results of FRC-2PEG binding to CD25 and CD122 in Example 4 of the present invention.

[0034] In a, b, c and d, the concentration of the curves decreases by 2 times from top to bottom (the corresponding concentrations are: 2μM, 1μM, 0.5μM, 0.25μM, 0.125μM, 0.062μM, 0.031μM, 0.015μM, 0.0078μM and 0.0039μM).

[0035] FIG11 is a schematic diagram showing the activation effects of IL-2 and FRC-2PEG on different cells in Example 4 of the present invention.

[0036] FIG12 shows a schematic diagram of the PK of FRC and FRC-2PEG in Example 5 of the present invention in mice.

[0037] FIG13 is a schematic diagram showing the IL-5 concentration in the plasma of different dosing groups at the first blood sampling point in Example 6 of the present invention.

[0038] FIG14 is a schematic diagram showing the IL-5 concentration in the plasma of different dosing groups at the second blood sampling point in Example 6 of the present invention.

[0039] FIG15 shows a schematic diagram of analysis of different cell ratios in peripheral blood and spleen lymphocytes in Example 6 of the present invention.

[0040] FIG16 shows a schematic diagram of the analysis of the in vivo tumor inhibition effect of FRC-2PEG in Example 7 of the present invention.

[0041] FIG17 shows a schematic diagram of analysis of different cell ratios in peripheral blood, spleen, and tumor lymphocytes in Example 7 of the present invention. DETAILED DESCRIPTION

[0042] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0043] As mentioned in the background art, low doses of IL-2 can activate and promote the proliferation of Treg cells, leading to immunosuppression, while high doses of IL-2 can cause severe vascular permeability syndrome (VLS), leading to damage such as water accumulation in human organs. Therefore, the toxic side effects caused by the dose window greatly limit the further clinical application of IL-2 related immunotherapy. Since different concentrations of IL-2 have different activation degrees for different lymphocytes, the development of IL-2 with a preference for selectively stimulating the proliferation of target lymphocytes is the key to solving the limitations of IL-2. However, in the existing technology for modifying IL-2, whether by reducing the affinity of IL-2 to the IL-2Rαβγ trimer receptor or by enhancing the affinity of IL-2 to the IL-2Rβγ dimer receptor, the non-site-specific foreign conjugates or non-natural amino acid modifications introduced therein all bring certain hidden dangers to the safety of the modified IL-2 protein production, preparation and clinical use.

[0044] Therefore, in this application, the inventors attempted to solve the toxic side effects of IL-2 by constructing a mutant and a PEG conjugate, making it have a stronger cell (NK cell and CD8+T cell) bias, which can significantly weaken the activation effect of Treg on the IL-2Rαβγ trimer receptor, and thus better use it in tumor treatment. On this basis, the applicant proposed a series of protection schemes of this application.

[0045] In a first typical embodiment of the present application, an IL-2 mutant is provided, comprising an amino acid mutation at position 125 of a protein having an amino acid sequence as shown in SEQ ID NO: 1 and an amino acid mutation at at least one of the following sites: F42 or R38, wherein the protein having the amino acid sequence shown in SEQ ID NO: 1 is a wild-type IL-2, the IL-2 mutant has a lower ability to activate IL-2Rαβγ than the wild-type IL-2, and there is no significant difference between the IL-2 mutant and the wild-type IL-2 in their ability to activate IL-2Rβγ.

[0046] The sequence of SEQ ID NO: 1 (wild type) is as follows:

[0047] These mutants are characterized by their higher selectivity in stimulating cytotoxic effector CD8+ T cells and NK cells than in stimulating Treg cells, thus avoiding the immunosuppression caused by low-dose natural IL-2 activating Tregs; at the same time, the high-dose IL-2 variants of the present invention also do not induce VLS induced by high-dose natural IL-2 due to activation of CD31+ endothelial cells; in addition, the IL-2 mutants of the present invention significantly prolong the time (half-life) of IL-2 in the body. The IL-2 mutants of the present invention, with mutations in C125, F42, and / or R38, solve the problems of low-dose IL-2-induced immunosuppression and high-dose-induced VLS that plague tumor treatment in the prior art. Not only is the preparation process simple, but the frequency of administration can also be greatly reduced during clinical use, thereby improving patient compliance with medication.

[0048] In a preferred embodiment of the present invention, the mutations in the IL-2 mutant are each independently selected from the following: F42X+C125J, R38X+C125J, or F42X+R38X+C125J, wherein the letter before the number represents the original amino acid, the letter after the number represents the mutant amino acid, the amino acid represented by X is any natural or synthetic amino acid containing a thiol group, and J represents any of the following amino acids: G, A, S, T, or V. In a preferred embodiment, the thiol-containing amino acid is cysteine ​​(Cys); in a preferred embodiment, upon the mutation of position 125 from C to any of G, A, S, T, or V, F42C or R38C is independently cys-ylated; in a preferred embodiment, upon the mutation of position 125 from C to any of G, A, S, T, or V, F42C and R38C are simultaneously cys-ylated.

[0049] In a preferred embodiment, the IL-2 mutant is F42C+R38C+C125S; in a preferred embodiment, the IL-2 mutant is an IL-2 mutant in which C125 is mutated to S (i.e., the C at position 125 is mutated to S) and F42X and R38X directly form an intramolecular disulfide bond; in a preferred embodiment, the IL-2 mutant is an IL-2 mutant in which C125 is mutated to S and the sulfhydryl groups on F42X and R38X are modified; in a preferred embodiment, the IL-2 mutant is an IL-2 mutant in which C125 is mutated to S and the sulfhydryl groups on F42X and R38X are modified with DCA; the structure in which the sulfhydryl groups on F42X and R38X are modified with DCA is shown in Formula I:

[0050] The activation ability of the wild-type IL-2 on the IL-2Rαβγ complex was recorded as the first EC 50 The activation ability of the above IL-2 mutants on the IL-2Rαβγ complex was recorded as the second EC 50 Value, second EC 50 Value and first EC 50 The ratio of the values ​​is denoted as n, where n ≥ 74, and preferably n ≥ 256. It can be seen that the IL-2 mutants with mutations at F42 and / or R38 significantly reduce their ability to activate IL-2Rαβγ compared to wild-type IL-2. IL-2 mutants that meet the above conditions not only have lower toxic side effects but also have stronger cell (CD8+ T cell and / or NK cell) tropism, making them more suitable for tumor treatment.

[0051] More preferably, the ability of IL-2 to activate the IL-2Rβγ complex is expressed as EC 50 The activation ability of wild-type IL-2 on IL-2Rβγ complex was recorded as the third EC 50 The activation ability of IL-2 mutants on IL-2Rβγ complex was recorded as the fourth EC 50 Value, Fourth EC 50 Value and third EC 50 The ratio of the values ​​is recorded as m. When m≤15, it is considered that there is no significant difference in the activation ability of IL-2 wild type and IL-2 mutant on the IL-2Rβγ complex.

[0052] Modification of either or both of the above two sites to any amino acid with a thiol group facilitates coupling with PEG using the thiol group. The specific type of such amino acid is not particularly limited and can be Cys or other natural or unnatural amino acids. The type of mutation can be appropriately selected based on the selected amino acids.

[0053] In a second typical embodiment of the present application, an IL-2 mutant conjugate is provided, which is an IL-2 protein-polyethylene glycol (PEG) conjugate obtained by PEG conjugation based on the above-mentioned IL-2 mutant protein.

[0054] The PEG in the aforementioned IL-2 mutant conjugate is PEG modified with a chemical modifier, the IL-2 protein is an IL-2 mutant having F42X and / or R38X mutation sites and C125J, and the thiol groups of F42X and / or R38X are coupled to the PEG via a chemical coupling agent in the PEG. The letters preceding the numbers represent the original amino acids, the letters following the numbers represent the mutant amino acids, the amino acid represented by X is any amino acid with a thiol group, and J represents any of the following amino acids: G, A, S, T, or V. In a preferred embodiment, the chemical coupling agent is a compound having a hydroxylamino group or a hydrazide group; in a preferred embodiment, the compound having a hydroxylamino group is selected from any of the following: maleimide and succinimide; in another preferred embodiment, the substituent having a hydrazide group is selected from an alkyl group, an aryl group, or a heteroaryl group, wherein the number of carbon atoms in the alkyl group is selected from 1 to 8, and the number of carbon atoms in the aryl or heteroaryl group is selected from 5 to 10.

[0055] The above-mentioned PEG coupling method is not limited, and any method that can achieve this purpose is applicable to the present application. In a preferred embodiment, the coupling method of FRC-2PEG includes treating the above-mentioned IL-2 mutant with TCEP (tris(2-carboxyethyl)phosphine), incubating with 10 moles of mal-PEG at 37°C for 2h, coupling with the cysteine ​​of F42C and R38C in IL-2 through its maleimide, and forming a SC covalent bond through the sulfhydryl and maleimide. The chemical reaction is shown in the following formula. After passing through a secondary nickel column, the unreacted mal-PEG is removed, and the purified coupling product FRC-2PEG is directly obtained by passing through a molecular sieve. The product obtained after the above-mentioned mutant is coupled with PEG by sulfhydryl has low toxicity and side effects and is cell-biased.

[0056] The activation ability of wild-type IL-2 on the IL-2Rαβγ complex was recorded as the first EC 50 The activation ability of the above IL-2 mutant conjugates on the IL-2Rαβγ complex was recorded as the second EC 50 Value, second EC 50 Value and first EC 50 The ratio of the values ​​is recorded as n, where ≥ 2140. It can be seen that the IL-2 mutant conjugate further reduces the activation ability of the IL-2 mutant on the IL-2Rαβγ complex.

[0057] In a preferred embodiment, the FRC-PEG coupling method includes expressing and purifying the aforementioned IL-2 mutant in E. coli BL21, adding TCEP to a final concentration of 0.1 mM to the purified FRC, incubating the mixture in a 37°C water bath for 2 hours, and then adding approximately 1 / 3 volume of 200 mM NaHCO3 buffer. Four molar equivalents of 1,3-dichloroacetone (DCA) are added to the reduced FRC, mixed thoroughly, and reacted at 4°C overnight. After filtration through a desalting column, NH2O-PEG is added to the FRC-DCA for orthogonal carbonyl coupling. The chemical reaction is shown below. After one day of reaction at room temperature, the coupled product, FRC-PEG, is separated by molecular sieves.

[0058] The IL-2 mutant FRC (F42C+R38C+C125S) of the present invention exhibits strong cell tropism, namely, reduced ability to activate CD25 while substantially retaining the ability to activate the IL-2Rβγ complex, thereby reducing the ability to activate the IL-2Rαβγ complex. To further enhance the cell tropism of this mutant, the inventors modified FRC with DCA. DCA modification further stabilizes the Cys structure and provides a group that facilitates PEG conjugation to the IL-2 mutant. Both direct PEG conjugation and DCA-modified PEG conjugation help further reduce the binding of FRC to CD25 (IL-2Rα) by increasing steric hindrance. The PEG-conjugated IL-2 mutant conjugate lacks the ability to bind to CD25 or activate the IL-2Rαβγ complex. Furthermore, PEG conjugation further extends the in vivo half-life of the IL-2 mutant, which can reduce the frequency of dosing in clinical use and improve patient compliance.

[0059] In a third typical embodiment of the present application, a DNA molecule is provided, which encodes the above-mentioned IL-2 mutant.

[0060] In a fourth typical embodiment of the present application, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.

[0061] In a fifth exemplary embodiment of the present application, a host cell is provided, wherein the host cell is transformed with the aforementioned recombinant plasmid. Utilizing the aforementioned host cell, the recombinant plasmid can be replicated within the host cell, and the DNA molecules carried by the recombinant plasmid can be transcribed and translated to obtain a large number of IL-2 mutants.

[0062] In a preferred embodiment, the host cells are BL21 cells, transformed with the recombinant plasmid, cultured at 30°C to an OD600 of 0.4-0.6, and induced at 42°C. The inclusion bodies are purified by high-pressure disruption, solubilized with guanidine hydrochloride as a denaturant, and dialyzed into TRIS buffer (pH 8.5). The dialyzate is purified by nickel column, and the eluate is concentrated and directly passed through molecular sieves to obtain the pure IL-2 mutant (FRC).

[0063] In a sixth exemplary embodiment of the present application, there is provided a use of an IL-2 mutant or an IL-2 mutant conjugate in the preparation of a drug or formulation for treating cancer, including any of the following cancers: renal cancer, malignant melanoma, pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, mesothelioma, leukemia, multiple myeloma, lymphoma, liver cancer, sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary central nervous system lymphoma, primitive neuroectodermal tumor, bladder cancer, esophageal cancer, uterine cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer. The drug containing the IL-2 mutant of the present application has minimal toxicity and side effects, and can significantly reduce or even completely eliminate the activation of Treg cells of the IL-2Rαβγ trimer receptor, thereby being better used for tumor treatment.

[0064] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0065] Example 1 Preparation of IL2 mutant FRC and its staple product

[0066] To create an IL-2 mutant that completely blocks its interaction with CD25, we selected the most critical sites in the IL-2-CD25 interaction epitope and mutated them to Cys. We also mutated the remaining Cys at position 125. We then site-specifically coupled PEG to block the interaction through steric hindrance. Using alphafold structural modeling of IL-2 and its mutants, we found that mutating R38 and F42 to Cysteine ​​and C125 to Ser exposed the side chains of these residues on the protein surface and pointed toward CD25 (Figure 1).

[0067] We observed that the side chains of the two Cys residues on the mutant FRC were relatively close. To stabilize the two Cys residues, we used 1,3-dichloroacetone (DCA) to staple them together into a peptide structure and simultaneously introduced a carbonyl group (Figure 2). The carbonyl group can serve as an active group for subsequent coupling.

[0068] Through point mutagenesis, an expression plasmid for the IL-2 mutant F42C+R38C+C125S (hereinafter referred to as FRC) was successfully constructed. After transformation into the BL21 expression strain, the cells were cultured at 30°C to an OD600 of 0.4-0.6 and induced at 42°C. The inclusion bodies were purified by high-pressure fragmentation and solubilized with 8M guanidine hydrochloride containing 2mM cysteine ​​as a denaturant, followed by dialysis into TRIS buffer (pH 8.5). The dialyzate was purified by nickel column, and the eluate was concentrated and directly passed through molecular sieves to obtain pure FRC. Mass spectrometry revealed that FRC has two molecular weights, corresponding to the two states of the two Cys residues on FRC: one is that C38 and C42 directly form a pair of disulfide bonds (mass spectrometry molecular weight 15433.11 Da), and the other is that both C38 and C42 residues are Cys-ylated (mass spectrometry molecular weight 15673.40 Da). It is speculated that the free Cys in FRC may react with the Cys in the solution during the renaturation process (Figure 3).

[0069] TCEP was added to the prepared FRC at a final concentration of 0.1 mM. After incubating in a 37°C water bath for 2 hours, approximately 1 / 3 of the volume of 200 mM NaHCO3 buffer was added. 4 molar equivalents of 1,3-dichloroacetone (DCA) were added to the reduced FRC, mixed thoroughly, and reacted at 4°C overnight. The next day, TCEP, reduced Cys, and unreacted DCA were removed from the solution directly through a desalting column. The prepared FRC-DCA was subjected to mass spectrometry, and we found that the entire process was very complete. The mass spectrometry data showed only a single molecular weight peak, which corresponded to FRC-DCA, with no unreacted FRC molecular weight remaining (Figure 4).

[0070] Based on this, we added NH2O-PEG to FRC-DCA for orthogonal carbonyl coupling to prepare the PEGylated product. After one day of reaction at room temperature, we directly isolated the coupled product FRC-PEG using molecular sieves (Figure 5).

[0071] Example 2 IL-2 mutants weaken the interaction between IL-2 and CD25, thereby reducing the interaction with IL-2Rαβγ

[0072] To test the stimulatory activity of FRC, FRC-DCA, and FRC-PEG on T cells expressing different proteins, we tested the proliferation effects of IL-2, FRC, FRC-DCA, and FRC-PEG on CTLL2 cells expressing the trimeric receptor IL-2Rαβγ and MO7E cells expressing the dimeric receptor IL-2Rβγ, respectively, based on Example 1. As shown in Figure 6, compared with IL-2, the EC of FRC not coupled with PEG was 50It showed a significant shift (shifted to the right by about 256 times), indicating that when containing the C125S mutation, mutating the F42 and R38 sites to Cys (whether F42C and R38C form an intramolecular disulfide bond or F42C and R38C are modified by Cys) can significantly reduce its interaction with CD25, thereby weakening the activation effect on CTLL2 cells. 50 The shift to the right is 74 times, which may be because the steric hindrance of FRC-DCA is smaller than that of the two Cys-Cys on FRC. After coupling with PEG, the EC of FRC-PEG 50 The inhibitory effect of PEG was significantly stronger than that of Thor-70, which was 7800 times stronger than that of IL-2Rβγ. 50 It is only shifted to the right by about 3.45 times, which means that by "staple" the F42 and R38 sites and then coupling them with PEG, it will hardly have any effect on its binding to the IL-2Rβγ receptor, but it can effectively block the interaction between FRC and IL-2Rα.

[0073] Example 3 Preparation and Activity Test of FRC-2PEG

[0074] Considering the complex preparation process of FRC-PEG, which requires a two-step coupling process, and despite the inclusion of the C125S mutation and the selection of point mutations at sites F42 and R38, the blocking effect of coupling PEG after preparing FRC-DCA was not significantly different from that of coupling with mono-PEG. Therefore, we considered directly preparing the bis-PEG coupling product FRC-2PEG through a one-step coupling based on FRC, hoping that the bis-PEG would provide a stronger blocking effect on FRC. Therefore, we added TCEP to a final concentration of 0.1 mM to the FRC prepared in Example 1. After reacting at 37°C for 2 hours, 12 molar equivalents of maleimide-PEG were directly added. After thorough mixing, the reaction was incubated at 4°C overnight. The bis-PEG-coupled FRC-2PEG can be directly prepared by molecular sieve separation (Figures 7-9). Therefore, compared to FRC-PEG, FRC-2PEG requires only a single coupling step to prepare, resulting in a simpler preparation process.

[0075] Example 4 IL-2 mutants retain affinity for CD122 (IL-2Rβ)

[0076] Surface plasmon resonance (SPR) technology was used to detect the affinity of IL-2 mutants to CD25 and CD122: CD25-Fc and CD122-Fc were diluted to 50 μg / mL with immobilization buffer (10 mM NaAc pH 5.0), and the CM5 chip was activated with 400 mM EDC and 100 mM NHS. The diluted CD25-Fc and CD122-Fc were then immobilized on the CM5 chip at a flow rate of 10 μL / min until the RU value reached approximately 2000 RU. The immobilized CM5 chip was then blocked with ethanolamine.

[0077] IL-2 and FRC-2PEG were diluted with working buffer (1XHEPES 0.005% Tween-20 pH 7.5) to different concentration gradients, loaded at a flow rate of 30 μL / min, and the corresponding binding dissociation constants were calculated based on the curves.

[0078] As shown in Figure 10, on the channel of CD25-Fc (a in Figure 10), IL-2 exhibited fast binding and slow dissociation kinetics, while FRC-2PEG (b in Figure 10) did not show interaction with CD25 at up to 2 μM. On the channel of CD122-Fc (c in Figure 10), IL-2 and FRC-2PEG (d in Figure 10) showed similar interactions with CD122. Therefore, selecting F42 and R38 sites for coupling PEG can almost completely block its interaction with CD25, but retain its interaction with CD122.

[0079] Based on this experiment, we tested the proliferative effects of IL-2 and FRC-2PEG on CTLL2 cells expressing trimeric receptors and MO7E cells expressing dimeric receptors. As shown in Figure 11, compared with IL2, FRC-2PEG coupled with two 10k-long polyethylene glycols still had no activating effect on CTLL2 at a working concentration of up to 100nM. However, compared with IL2, FRC-2PEG had a significant effect on the EC of MO7E cells. 50 It is only shifted to the right by 12.84 times, so the coupling of bis-PEG has little effect on lymphocytes expressing dimeric receptors.

[0080] Example 5 IL-2 mutants extend plasma half-life

[0081] Female C57BL mice were randomly divided into two groups (6 mice / group). Equal doses of IL-2 or FRC-2 PEG were injected via the tail vein. Blood was collected from the tail vein at different time points. IL-2 and FRC-2 PEG concentrations in each sample were determined by ELISA, and the data were processed using GraphPad Prism.

[0082] As shown in Figure 12, the half-life of FRC-2PEG was significantly longer than that of IL-2, with the concentration of IL-2 falling close to the detection limit after 8 hours, while FRC-2PEG could still be detected in plasma after the tenth day.

[0083] Example 6 IL-2 mutants significantly promote the proliferation of NK cells and CD8+ T cells without causing VLS doses.

[0084] C57BL6 mice were intraperitoneally injected with PBS, IL-2, and FRC-2PEG. Blood was collected at various times. The PBS and FRC-2PEG groups received a single dose at 0 hours, while the IL-2 group received continuous dosing at 0, 12, 24, 36, and 48 hours. Mice were euthanized at 54 hours, and whole blood and spleen samples were collected for flow cytometry analysis of T cell and NK cell counts.

[0085] The results are shown in Figures 13-14. At different time points, the IL-5 levels in the IL-2 group were significantly higher than those in the PBS and FRC-2PEG groups, and the FRC-2PEG group was essentially equivalent to the PBS group. This indicates that compared to high-frequency, high-dose injections of IL-2, a single dose of FRC-2PEG does not induce VLS. However, a single dose of FRC-2PEG can significantly promote the proliferation of NK cells and CD8 T cells in the peripheral blood and spleen of mice, but has a less pronounced effect on the proliferation of Treg cells (Figure 15).

[0086] Example 7 IL-2 mutants significantly inhibit tumor growth by promoting the proliferation of NK cells and CD8+ T cells

[0087] Female C57BL / 6N mice were randomly divided into PBS group, IL-2 group and FRC-2PEG group, with 5 mice in each group, and subcutaneously injected with B16F1. 3 Four days later, the second administration was carried out in the same way, and the tumor volume of mice was recorded every two days. When the tumor volume of mice in the PBS group reached 1500mm 3 At the same time, the mice were euthanized, and the whole blood, spleen, and tumor tissue were collected to analyze the lymphocyte components.

[0088] As shown in Figure 16, compared with the PBS group and the IL-2 group, FRC-2PEG can significantly inhibit tumor growth, while IL-2 has almost no effect in delaying tumor growth. Flow cytometry results show (Figure 17) that FRC-2PEG can significantly reduce the proportion of CD4+T cells in peripheral blood and spleen, and increase the proportion of CD8+T cells and NK cells in lymphocytes. At the same time, in the flow cytometry of tumor tissue (Figure 17), we can detect that the FRC-2PEG group can enhance the infiltration of lymphocytes and significantly increase the proportion of CD8+T cells and NK cells.

[0089] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: FRC-2PEG in this application can almost completely block its interaction with CD25, but retains its interaction with CD122. Because its interaction with CD25 is significantly reduced, the activation effect of lymphocytes on the trimeric receptor can be significantly weakened. In addition, FRC-2PEG has a long half-life in mice. In addition, the efficacy of FRC-2PEG in mice was evaluated, and it was found that a single dose of FRC-2PEG did not cause VLS, and could significantly promote the proliferation of NK cells and CD8T cells in the peripheral blood and spleen of mice, but the effect on the proliferation of Treg cells was not obvious. Therefore, compared with IL-2, which has almost no effect on delaying tumor growth, FRC-2PEG can significantly inhibit tumor growth. And in the flow cytometry of tumor tissue, it can be detected that the FRC-2PEG group can enhance the infiltration of lymphocytes and significantly increase the proportion of CD8+T cells and NK cells. This shows that the IL-2 mutant and its PEG conjugate of the present application have strong cell bias and provide a positive impact on the treatment of tumors.

[0090] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An interleukin-2 (IL-2) mutant, characterized in that include: The amino acid sequence of the protein shown in SEQ ID NO: 1 has an amino acid mutation at position 125 and at least one of the following positions has an amino acid mutation: F42 or R38, Among them, the protein having the amino acid sequence shown in SEQ ID NO: 1 is the IL-2 wild type, the IL-2 mutant has a lower ability to activate IL-2Rαβγ than the IL-2 wild type, and there is no obvious difference between the IL-2 mutant and the IL-2 wild type in their ability to activate IL-2Rβγ.

2. The IL-2 mutant according to claim 1, characterized in that The mutations of the IL-2 mutant are each independently selected from the following: F42X+C125J, R38X+C125J, or F42X+R38X+C125J, wherein the letters before the numbers represent the original amino acids, the letters after the numbers represent the mutated amino acids, the amino acid represented by X is any amino acid with a sulfhydryl group, and J represents any of the following amino acids: G, A, S, T, or V; Preferably, the IL-2 mutant is F42C+R38C+C125S; Preferably, the IL-2 mutant is an IL-2 mutant in which C125 is mutated to S and F42X and R38X directly form an intramolecular disulfide bond; Preferably, the IL-2 mutant is an IL-2 mutant in which C125 is mutated to S and the sulfhydryl groups on F42X and R38X are modified; Preferably, the IL-2 mutant is an IL-2 mutant in which C125 is mutated to S and the sulfhydryl groups on F42X and R38X are modified by DCA; Preferably, the structure in which the thiol groups on F42X and R38X are modified by DCA is as shown in Formula I:

3. The IL-2 mutant according to claim 2, characterized in that The activation ability of the wild type IL-2 on the IL-2Rαβγ complex was recorded as the first EC 50 The activation ability of the IL-2 mutant on the IL-2Rαβγ complex was recorded as the second EC 50 Value, the second EC 50 Value and the first EC 50 The ratio of the values ​​is recorded as n, where n≥74, preferably n≥256.

4. An IL-2 mutant conjugate, characterized in that The conjugate is an IL-2 protein-polyethylene glycol (PEG) conjugate obtained by performing PEG conjugation on the IL-2 mutant protein according to any one of claims 1 to 3.

5. The IL-2 mutant conjugate according to claim 4, characterized in that The PEG is PEG modified with a chemical coupling agent, the IL-2 protein is an IL-2 mutant having F42X and / or R38X mutation sites and C125J, and the sulfhydryl groups of the F42X and / or R38X are coupled to the PEG via the chemical coupling agent. The letters before the numbers represent the original amino acids, the letters after the numbers represent the mutant amino acids, the amino acids represented by X are any amino acids with sulfhydryl groups, and J represents any of the following amino acids: G, A, S, T, or V; Preferably, the chemical coupling agent is a compound with a hydroxylamino group or a hydrazide group; Preferably, the compound having a hydroxylamino group is selected from any one of the following: maleimide, succinimide; Preferably, the substituent having a hydrazide group is selected from an alkyl group, an aryl group or a heteroaryl group, the number of carbon atoms of the alkyl group is selected from 1 to 8, and the number of carbon atoms of the aryl group or the heteroaryl group is selected from 5 to 10.

6. A DNA molecule, characterized in that The DNA molecule encodes the IL-2 mutant according to any one of claims 1 to 3.

7. A recombinant plasmid, characterized in that The recombinant plasmid is connected to the DNA molecule according to claim 6.

8. A host cell, characterized in that The host cell is transformed with the recombinant plasmid according to claim 7.

9. Use of the IL-2 mutant according to any one of claims 1 to 3 or the IL-2 mutant conjugate according to claim 4 or 5 in the preparation of a medicament or preparation for treating cancer.

10. The use according to claim 9, characterized in that The cancer is selected from any one of the following: renal cancer, malignant melanoma, pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, mesothelioma, leukemia, multiple myeloma, lymphoma, liver cancer, sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary central nervous system lymphoma, primitive neuroectodermal tumor, bladder cancer, esophageal cancer, uterine cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer and gastric cancer.

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