WO2024104444A1 - Il-2 mutant and use thereof - Google Patents

Abstract

The present invention provides an IL-2 mutant and a use thereof. The IL-2 mutant comprises an amino acid mutation in position 125 of a protein represented by SEQ ID NO: 1 and an amino acid mutation in at least one of the following sites: F42 or R38, wherein the protein having the amino acid sequence represented by SEQ ID NO: 1 is wild-type IL-2, the activation ability of the IL-2 mutant to IL-2Rαβγ is lower than the activation ability of wild-type IL-2 to IL-2Rαβγ, and there is no obvious difference between the activation ability of the IL-2 mutant to IL-2Rαβγ and the activation ability of wild-type IL-2 to IL-2Rαβγ. The IL-2 mutant improves cell bias, can selectively activate CD8+T effector cells and NK cells, avoids immunosuppression caused by activation of Treg cells, and effectively solves the problem of low cell bias in the prior art.

Description

IL-2 mutants and their applications

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

The present invention relates to the field of genetic engineering, and in particular to an IL-2 mutant and application thereof.

Background technique

In recent years, with the PD-L1/PD-1 and CTLA-4 target research winning the 2018 Nobel Prize in Physiology or Medicine, immunotherapy has become one of the hot spots in current drug research. In November 1984, a female melanoma patient's whole body tumors disappeared only a few months after receiving high-dose IL-2 treatment [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 effective rate 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 induces Treg activation, inhibits immune response, and promotes tumor escape. Therefore, low-dose IL-2 cannot be used to treat tumors. The dosage window greatly limits the further clinical application of IL-2-related immunotherapy.

The receptor of IL-2 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 necessary 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, high expression of CD25 on their surface will be induced. 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 will undergo a subtle repositioning, which will greatly enhance its interaction with the CD122 and CD132 heterodimeric receptors and form a high-affinity tertiary complex (Kd≈10-50pM) [Science 2005; 310(5751): 1159-1163; Nature 2012; 484(7395): 529-533].

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, which is 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 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 medium-affinity IL-2Rβγ receptors 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 Treg, 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 trigger 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.

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 to allow higher dosages; (2) enhancing the affinity of IL-2 for the dimeric receptor, making it easier to activate effector T cells or NK cells to allow lower dosages. For example, NKTR-214 developed by Nektar Therapeutics modified IL-2 with 6 PEGs by non-site-specific conjugation to make it a prodrug, and it is assumed that it will eventually degrade into an active form with only one PEG or no PEG by gradual hydrolysis in the body. Since PEG conjugation will hinder the interaction between IL-2 and CD25, but retain its function of stimulating 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]. Since it is non-site-specific conjugation, it brings great challenges to the preparation and quality control of NKTR-214.

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, which has a strong anti-tumor effect. In terms of safety, even 1000ug/kg of THOR 707 will not cause VLS [WO2019028419A1].

However, the introduction of unnatural amino acids will affect the yield of proteins and increase the overall cost. At the same time, compared with wild-type IL-2, the EC ₅₀ of THOR707 on the proliferation activity of CTLL2 cells shifted by about 800 times, and the selected coupling site may not be the optimal site. In addition, through directed evolution, LEVIN et al. screened out the super mutant H9 of IL-2, which has a significantly improved affinity for CD122 compared with wild-type IL-2 [Nature 2012; 484(7395): 529-533].

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

Summary of the invention

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

To achieve the above-mentioned purpose, according to the first aspect of the present invention, there is provided an interleukin-2 (IL-2) mutant, comprising: an amino acid mutation at position 125 of the protein having the 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 the IL-2 wild type, the IL-2 mutant has a lower activation ability for IL-2Rαβγ than the IL-2 wild type, and there is no obvious difference between the activation ability of the IL-2 mutant for IL-2Rβγ and the activation ability of the IL-2 wild type for IL-2Rβγ.

Further, the mutations of the IL-2 mutants 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, and X represents The amino acid represented is any amino acid with a thiol group, and J represents any one 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 thiol 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 thiol 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:

Furthermore, the activation ability of wild-type IL-2 on the IL-2Rαβγ complex is recorded as the first _EC50 value, the activation ability of the above-mentioned IL-2 mutant on the IL-2Rαβγ complex is recorded as the second _EC50 value, and the ratio of the second _EC50 value to the first _EC50 value is recorded as n, wherein n≥74, preferably n≥256.

In order to achieve the above 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.

Furthermore, in the above-mentioned IL-2 mutant conjugate, PEG is PEG modified by 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 PEG through a chemical coupling agent in PEG, wherein 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 thiol 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 with a hydrazide group; preferably, the compound with a hydroxylamino group is selected from any 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 C atoms of the alkyl group is selected from 1 to 8, and the number of C atoms of the aryl group or the heteroaryl group is selected from 5 to 10.

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.

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 with the above DNA molecule.

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

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.

Furthermore, the 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 Malignant tumors, 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.

By applying the technical solution of the present invention, a site-directed mutagenesis is performed on the basis of the existing IL-2 to obtain a double cys mutant (FRC). The obtained 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, which means that the IL-2 mutant in the present invention can avoid the immunosuppression caused by the activation of Treg by the 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

The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

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

Fig. 2 is a schematic diagram showing the preparation process of FRC-DCA in Example 1 of the present invention, wherein "STAPLE" means staple.

FIG. 3 is a schematic diagram showing mass spectrum data of FRC in Example 1 of the present invention.

FIG. 4 is a schematic diagram showing mass spectrum data of FRC-DCA in Example 1 of the present invention.

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

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. The "Ratio" in Figure (d) means ratio.

FIG. 7 shows a schematic diagram of SDS-PAGE of FRC and FRC-2PEG in Example 3 of the present invention.

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

FIG. 9 shows a schematic diagram of size exclusion chromatography of FRC-2PEG in Example 3 of the present invention.

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

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).

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.

FIG. 12 shows a schematic diagram of the PK of FRC and FRC-2PEG in Example 5 of the present invention in mice.

FIG. 13 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.

FIG. 14 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.

FIG. 15 is a schematic diagram showing analysis of different cell ratios in peripheral blood and spleen lymphocytes in Example 6 of the present invention.

FIG. 16 shows a schematic diagram of the in vivo tumor inhibition effect analysis of FRC-2PEG in Example 7 of the present invention.

FIG. 17 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 ways

It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present 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.

As mentioned in the background technology, 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 transforming 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-point-specific foreign conjugates or non-natural amino acid modifications introduced therein all bring certain hidden dangers to the safety of the production, preparation and clinical use of the transformed IL-2 protein.

Therefore, in the present 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 the present application.

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 as shown in SEQ ID NO: 1 is a wild-type IL-2, the activation ability of the IL-2 mutant on IL-2Rαβγ is lower than that of the wild-type IL-2, and there is no obvious difference between the activation ability of the IL-2 mutant on IL-2Rβγ and that of the wild-type IL-2.

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

These mutants are characterized in that they stimulate cytotoxic effector CD8+ T cells and NK cells with higher selectivity than stimulating Treg cells, thus avoiding the immunosuppression caused by low-dose natural IL-2 activating Tregs; at the same time, high-dose IL-2 of the present invention The variant also does not induce VLS induced by high-dose natural IL-2 due to activation of CD31+ endothelial cells; in addition, the IL-2 mutant of the present invention significantly prolongs the time (half-life) of IL-2 in the body. The mutations of the IL-2 mutant of the present invention on 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 be greatly reduced during clinical use, thereby improving the patient's compliance with medication.

In a preferred embodiment of the present invention, the mutations of the IL-2 mutant are 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 mutant amino acids, the amino acids represented by X are any natural amino acids or synthetic amino acids with thiol groups, and J represents any of the following amino acids: G, A, S, T or V. In a preferred embodiment, the amino acid with thiol groups is cysteine (Cys); in a preferred embodiment, based on the mutation of C at position 125 to any one of G, A, S, T or V, F42C or R38C is independently Cys-ylated; in a preferred embodiment, based on the mutation of C at position 125 to any one of G, A, S, T or V, F42C and R38C are simultaneously Cys-ylated.

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., 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 by DCA; the structure in which the sulfhydryl groups on F42X and R38X are modified by DCA is shown in Formula I:

The activation ability of the above IL-2 wild type on the IL-2Rαβγ complex is recorded as the first EC ₅₀ value, the activation ability of the above IL-2 mutant on the IL-2Rαβγ complex is recorded as the second EC ₅₀ value, and the ratio of the second EC ₅₀ value to the first EC ₅₀ value is recorded as n, wherein n≥74, preferably n≥256. It can be seen that the activation ability of the IL-2 mutant on F42 and/or R38 is significantly reduced compared with the IL-2 wild type. The IL-2 mutant that meets the above conditions not only has lower toxic side effects, but also has stronger cell (CD8+T cells and/or NK cells) bias, and is more suitable for tumor treatment.

More preferably, the activation ability of IL-2 on the IL-2Rβγ complex is characterized by an EC ₅₀ value, wherein the activation ability of the IL-2 wild type on the IL-2Rβγ complex is recorded as the third EC ₅₀ value, the activation ability of the IL-2 mutant on the IL-2Rβγ complex is recorded as the fourth EC ₅₀ value, and the ratio of the fourth EC ₅₀ value to the third EC ₅₀ value is recorded as m. Then, when m≤15, it is considered that there is no obvious difference in the activation ability of the IL-2 wild type and the IL-2 mutant on the IL-2Rβγ complex.

Any one or both of the above two sites can be modified to any amino acid with a thiol group, which is convenient for coupling with PEG by using the thiol group. As for the specific type of such amino acid, there is no special limitation, and it can be directly Cys, or other natural or non-natural amino acids. According to the different amino acids selected, the type of variation can be reasonably selected.

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.

Wherein, the PEG in the above IL-2 mutant conjugate is PEG modified by a chemical modifier, the IL-2 protein is an IL-2 mutant having F42X and/or R38X mutation sites and C125J, the thiol groups of F42X and/or R38X are coupled to PEG via a chemical coupling agent in PEG, wherein 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 thiol groups, 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 with a hydroxylamino group or a hydrazide group; in a preferred embodiment, the compound with a hydroxylamino group is selected from any of the following: maleimide, succinimide; in a preferred embodiment, the substituent with a hydrazide group is selected from an alkyl group, an aryl group or a heteroaryl group, the number of C atoms of the alkyl group is selected from 1 to 8, and the number of C atoms of the aryl group or the heteroaryl group is selected from 5 to 10.

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 (tri(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 a secondary nickel column, the unreacted mal-PEG is removed, and a molecular sieve is passed to directly obtain the purified coupling product FRC-2PEG. The product obtained after the above-mentioned mutant is coupled with PEG by sulfhydryl has small toxicity and side effects, and has cell bias.

The activation ability of the IL-2 wild type on the IL-2Rαβγ complex is recorded as the first EC ₅₀ value, the activation ability of the above IL-2 mutant conjugate on the IL-2Rαβγ complex is recorded as the second EC ₅₀ value, and the ratio of the second EC ₅₀ value to the first EC ₅₀ value 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.

In a preferred embodiment, the coupling method of FRC-PEG includes, after the above IL-2 mutant is expressed and purified by E. coli BL21, TCEP with a final concentration of 0.1mM is added to the purified FRC, and after a warm water bath at 37°C for 2h, about 1/3 volume of 200mM NaHCO3 buffer is added. 4 molar equivalents of 1,3-dichloroacetone (DCA) are added to the reduced FRC, and after sufficient mixing, the reaction is carried out at 4°C overnight. NH2O-PEG is added to the FRC-DCA filtered through a desalting column for orthogonal coupling of the carbonyl group. The chemical reaction is shown in the following formula. After reacting at room temperature for 1 day, the coupling product FRC-PEG is obtained by molecular sieve separation.

The IL-2 mutant FRC (F42C+R38C+C125S) of the present invention exhibits a strong cell bias, that is, it has a reduced ability to activate CD25, while substantially retaining the ability to activate the IL-2Rβγ complex, and thus has a reduced ability to activate the IL-2Rαβγ complex. In order to further improve the cell bias of the mutant, the inventors modified the FRC with DCA, which further stabilized the structure of Cys and provided a group that facilitated the coupling of PEG with the IL-2 mutant. Whether it is direct PEG coupling or PEG coupling modified by DCA, it helps to further reduce the binding of FRC with CD25 (IL-2Rα) by increasing steric hindrance; the IL-2 mutant conjugate after PEG coupling has no ability to bind CD25, nor has it the ability to activate the IL-2Rαβγ complex; in addition, PEG coupling also further prolongs the half-life of the IL-2 mutant in vivo, which can reduce the frequency of administration in clinical use and improve the compliance of patients with medication.

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

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.

In a fifth typical embodiment of the present application, a host cell is provided, in which the above-mentioned recombinant plasmid is transformed. The above-mentioned host cell can be used to replicate the recombinant plasmid in the host cell, and the DNA molecules carried on the recombinant plasmid can also be transcribed and translated to obtain a large number of IL-2 mutants.

In a preferred embodiment, the host cell is BL21, and the recombinant plasmid is transformed therein, cultured at 30°C until OD600 = 0.4-0.6, and induced at 42°C. After high-pressure crushing, the inclusion bodies are purified, and after dissolving the inclusion bodies with guanidine hydrochloride denaturant, they are dialyzed into TRIS ph8.5 buffer. After the dialysate is purified by a nickel column, the eluate is concentrated and directly passed through a molecular sieve to obtain a pure IL-2 mutant (FRC).

In the sixth typical 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 preparation 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 malignant tumors, 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 small toxic and side effects, and can significantly weaken or even completely eliminate the activation effect of Treg cells on the IL-2Rαβγ trimer receptor, and can be better used for tumor treatment.

The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

Example 1 Preparation of IL2 mutant FRC and its staple product

In order to prepare an IL-2 mutant that can completely block the interaction with CD25, we selected the most critical site in the interaction epitope between IL-2 and CD25, mutated it to Cys, and mutated the remaining Cys site at position 125, and then site-specifically coupled PEG to block the interaction between the two through the steric hindrance of PEG. We performed structural simulations of the protein and found that after mutations of R38 and F42 to Cysteine and C125 to Ser, the side chains of these residues were exposed on the protein surface and pointed toward CD25 (Figure 1).

We observed that the side chains of the two Cys on the mutant FRC were relatively close. In order to make the two Cys more stable, we planned to use 1,3-dichloroacetone (DCA) to staple the two Cys into a peptide structure and introduce a carbonyl group (Figure 2). The introduction of the carbonyl group can serve as an active group for further coupling.

Through point mutation, the expression plasmid of IL-2 mutant F42C+R38C+C125S (hereinafter referred to as FRC) was successfully constructed. After transformation into BL21 expression strain, it was cultured at 30℃ until OD600=0.4-0.6 and induced at 42℃. High-pressure crushing, inclusion body purification, using 8M guanidine hydrochloride denaturant containing 2mM cysteine to dissolve the inclusion body, and then dialyzed into TRIS ph8.5 buffer. After the dialysate was purified by nickel column, the eluate was concentrated and directly passed through molecular sieves to obtain pure FRC. Through mass spectrometry, we found that FRC has two molecular weights, corresponding to the two states of the two Cys on FRC: one is that C38 and C42 directly formed a pair of disulfide bonds (mass spectrometry molecular weight 15433.11Da), and the other is that both C38 and C42 residues were Cys-ylated (mass spectrometry molecular weight 15673.40Da). It is speculated that the free Cys in FRC may react with the Cys in the solution during the renaturation process (Figure 3).

Add TCEP to a final concentration of 0.1 mM to the prepared FRC, warm it in 37°C water for 2 hours, and then add about 1/3 volume of 200 mM NaHCO ₃ buffer. Add 4 molar equivalents of 1,3-dichloroacetone (DCA) to the reduced FRC, mix thoroughly, and react overnight at 4°C. The next day, directly remove TCEP, reduced Cys, and unreacted DCA from the solution through a desalting column. The prepared FRC-DCA was subjected to mass spectrometry detection, and we found that the whole process reacted very fully. There was only a single molecular weight peak in the mass spectrometry data, and the molecular weight corresponded to FRC-DCA, and there was no unreacted FRC molecular weight remaining (Figure 4).

On this basis, we added NH ₂ O-PEG to FRC-DCA for orthogonal coupling of carbonyl groups to prepare PEGylated products. After one day of reaction at room temperature, we directly separated the coupling product FRC-PEG using molecular sieves (Figure 5).

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

To test the stimulatory activity of FRC, FRC-DCA, and FRC-PEG on different T cells, based on Example 1, we tested the proliferative 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. As shown in Figure 6, compared with IL-2, the EC ₅₀ of FRC without PEG showed a significant shift (shifted to the right by about 256 times), indicating that while containing the C125S mutation, the mutation of the two sites of F42 and R38 to Cys (whether F42C and R38C form an intramolecular disulfide bond, or F42C and R38C are modified by Cys respectively) can significantly reduce its interaction with CD25, thereby weakening the activation of CTLL2 cells. The EC ₅₀ of FRC-DCA shifted 74 times to the right, probably because the steric hindrance effect of FRC-DCA is smaller than that of the two Cys-modified Cys on FRC. After coupling with PEG, the EC ₅₀ of FRC-PEG shifted directly to the right by 2140 times, which was significantly stronger than the blocking effect of Thor-70 by 7800 times. For MO7E cells expressing IL-2Rβγ, even after coupling with PEG, its EC ₅₀ only shifted to the right by about 3.45 times, which means that by "staple" the F42 and R38 sites and then coupling with PEG, it has almost no effect on the binding of its IL-2Rβγ receptor, but can effectively block the interaction between FRC and IL-2Rα.

Example 3 Preparation and Activity Test of FRC-2PEG

Considering that the preparation process of FRC-PEG is relatively complicated, it needs to be prepared by a two-step coupling method. Although we selected F42 and R38 for point mutation while containing C125S mutation, the blocking effect of coupling PEG after preparing FRC-DCA is not particularly different from that of coupling single PEG. Therefore, we consider directly preparing the coupling product FRC-2PEG of double PEG by one-step coupling on the basis of FRC, hoping that double PEG can bring stronger blocking effect to FRC. Therefore, we added TCEP with a final concentration of 0.1mM to the FRC prepared in Example 1, reacted at 37°C for 2h, and then directly added 12 molar equivalents of Maleimide-PEG, and reacted at 4°C overnight after sufficient mixing. FRC-2PEG coupled with double PEG can be directly prepared by molecular sieve separation (Figure 7-9). Therefore, compared with FRC-PEG, FRC-2PEG only needs one-step coupling to prepare, and the preparation process is simpler.

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

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 fixed 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 fixed on the CM5 chip at a flow rate of 10 μL/min until the RU value reached about 2000RU. The fixed CM5 chip was blocked with ethanolamine.

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

As shown in Figure 10, on the channel of CD25-Fc (a in Figure 10), IL-2 shows fast binding and slow dissociation kinetics, while FRC-2PEG (b in Figure 10) still does 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) show similar interactions to CD122. Therefore, selecting F42 and R38 sites to couple PEG can almost completely block its interaction with CD25, but retain its interaction with CD122.

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-length polyethylene glycols still had no activation effect on CTLL2 at a working concentration of up to 100nM. However, compared with IL2, the EC ₅₀ of FRC-2PEG for MO7E cells was only shifted to the right by 12.84 times, so the coupling of double PEG had little effect on lymphocytes expressing dimeric receptors.

Example 5 IL-2 mutants extend plasma half-life

Female C57BL mice were randomly divided into two groups (6 mice/group). Equal doses of IL-2 or FRC-2PEG were injected through the tail vein. Venous blood was collected from the tail vein at different time points. The concentrations of IL-2 and FRC-2PEG in each sample were detected by ELISA, and the data were processed by GraphPad Prism.

As shown in Figure 12, the half-life of FRC-2PEG is significantly better than that of IL-2, and the concentration of IL-2 has fallen close to the detection limit at 8 hours. However, FRC-2PEG can still be detected in plasma after the tenth day.

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

PBS, IL-2, and FRC-2PEG were injected into C57BL6 mice by intraperitoneal injection, and blood was collected at different times. The PBS group and the FRC-2PEG group were only given a single dose at the 0h time point, and the IL-2 group was continuously given at 0h, 12h, 24h, 36h, and 48h. The mice were euthanized at 54h, and whole blood and spleen were collected to analyze the content of T cells and NK cells by flow cytometry.

The results are shown in Figures 13-14. In the blood samples at different time points, the IL-5 in the IL-2 group was significantly higher than that in the PBS group and the FRC-2PEG group, and the FRC-2PEG group was basically equivalent to the PBS group. This shows that compared with high-frequency and high-dose injections of IL-2, a single dose of FRC-2PEG does not cause VLS. However, a single dose of FRC-2PEG can 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 is not obvious (Figure 15).

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

Female C57BL/6N mice were randomly divided into PBS group, IL-2 group and FRC-2PEG group, 5 mice in each group, and subcutaneously injected with B16F1. When the tumor size reached a volume of about ^50mm3 , the drug was administered by intraperitoneal injection. Four days later, the second administration was performed in the same way, and the tumor volume of the mice was recorded every two days. When the tumor volume of the mice in the PBS group reached ^1500mm3 , the mice were euthanized, and the whole blood, spleen, and tumor tissue were taken to analyze the lymphocyte components.

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. The 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.

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 the present application can almost completely block its interaction with CD25, but retains the interaction with CD122. Because its interaction with CD25 is significantly reduced, the activation of lymphocytes of the trimer 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 proliferation effect on 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 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. It can be seen that the IL-2 mutant and its PEG conjugate of the present application have strong cell bias and provide a positive effect on the treatment of tumors.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. An interleukin-2 (IL-2) mutant, characterized by comprising:

   The protein with the amino acid sequence 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 wild type of IL-2, the activation ability of the IL-2 mutant on IL-2Rαβγ is lower than that of the wild type of IL-2, and there is no obvious difference between the activation ability of the IL-2 mutant on IL-2Rβγ and that of the wild type of IL-2.
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 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;

   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 is characterized in that the activation ability of the IL-2 wild type on the IL-2Rαβγ complex is recorded as a first _EC50 value, the activation ability of the IL-2 mutant on the IL-2Rαβγ complex is recorded as a second _EC50 value, and the ratio of the second _EC50 value to the first _EC50 value is recorded as n, wherein 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 PEG conjugation based 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 a PEG modified by 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 recombinant plasmid according to claim 7 is transformed into the host cell.
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 is 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 malignant tumors, 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.