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WO2024104444A1 - Il-2突变体及其应用 - Google Patents

Il-2突变体及其应用 Download PDF

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WO2024104444A1
WO2024104444A1 PCT/CN2023/132133 CN2023132133W WO2024104444A1 WO 2024104444 A1 WO2024104444 A1 WO 2024104444A1 CN 2023132133 W CN2023132133 W CN 2023132133W WO 2024104444 A1 WO2024104444 A1 WO 2024104444A1
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mutant
cancer
frc
peg
group
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PCT/CN2023/132133
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English (en)
French (fr)
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王峰
董超
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南通壹宸生物医药科技有限公司
中国科学院生物物理研究所
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Priority to CN202380078614.9A priority Critical patent/CN120225547A/zh
Publication of WO2024104444A1 publication Critical patent/WO2024104444A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2

Definitions

  • the present invention relates to the field of genetic engineering, and in particular to an IL-2 mutant and application thereof.
  • IL-2 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.].
  • VLS vascular permeability syndrome
  • 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-2R ⁇ (CD25), IL-2R ⁇ (CD122), and IL-2R ⁇ (CD132).
  • 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].
  • IL-2 interacts with receptors on different cells expressing different subunits, producing different biological activities.
  • regulatory T cells constitutively express the high-affinity trimeric receptor IL-2R ⁇ , 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.
  • autoimmune diseases such as vasculitis, inflammatory myopathy, and systemic lupus erythematosus (SLE). Effector T cells and natural killer cells express medium-affinity IL-2R ⁇ receptors and require higher concentrations of IL-2 to be effectively stimulated.
  • IL-2 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].
  • CD31+ pulmonary endothelial cells express low to moderate levels of IL-2R ⁇ 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.
  • 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.
  • 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].
  • 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.
  • 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-2R ⁇ than the IL-2 wild type, and there is no obvious difference between the activation ability of the IL-2 mutant for IL-2R ⁇ and the activation ability of the IL-2 wild type for IL-2R ⁇ .
  • 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
  • the activation ability of wild-type IL-2 on the IL-2R ⁇ complex is recorded as the first EC50 value
  • the activation ability of the above-mentioned IL-2 mutant on the IL-2R ⁇ complex is recorded as the second EC50 value
  • the ratio of the second EC50 value to the first EC50 value is recorded as n, wherein n ⁇ 74, preferably n ⁇ 256.
  • an IL-2 mutant conjugate which is an IL-2 protein-polyethylene glycol (PEG) conjugate obtained by PEG conjugation based on the above-mentioned IL-2 mutant protein.
  • PEG polyethylene glycol
  • the IL-2 protein 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 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
  • 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
  • a DNA molecule which encodes the above IL-2 mutant.
  • a recombinant plasmid is provided, wherein the recombinant plasmid is connected with the above DNA molecule.
  • a host cell wherein the above recombinant plasmid is transformed into the host cell.
  • 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.
  • 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-2R ⁇ complex.
  • 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.
  • 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
  • the concentration of the curves decreases by 2 times from top to bottom (the corresponding concentrations are: 2 ⁇ M, 1 ⁇ M, 0.5 ⁇ M, 0.25 ⁇ M, 0.125 ⁇ M, 0.062 ⁇ M, 0.031 ⁇ M, 0.015 ⁇ M, 0.0078 ⁇ M and 0.0039 ⁇ M).
  • 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.
  • 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.
  • 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-2R ⁇ trimer receptor, and thus better use it in tumor treatment.
  • the applicant proposed a series of protection schemes of the present application.
  • an IL-2 mutant 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-2R ⁇ 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-2R ⁇ and that of the wild-type IL-2.
  • SEQ ID NO: 1 wild type
  • 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.
  • 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.
  • 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.
  • Cys cysteine
  • 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-2R ⁇ complex is recorded as the first EC 50 value
  • the activation ability of the above IL-2 mutant on the IL-2R ⁇ complex is recorded as the second EC 50 value
  • 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.
  • the activation ability of IL-2 on the IL-2R ⁇ complex is characterized by an EC 50 value, wherein the activation ability of the IL-2 wild type on the IL-2R ⁇ complex is recorded as the third EC 50 value, the activation ability of the IL-2 mutant on the IL-2R ⁇ 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-2R ⁇ 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.
  • a thiol group which is convenient for coupling with PEG by using the thiol group.
  • 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.
  • an IL-2 mutant conjugate which is an IL-2 protein-polyethylene glycol (PEG) conjugate obtained by PEG conjugation based on the above-mentioned IL-2 mutant protein.
  • PEG polyethylene glycol
  • 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
  • J represents any of the following amino acids: G, A, S, T or V.
  • 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 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.
  • TCEP tri(2-carboxyethyl)phosphine
  • 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-2R ⁇ complex is recorded as the first EC 50 value
  • the activation ability of the above IL-2 mutant conjugate on the IL-2R ⁇ complex is recorded as the second EC 50 value
  • 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-2R ⁇ complex.
  • 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.
  • DCA 1,3-dichloroacetone
  • 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-2R ⁇ complex, and thus has a reduced ability to activate the IL-2R ⁇ complex.
  • 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.
  • a DNA molecule which encodes the above-mentioned IL-2 mutant.
  • a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.
  • a host cell 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.
  • 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).
  • 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
  • FRC expression plasmid of IL-2 mutant F42C+R38C+C125S
  • 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).
  • Example 2 IL-2 mutants weaken the interaction between IL-2 and CD25, thereby reducing the interaction with IL-2R ⁇
  • 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.
  • CD25-Fc and CD122-Fc were diluted to 50 ⁇ g/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 ⁇ L/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 ⁇ L/min, and the corresponding binding dissociation constants were calculated based on the curve.
  • Example 5 IL-2 mutants extend plasma half-life
  • mice 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.
  • FRC-2PEG 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.
  • Example 7 IL-2 mutants significantly inhibit tumor growth by promoting the proliferation of NK cells and CD8+T cells
  • mice 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.
  • the tumor size reached a volume of about 50mm3
  • the drug was administered by intraperitoneal injection.
  • the second administration was performed in the same way, and the tumor volume of the mice was recorded every two days.
  • the mice were euthanized, and the whole blood, spleen, and tumor tissue were taken to analyze the lymphocyte components.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

本发明提供了一种IL-2突变体及其应用。该IL-2突变体包括如SEQ ID NO：1所示的氨基酸序列的蛋白的第125位发生氨基酸突变以及如下至少一个位点发生氨基酸突变：F42或R38，其中，具有SEQ ID NO：1所示的氨基酸序列的蛋白为IL-2野生型，IL-2突变体对IL-2Rαβγ的激活能力低于IL-2野生型对IL-2Rαβγ的激活能力，IL-2突变体对IL-2Rβγ的激活能力与IL-2野生型对IL-2Rβγ的激活能力无明显区别。该IL-2突变体提高细胞偏向性，可选择性激活CD8+T效应细胞及NK细胞，避免了对Treg细胞激活带来的免疫抑制，有效解决了现有技术中细胞偏向性较低的问题。

Description

IL-2突变体及其应用
本申请是以CN申请号为202211442050.6，申请日为2022年11月17日的中国申请为基础，并主张其优先权，该CN申请的公开内容再次作为整体引入本申请中。
技术领域
本发明涉及基因工程领域，具体而言，涉及一种IL-2突变体及其应用。
背景技术
近年来，随着PD-L1/PD-1和CTLA-4靶点研究获得2018年诺贝尔生理学或医学奖，免疫治疗成为当前药物研究的热点之一。1984年11月，一位女性黑色素瘤患者在接受大剂量IL-2治疗仅几个月后，全身肿瘤消失[Rosenberg SA etal.N Engl J Med 1985；313(23)1485-92]。基于IL-2在治疗癌症上的显著疗效，FDA分别在1992年和1998年批准了高剂量IL-2用于治疗晚期肾癌和恶性黑色素瘤，其有效率为15％-20％。虽然IL-2的使用剂量越高，抗肿瘤越好，但高剂量的IL-2会引起严重的血管渗透综合征(VLS)，导致人体器官的积水，引发肺水肿和肝脏细胞损伤等[DF McDermott，Oncoimmunology.2016 Jun；5(6)：e1163462.]。近年来的研究发现低剂量的IL-2会优先诱导Treg的激活，抑制免疫反应，促进肿瘤逃逸。因此低剂量的IL-2不能被用于治疗肿瘤。剂量窗口极大地限制了IL-2相关免疫疗法的进一步临床应用。
IL-2的受体主要由IL-2Rα(CD25)，IL-2Rβ(CD122)，IL-2Rγ(CD132)三个亚基组成。其中CD25能与IL-2发生低亲和力的结合(Kd≈10nM)，CD25不是信号传导所必须的[Ye CX，et al.Siganl Transduct Target Ther 2018；3：2]。静息状态下的T细胞表达很低水平的CD25，一旦T细胞被激活，会诱导其表面CD25的高表达。CD122与CD132构成IL-2中等亲和力(Kd≈1nM)的异二聚体受体，对下游增殖信号至关重要[Math Med Biol 2018；35(1)：79-119]。当IL-2与CD25结合后，IL-2的构象会发生轻微的变化(subtle repositioning)，这将会极大提高其与CD122，CD132异二聚体受体的相互作用，并形成高亲和力的三级复合物(Kd≈10-50pM)[Science 2005；310(5751)：1159-1163；Nature 2012；484(7395)：529-533]。
IL-2与表达不同亚基的不同细胞上的受体相互作用，产生不同的生物学活性。例如，调节性T细胞组成型表达高亲和力的三聚体受体IL-2Rαβγ，对低浓度的IL-2更为敏感，因此低剂量IL-2可活化并促进Treg细胞的增殖，抑制免疫系统过度激活进而调节免疫稳态，目前已有多项临床结果表明，低剂量IL-2对于多种自身免疫性疾病，如低剂量IL-2对于多种自身免疫性疾病，如血管炎，炎性肌病及系统性红斑狼疮(systemic lupus erythematosus，SLE)及等有效。效应T细胞和自然杀伤细胞表达中等亲和力的IL-2Rβγ受体，需要较高浓度的IL-2才能有效刺激。因此，在利用IL-2进行免疫治疗的时候，必须使用高剂量才能尽可能地激活Teff和NK细胞[Nat Rev Immunol 2018；18(10)：648-659]。然而，除了Treg，CD31+肺内皮细胞上表达有低至中等水平的IL-2Rαβγ三聚体受体，因此高剂量的IL-2又会不可避免地与其相互作用，引发 严重的VLS[Proc Natl Acad Sci USA2010；107(26)：11906-11911]。研发具有细胞选择性刺激淋巴细胞增殖的IL-2是解决其毒副作用的关键。
现有的改造方案一般从两种互补的方式入手：(1)降低IL-2对三聚体受体的亲和力或者降低IL-2与其受体CD25的相互作用，使其成为一种毒性较小的分子，以允许更高的给药剂量；(2)增强IL-2与二聚体受体的亲和力，使其更易活化效应T细胞或NK细胞，以允许更低的给药剂量。例如Nektar Therapeutics公司研发的NKTR-214，它通过非定点偶连的方式在IL-2上修饰了6个PEG将其做成前药，并假设通过在体内逐渐水解的方式，最终降解为只有一个PEG或者没有PEG的活性形式。由于PEG的偶连会阻碍IL-2与CD25的相互作用，但保留了它偏向激动CD122的功能，使其更偏向于激活CD8 T及NK细胞起到杀伤肿瘤的作用[Clin Cancer Res 2016；22(3)：680-690；Plos One 2017；12(7)：e0179431]。由于是非定点偶连，给NKTR-214的制备和质控带来很大的挑战。
其次，只有当蛋白降解至一个PEG的状态下才具有活性，导致蛋白有效浓度降低，严重影响其抗肿瘤活性。Synthorx公司的THOR-707在此基础上，通过非天然氨基酸技术，在IL-2第65位上的脯氨酸上插入带有叠氮基团的赖氨酸衍生物，然后通过点击化学的手段，定点偶连30k长度的聚乙二醇高分子[Nature Communication 2021；12(1)：1-14]。定点偶连的PEG不仅显著延长了IL-2的半衰期，同时有效阻挡了IL-2与CD25的相互作用，使其偏向于促进CD8 T细胞的扩增，增加CD8+T的肿瘤浸润，起到很强的抗肿瘤效果，并且在安全性方面，即使1000ug/kg的THOR 707也不会引起VLS[WO2019028419A1]。
然而，引入非天然氨基酸，会影响蛋白的产量，增加整体的成本。同时，相比于野生型的IL-2，THOR707对CTLL2细胞的促增殖活性EC50偏移了约800倍，选取的偶连位点可能并非是最佳位点。除此之外，通过定向进化的方式，LEVIN等人筛选出了IL-2的超级突变体H9，相比于野生型IL-2，H9对CD122的亲和力有了显著的提高[Nature 2012；484(7395)：529-533]。
因此构建一个具有更强细胞偏向性的IL-2突变体已成为领域内研究的热点。
发明内容
本发明的主要目的在于提供一种IL-2突变体及其应用，以解决现有技术中改造的IL-2的细胞偏向性较低的问题。
为了实现上述目的，根据本发明的第一个方面，提供了一种白细胞介素-2(IL-2)突变体，包括：如SEQ ID NO：1所示的氨基酸序列的蛋白的第125位发生氨基酸突变以及如下至少一个位点发生氨基酸突变：F42或R38，其中，具有SEQ ID NO：1所示的氨基酸序列的蛋白为IL-2野生型，IL-2突变体对IL-2Rαβγ的激活能力低于IL-2野生型对IL-2Rαβγ的激活能力，IL-2突变体对IL-2Rβγ的激活能力与IL-2野生型对IL-2Rβγ的激活能力无明显区别。
进一步地，IL-2突变体的突变各自独立地选自如下：F42X+C125J、R38X+C125J或F42X+R38X+C125J，其中，数字前字母代表原始氨基酸，数字后字母代表突变氨基酸，X所 代表的氨基酸为任意带有巯基的氨基酸，J代表如下任意一种氨基酸：G、A、S、T或V；优选地，IL-2突变体为F42C+R38C+C125S；优选地，IL-2突变体为C125突变为S且F42X和R38X直接形成分子内二硫键的IL-2突变体；优选地，IL-2突变体为C125突变为S且F42X和R38X上的巯基被修饰的IL-2突变体；优选地，IL-2突变体为C125突变为S且F42X和R38X上的巯基被DCA所修饰的IL-2突变体；优选地，F42X和R38X上的巯基被DCA所修饰的结构如式I所示：
进一步地，IL-2野生型对IL-2Rαβγ复合物的激活能力记为第一EC50值，上述IL-2突变体对IL-2Rαβγ复合物的激活能力记为第二EC50值，第二EC50值与第一EC50值的比值记为n，其中，n≥74，优选n≥256。
为了实现上述目的，根据本发明的第二个方面，提供了一种IL-2突变体偶联物，该偶联物为在上述的IL-2突变体蛋白质基础上，进行PEG偶联，所获得的IL-2蛋白-聚乙二醇(PEG)偶联物。
进一步地，上述IL-2突变体偶联物中PEG为化学修饰剂修饰的PEG，IL-2蛋白为具有F42X和/或R38X突变位点以及C125J的IL-2突变体，F42X和/或R38X的巯基通过PEG中的化学偶联剂与PEG偶联，其中，数字前字母代表原始氨基酸，数字后字母代表突变氨基酸，X所代表的氨基酸为任意带有巯基的氨基酸，J代表如下任意一种氨基酸：G、A、S、T或V；优选地，化学偶联剂为带有羟氨基或带有酰肼基的化合物；优选地，带有羟氨基的化合物选自如下任意一种：马来酰亚胺、琥珀酰亚胺；优选地，带有酰肼基的取代基选自烷基、芳基或杂芳基，烷基的C原子数选自1～8，芳基或杂芳基的C原子数选自5～10。
为了实现上述目的，根据本发明的第三个方面，提供了一种DNA分子，该DNA分子编码上述的IL-2突变体。
为了实现上述目的，根据本发明的第四个方面，提供了一种重组质粒，该重组质粒连接有上述的DNA分子。
为了实现上述目的，根据本发明的第五个方面，提供了一种宿主细胞，该宿主细胞内转化有上述的重组质粒。
为了实现上述目的，根据本发明的第六个方面，提供了一种上述IL-2突变体或IL-2突变体偶联物在制备治疗癌症的药物或制剂中的用途。
进一步地，上述癌症选自如下任意一种：肾癌、黑色素瘤、胰腺癌、骨癌、前列腺癌、小细胞肺癌、非小细胞肺癌、间皮瘤、白血病、多发性骨髓瘤、淋巴瘤、肝癌、肉瘤、B细胞 恶性肿瘤、乳腺癌、卵巢癌、结直肠癌、神经胶质瘤、多形性胶质母细胞瘤、脑膜瘤、垂体腺瘤、前庭神经鞘瘤、原发性中枢神经系统淋巴瘤、原始神经外胚层肿瘤、膀胱癌、食道癌、子宫癌、脑癌、头颈癌、宫颈癌、睾丸癌、甲状腺癌和胃癌。
应用本发明的技术方案，在现有IL-2的基础上进行定点突变，获得双cys突变体(FRC)，所得FRC具有较高的细胞偏向性，其具有降低的激活CD25的能力，同时基本保留激活IL-2Rβγ复合物的能力。由于大大减少其与CD25的相互作用，且保留了激活IL-2Rβγ复合物的能力，因此FRC对IL-2Rαβγ三聚体的激活能力大大减弱，这意味着本发明中的IL-2突变体可避免天然IL-2低剂量使用激活Treg激活而导致的免疫抑制，选择性激活CD8+T细胞和/或NK细胞，在临床中可以高剂量使用而达到肿瘤治疗的效果，对肿瘤的治疗提供了积极影响。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了本发明的实施例1中通过Alphafold预测的F42C+R38C+C125S突变的结构示意图。
图2示出了本发明的实施例1中FRC-DCA制备过程的示意图。其中的“STAPLE”意为订书钉。
图3示出了本发明的实施例1中的FRC的质谱数据的示意图。
图4示出了本发明的实施例1中的FRC-DCA的质谱数据的示意图。
图5示出了本发明的实施例1中纯化出的FRC和FRC-PEG蛋白鉴定的示意图。
图6示出了本发明的实施例2中IL-2、FRC、FRC-DCA对不同细胞的激活效果示意图。其中(d)图中的“Ratio”意为比值。
图7示出了本发明的实施例3中的FRC与FRC-2PEG的SDS-PAGE示意图。
图8示出了本发明的实施例3中的FRC的排阻层析示意图。
图9示出了本发明的实施例3中的FRC-2PEG的排阻层析示意图。
图10示出了本发明的实施例4中的FRC-2PEG与CD25、CD122结合的SPR检测结果，
其中在a、b、c和d中，从上到下曲线浓度依次按2倍递减(所对应的浓度分别为：2μM、1μM、0.5μM、0.25μM、0.125μM、0.062μM、0.031μM、0.015μM、0.0078μM及0.0039μM)。
图11示出了本发明的实施例4中的IL-2、FRC-2PEG对不同细胞的激活效果的示意图。
图12示出了本发明的实施例5中的FRC和FRC-2PEG在小鼠体内PK示意图。
图13示出了本发明的实施例6中的第一采血点不同给药组血浆中IL-5浓度示意图。
图14示出了本发明的实施例6中的第二采血点不同给药组血浆中IL-5浓度示意图。
图15示出了本发明的实施例6中的外周血和脾脏淋巴细胞中不同细胞比例分析示意图。
图16示出了本发明的实施例7中的FRC-2PEG体内抑瘤效果分析示意图。
图17示出了本发明的实施例7中的外周血、脾脏、和肿瘤淋巴细胞中不同细胞比例分析示意图。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
如背景技术所提到的，低剂量的IL-2可活化并促进Treg细胞的增殖，导致免疫抑制，而高剂量的IL-2会引起严重的血管渗透综合征(VLS)，导致人体器官的积水等损伤，因此剂量窗口导致的毒副作用极大地限制了IL-2相关免疫疗法的进一步临床应用。由于不同浓度的IL-2对不同的淋巴细胞的激活程度不同，因此研发具有偏好选择性刺激目标淋巴细胞增殖的IL-2是解决IL-2局限性的关键。但是在改造IL-2的现有技术中，无论是通过降低IL-2与IL-2Rαβγ三聚体受体的亲和力还是通过增强IL-2与IL-2Rβγ二聚体受体的亲和力，其引入的非定点外来偶联物或是非天然氨基酸改造，都对改造后的IL-2蛋白生产、制备及临床使用的安全性带来一定的隐患问题。
因此，在本申请中发明人尝试通过构建突变体与PEG偶联物，进而解决IL-2的毒副作用，使其具有更强的细胞(NK细胞和CD8+T细胞)偏向性，可明显减弱对IL-2Rαβγ三聚体受体的Treg的活化作用，进而更好地用于肿瘤治疗中，在此基础上，申请人提出了本申请的一系列保护方案。
在本申请第一种典型的实施方式中，提供了一种IL-2突变体，包括如SEQ ID NO：1所示的氨基酸序列的蛋白的第125位发生氨基酸突变以及如下至少一个位点发生氨基酸突变：F42或R38，其中，具有SEQ ID NO：1所示的氨基酸序列的蛋白为IL-2野生型，IL-2突变体对IL-2Rαβγ的激活能力低于IL-2野生型对IL-2Rαβγ的激活能力，IL-2突变体对IL-2Rβγ的激活能力与IL-2野生型对IL-2Rβγ的激活能力无明显区别。
SEQ ID NO：1(野生型)的序列如下：
这些突变体的特征在于它们刺激细胞毒性效应CD8+T细胞和NK细胞比刺激Treg细胞具有更高的选择性，避免了低剂量天然IL-2激活Treg导致的免疫抑制；同时本发明高剂量的IL-2 变体也未诱发高剂量天然IL-2由于激活CD31+内皮细胞诱发的VLS；另外，本发明的IL-2突变体显著延长了IL-2在体内的时间(半衰期)。本发明的IL-2突变体在C125以及F42和/或R38上的突变解决了现有技术中低剂量IL-2导致的免疫抑制、高剂量导致的VLS等困扰肿瘤治疗的问题，不仅制备过程简便，同时在临床使用过程中可以大大降低给药频次，提高患者用药的依从性。
本发明的一种优选实施例中，IL-2突变体的突变各自独立地选自如下：F42X+C125J、R38X+C125J或F42X+R38X+C125J，其中，数字前字母代表原始氨基酸，数字后字母代表突变氨基酸，X所代表的氨基酸为任意带有巯基的天然氨基酸或合成氨基酸，J代表如下任意一种氨基酸：G、A、S、T或V。在一个优选的实施方案中，带有巯基的氨基酸为半胱氨酸(Cys)；在一个优选的实施方案中在第125位由C突变为G、A、S、T或V任一种氨基酸的基础上，F42C或R38C独立地被Cys化；在一个优选的实施方案中，在第125位由C突变为G、A、S、T或V任一种氨基酸的基础上，F42C和R38C同时被Cys化。
在一个优选的实施方案中，IL-2突变体为F42C+R38C+C125S；在一个优选的实施方案中，IL-2突变体为C125突变为S(即第125为的C突变为S)且F42X和R38X直接形成分子内二硫键的IL-2突变体；在一个优选的实施方案中，IL-2突变体为C125突变为S且F42X和R38X上的巯基被修饰的IL-2突变体；在一个优选的实施方案中，IL-2突变体为C125突变为S且F42X和R38X上的巯基被DCA所修饰的IL-2突变体；F42X和R38X上的巯基被DCA所修饰的结构如式I所示：
上述IL-2野生型对IL-2Rαβγ复合物的激活能力记为第一EC50值，上述IL-2突变体对IL-2Rαβγ复合物的激活能力记为第二EC50值，第二EC50值与第一EC50值的比值记为n，其中，n≥74，优选n≥256。可以看出，IL-2突变体在F42和/或R38上的突变对IL-2Rαβγ的激活能力与IL-2野生型相比显著降低。符合上述条件的IL-2突变体不仅具有较低的毒副作用，而且还具有更强的细胞(CD8+T细胞和/或NK细胞)偏向性，更适合应用于肿瘤治疗。
更优选地，将IL-2对IL-2Rβγ复合物的激活能力以EC50值来表征，其中，IL-2野生型对IL-2Rβγ复合物的激活能力记为第三EC50值，IL-2突变体对IL-2Rβγ复合物的激活能力记为第四EC50值，第四EC50值与第三EC50值的比值记为m，则，m≤15时，视为IL-2野生型与IL-2突变体对IL-2Rβγ复合物的激活能力无明显区别。
在上述两个位点中的任意一个或同时，改进为任意带有巯基的氨基酸，均便于利用巯基实现与PEG的偶联，至于此类氨基酸的具体类别并无特殊限定，可以直接是Cys，也可以是其他天然或非天然的氨基酸。根据所选取的氨基酸的不同，可以对变异的类型进行合理选择。
在本申请第二种典型的实施方式中，提供了一种IL-2突变体偶联物，该偶联物为在上述IL-2突变体蛋白质基础上，进行PEG偶联，所获得的IL-2蛋白-聚乙二醇(PEG)偶联物。
其中，上述IL-2突变体偶联物中的PEG为化学修饰剂修饰的PEG，IL-2蛋白为具有F42X和/或R38X突变位点以及C125J的IL-2突变体，F42X和/或R38X的巯基通过PEG中的化学偶联剂与PEG偶联，其中，数字前字母代表原始氨基酸，数字后字母代表突变氨基酸，X所代表的氨基酸为任意带有巯基的氨基酸，J代表如下任意一种氨基酸：G、A、S、T或V。在一种优选的实施例中，化学偶联剂为带有羟氨基或带有酰肼基的化合物；在一种优选的实施例中，带有羟氨基的化合物选自如下任意一种：马来酰亚胺、琥珀酰亚胺；在一优选的实施例中，带有酰肼基的取代基选自烷基、芳基或杂芳基，烷基的C原子数选自1～8，芳基或杂芳基的C原子数选自5～10。
上述PEG偶联方法不限，任何能够实现该目的的方式均适用于本申请。在一种优选的实施例中，FRC-2PEG的偶联方法包括，将上述IL-2突变体经过TCEP(三(2-羧乙基)膦)处理后，与10摩尔的mal-PEG在37℃孵育2h后，通过其马来酰亚胺与IL-2中F42C和R38C的半胱氨酸偶联，通过巯基和马来酰亚胺形成S-C共价键，该化学反应如下式所示。经过二次镍柱，除去未反应完的mal-PEG，通过分子筛以直接获得纯化的偶连产物FRC-2PEG。上述突变体经过巯基进行PEG偶联后得到的产物毒副作用小，且具有细胞偏向性。
将IL-2野生型对IL-2Rαβγ复合物的激活能力记为第一EC50值，上述IL-2突变体偶联物对IL-2Rαβγ复合物的激活能力记为第二EC50值，第二EC50值与第一EC50值的比值记为n，其中，≥2140。可以看出，IL-2突变体偶联物进一步降低了IL-2突变体对IL-2Rαβγ复合物的激活能力。
在一种优选的实施例中，FRC-PEG的偶联方法包括，将上述IL-2突变体经大肠杆菌BL21表达纯化后，在纯化后的FRC中加入终浓度0.1mM的TCEP，37℃温水浴2h后，加入约1/3体积的200mM NaHCO3缓冲液。向还原好的FRC中加入4摩尔当量的1，3-二氯丙酮(DCA)，充分混匀后，4℃反应过夜。在经脱盐柱过滤后的FRC-DCA中加入NH2O-PEG用于羰基的正交偶联，该化学反应如下式所示，常温反应1天后，通过分子筛分离得到偶联产物FRC-PEG。
本发明的IL-2突变体FRC(F42C+R38C+C125S)表现出较强的细胞偏向性，即具有降低的激活CD25的能力，同时基本保留激活IL-2Rβγ复合物的能力，进而具有降低的激活IL-2Rαβγ复合物的能力。为进一步地提高该突变体的细胞偏向性，发明人在FRC的基础上进行了DCA的修饰，DCA修饰则进一步稳定Cys的结构，同时提供便于PEG与IL-2突变体偶联的基团。无论是直接的PEG的偶联还是DCA修饰修改后的PEG偶联，均有助于通过增加空间位阻进一步降低FRC与CD25(IL-2Rα)的结合；经PEG偶联后的IL-2突变体偶联物无结合CD25的能力，也无激活IL-2Rαβγ复合物的能力；此外，PEG偶联也进一步延长了IL-2突变体在体内的半衰期，在临床使用中可减少给药频次，提高患者用药的依从性。
在本申请第三种典型的实施方式中，提供了一种DNA分子，该DNA分子编码上述IL-2突变体。
在本申请第四种典型的实施方式中，提供了一种重组质粒，该重组质粒连接有上述DNA分子。
在本申请第五种典型的实施方式中，提供了一种宿主细胞，该宿主细胞内转化有上述重组质粒。利用上述宿主细胞，能够在宿主细胞中进行重组质粒的复制，也能够将重组质粒上携带的DNA分子进行转录、翻译，获得大量IL-2突变体。
在一种优选的实施例中，上述宿主细胞为BL21，且在其中转化有上述重组质粒，在30℃培养至OD600＝0.4-0.6，42℃诱导。经高压破碎，包涵体精纯，使用盐酸胍变性剂溶解包涵体后，透析至TRIS ph8.5的buffer中。透析液通过镍柱纯化后，洗脱液浓缩直接经过分子筛，可得到纯的IL-2突变体(FRC)。
在本申请第六种典型的实施方式中，提供了一种IL-2突变体或IL-2突变体偶联物在制备治疗癌症的药物或制剂中的用途，包括癌症如下任意一种：肾癌、恶性黑色素瘤、胰腺癌、骨癌、前列腺癌、小细胞肺癌、非小细胞肺癌、间皮瘤、白血病、多发性骨髓瘤、淋巴瘤、肝癌、肉瘤、B细胞恶性肿瘤、乳腺癌、卵巢癌、结直肠癌、神经胶质瘤、多形性胶质母细胞瘤、脑膜瘤、垂体腺瘤、前庭神经鞘瘤、原发性中枢神经系统淋巴瘤、原始神经外胚层肿瘤、膀胱癌、食道癌、子宫癌、脑癌、头颈癌、宫颈癌、睾丸癌、甲状腺癌和胃癌。含有本申请的IL-2突变体的药物毒副作用小，且能明显减弱甚至完全消除对IL-2Rαβγ三聚体受体的Treg细胞的活化作用，进而能更好地用于肿瘤治疗。
以下结合具体实施例对本申请作进一步详细描述，这些实施例不能理解为限制本申请所要求保护的范围。
实施例1 IL2突变体FRC及其staple产物的制备
为了制备可以完全阻挡与CD25相互作用的IL-2突变体，我们在IL-2与CD25的相互作用表位选择最关键的位点，将其突变为Cys，并将第125位余存在Cys的位点突变掉，再定点偶连PEG，通过PEG的空间位阻来阻挡两者间的相互作用。通过alphafold对IL-2及其突变 体进行结构模拟，我们发现将R38和F42均突变为Cysteine，C125突变为Ser后，其残基侧链均暴露于蛋白表面并指向CD25(图1)。
我们观察到突变体FRC上两个Cys的侧链比较靠近，为了使这两个Cys更加稳定，我们准备使用1，3-二氯丙酮(DCA)将它两做成订书肽结构，同时引入羰基基团(图2)。羰基的引入可以作为后续进一步偶连的活性基团。
通过点突变，成功构建了IL-2的突变体F42C+R38C+C125S(后面简称FRC)的表达质粒。将其转化至BL21表达菌株后，30℃培养至OD600＝0.4-0.6，42℃诱导。高压破碎，包涵体精纯，使用含有2mM半胱氨酸的8M盐酸胍变性剂溶解包涵体后，透析至TRIS ph8.5的buffer中。透析液通过镍柱纯化后，洗脱液浓缩直接经过分子筛，可得到纯的FRC。经质谱检测我们发现FRC存在两种分子量，分别对应着FRC上两个Cys所处的两种状态：一种是C38和C42直接形成了一对二硫键(质谱分子量15433.11Da)，另外一种是C38和C42两个残基均被Cys化(质谱分子量15673.40Da)，推测可能是FRC在复性过程中游离的Cys与溶液中的Cys发生反应(图3)。
在制备的FRC中加入终浓度0.1mM的TCEP，37℃温水浴2h后，加入约1/3体积的200mM NaHCO3缓冲液。向还原好的FRC中加入4摩尔当量的1，3-二氯丙酮(DCA)，充分混匀后，4℃反应过夜。第二天直接通过脱盐柱除去溶液中的TCEP、还原下来的Cys和未反应的DCA。将制备好的FRC-DCA进行质谱检测，我们发现整个过程反应非常充分，质谱数据中只有单一分子量的峰，分子量对应着FRC-DCA，已没有未反应的FRC分子量残留(图4)。
在此基础上，我们向FRC-DCA中加入NH2O-PEG用于羰基的正交偶联，用来制备PEG化产物。常温反应1天后，我们直接用分子筛可分离出偶联产物FRC-PEG(图5)。
实施例2 IL-2突变体减弱了IL-2与CD25的相互作用，进而降低与IL-2Rαβγ的相互作用
为测试FRC、FRC-DCA、FRC-PEG对表达不同T细胞的刺激活性，在实施例1的基础上，我们分别测试了IL-2、FRC、FRC-DCA、FRC-PEG对表达三聚体受体IL-2Rαβγ的CTLL2细胞和表达二聚体受体IL-2Rβγ的MO7E细胞的促增殖效果。如图6所示，相比于IL-2，未偶连PEG的FRC的EC50表现出明显的偏移(向右偏移约256倍)，说明在含有C125S突变的同时，将F42、R38两个位点突变为Cys(无论是F42C和R38C形成分子内二硫键，还是F42C和R38C分别被Cys修饰)即可明显降低其与CD25的相互作用，从而减弱对CTLL2细胞的活化作用。FRC-DCA的EC50向右偏移了74倍，可能因为相比FRC上的两个Cys化的Cys，FRC-DCA位阻效应小一些。而偶联上PEG后，FRC-PEG的EC50直接向右偏移了2140倍，明显强于Thor-707800倍的阻挡作用。而对于表达IL-2Rβγ的MO7E细胞而言，即使偶联了PEG，其EC50也仅仅向右偏移约3.45倍，也就是说通过将F42和R38位点“装订”(staple)后偶联PEG，几乎不会对其IL-2Rβγ受体的结合产生任何影响，但却能够有效地阻挡FRC与IL-2Rα的相互作用。
实施例3 FRC-2PEG的制备及活性测试
考虑到FRC-PEG的制备流程较为复杂，需通过两步偶联的方式进行制备，而且虽然我们在含有C125S突变的同时，选择了F42、R38两个位点进行点突变，但通过制备FRC-DCA后再偶联PEG，其阻挡效果与偶联单PEG相比差异并不是特别大。因此我们考虑在FRC的基础上通过一步偶联，直接制备双PEG的偶联产物FRC-2PEG，希望双PEG能够给FRC带来更强的阻挡作用。因此，我们向实施例1制备好的FRC中加入终浓度0.1mM的TCEP，37℃反应2h后，直接加入12摩尔当量Maleimide-PEG，充分混匀后4℃反应过夜。偶联了双PEG的FRC-2PEG可直接通过分子筛分离制备(图7-9)。因此相较于FRC-PEG，FRC-2PEG只需要一步偶连即可制备，制备流程更为简单。
实施例4 IL-2突变体保留了对CD122(IL-2Rβ的亲和力)
用表面等离子共振(Surface plasmon resonance，SPR)技术检测IL-2突变体与CD25和CD122的亲和力：将CD25-Fc和CD122-Fc分别用固定缓冲液(10mM NaAc pH5.0)稀释至50μg/mL，CM5芯片用400mM EDC和100mM NHS活化后，将稀释好的CD25-Fc和CD122-Fc分别固定在CM5芯片上，10μL/min的流速，至RU值达到约2000RU，将固定好的CM5芯片用乙醇胺封闭。
将IL-2和FRC-2PEG分别用工作缓冲液(1XHEPES 0.005％Tween-20pH 7.5)稀释成不同浓度梯度，以30μL/min的流速上样，根据曲线计算出对应的结合解离常数。
如图10所示，在CD25-Fc的通道上(图10中的a)，IL-2表现出快结合慢解离的动力学，而FRC-2PEG(图10中的b)在高达2μM仍未表现出与CD25的相互作用。在CD122-Fc的通道上(图10中的c)，IL-2与FRC-2PEG(图10中的d)表现出对CD122类似的相互作用。因此，选择F42和R38两个位点偶连PEG，可几乎完全阻挡其与CD25的相互作用，但保留了它与CD122的相互作用。
在此实验基础上，我们分别测试了IL-2、FRC-2PEG对表达三聚体受体的CTLL2细胞和表达二聚体受体的MO7E细胞的促增殖效果。如图11所示，相比于IL2，偶连了两个10k长度聚乙二醇的FRC-2PEG在高达100nM的工作浓度下仍对CTLL2没任何活化作用。然而，相对于IL2，FRC-2PEG对于MO7E细胞的EC50仅向右偏移12.84倍，因此偶连双PEG对表达二聚体受体的淋巴细胞影响甚微。
实施例5 IL-2突变体延长了血浆半衰期
雌性C57BL小鼠随机分为两组(6只小鼠/组)。通过尾静脉注射等剂量的IL-2或FRC-2PEG。在不同时间点尾静脉采集静脉血。ELISA检测每个样品中IL-2及FRC-2PEG的浓度，并通过GraphPad Prism处理数据。
如图12所示，FRC-2PEG半衰期明显优于IL-2，IL-2在8h的浓度已跌近检测限。而FRC-2PEG在第十天后仍可在血浆中检测出FRC-2PEG。
实施例6 IL-2突变体在不引起VLS剂量下显著促进NK细胞和CD8+T的增殖，
将PBS，IL-2，FRC-2PEG通过腹腔注射的方式打到C57BL6小鼠体内，并在不同时间采血，其中PBS组及FRC-2PEG组仅在0h的时间点进行单剂量给药，IL-2组于0h，12h，24h，36h，48h共五个时间点连续给药。在第54h对小鼠进行安乐死处理，取全血及脾脏，利用流式分析T细胞及NK细胞的含量。
结果如图13-14所示，不同时间点的血样，IL-2组的IL-5均显著高于PBS组和FRC-2PEG组，FRC-2PEG组与PBS组基本相当。说明相比高频率高剂量的注射IL-2，单剂量的FRC-2PEG不会引起VLS，然而单剂量的FRC-2PEG却能显著促进小鼠外周血及脾脏中NK细胞和CD8T细胞的增殖，但对Treg细胞的增殖作用不太明显(图15)。
实施例7 IL-2突变体通过促进NK细胞和CD8+T细胞的增殖显著抑制肿瘤生长
将雌性C57BL/6N小鼠，随机分为PBS组IL-2组、FRC-2PEG组，每组5只，进行皮下注射B16F1。当种瘤大小达到约50mm3的体积，通过腹腔注射的方式进行给药。四天后，同样的方式进行第二次给药，每两天记录小鼠种瘤体积，当PBS组小鼠肿瘤体积达到1500mm3时，对小鼠进行安乐死，取全血，脾脏，瘤组织，对其中的淋巴细胞成分进行分析。
如图所16所示，相比于PBS组和IL-2组，FRC-2PEG可显著抑制肿瘤的生长，而IL-2几乎未起到任何延缓肿瘤生长的作用。流式结果表明(图17)，FRC-2PEG可显著降低外周血和脾脏中CD4+T细胞所占比例，提高CD8+T细胞和NK细胞在淋巴细胞中的比例。同时在肿瘤组织的流式中(图17)，我们可以检测到FRC-2PEG组可增强淋巴细胞的浸润，并显著提高CD8+T细胞及NK细胞所占比例。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：本申请中的FRC-2PEG可几乎完全阻挡其与CD25的相互作用，但保留了与CD122的相互作用。因其与CD25的相互作用明显降低，可明显减弱对三聚体受体的淋巴细胞的活化作用。此外，FRC-2PEG在小鼠体内的半衰期较长。另外对FRC-2PEG在小鼠体内的药效进行评价，发现单剂量的FRC-2PEG不会引起VLS，且能显著促进小鼠外周血及脾脏中NK细胞和CD8T细胞的增殖，但对Treg细胞的增殖作用不太明显。因而，相较几乎未起到任何延缓肿瘤生长的作用的IL-2来说，FRC-2PEG可显著抑制肿瘤的生长。且在肿瘤组织的流式中，可以检测到FRC-2PEG组可增强淋巴细胞的浸润，并显著提高CD8+T细胞及NK细胞所占比例。由此可见，本申请的IL-2突变体及其PEG偶联物的细胞偏向性较强且对肿瘤的治疗提供了积极影响。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种白细胞介素-2(IL-2)突变体，其特征在于，包括：
    如SEQ ID NO：1所示的氨基酸序列的蛋白的第125位发生氨基酸突变以及如下至少一个位点发生氨基酸突变：F42或R38，
    其中，具有SEQ ID NO：1所示的氨基酸序列的蛋白为IL-2野生型，所述IL-2突变体对IL-2Rαβγ的激活能力低于所述IL-2野生型对IL-2Rαβγ的激活能力，所述IL-2突变体对IL-2Rβγ的激活能力与所述IL-2野生型对IL-2Rβγ的激活能力无明显区别。
  2. 根据权利要求1所述的IL-2突变体，其特征在于，所述IL-2突变体的突变各自独立地选自如下：
    F42X+C125J、R38X+C125J或F42X+R38X+C125J，其中，数字前字母代表原始氨基酸，数字后字母代表突变氨基酸，X所代表的氨基酸为任意带有巯基的氨基酸，J代表如下任意一种氨基酸：G、A、S、T或V；
    优选地，所述IL-2突变体为F42C+R38C+C125S；
    优选地，所述IL-2突变体为C125突变为S且F42X和R38X直接形成分子内二硫键的IL-2突变体；
    优选地，所述IL-2突变体为C125突变为S且F42X和R38X上的巯基被修饰的IL-2突变体；
    优选地，所述IL-2突变体为C125突变为S且F42X和R38X上的巯基被DCA所修饰的IL-2突变体；
    优选地，所述F42X和R38X上的巯基被DCA所修饰的结构如式I所示：
  3. 根据权利要求2所述的IL-2突变体，其特征在于，所述IL-2野生型对IL-2Rαβγ复合物的激活能力记为第一EC50值，所述IL-2突变体对IL-2Rαβγ复合物的激活能力记为第二EC50值，所述第二EC50值与所述第一EC50值的比值记为n，其中，n≥74，优选n≥256。
  4. 一种IL-2突变体偶联物，其特征在于，所述偶联物为在权利要求1至3中任一项所述的IL-2突变体蛋白质基础上进行PEG偶联，所获得的IL-2蛋白-聚乙二醇(PEG)偶联物。
  5. 根据权利要求4所述的IL-2突变体偶联物，其特征在于，
    所述PEG为化学偶联剂修饰的PEG，所述IL-2蛋白为具有F42X和/或R38X突变位点以及C125J的IL-2突变体，所述F42X和/或R38X的巯基通过所述化学偶联剂与所述PEG偶联，
    其中，数字前字母代表原始氨基酸，数字后字母代表突变氨基酸，X所代表的氨基酸为任意带有巯基的氨基酸，J代表如下任意一种氨基酸：G、A、S、T或V；
    优选地，所述化学偶联剂为带有羟氨基或带有酰肼基的化合物；
    优选地，所述带有羟氨基的化合物选自如下任意一种：马来酰亚胺、琥珀酰亚胺；
    优选地，所述带有酰肼基的取代基选自烷基、芳基或杂芳基，所述烷基的C原子数选自1～8，所述芳基或杂芳基的C原子数选自5～10。
  6. 一种DNA分子，其特征在于，所述DNA分子编码权利要求1至3中任一项所述的IL-2突变体。
  7. 一种重组质粒，其特征在于，所述重组质粒连接有权利要求6所述的DNA分子。
  8. 一种宿主细胞，其特征在于，所述宿主细胞内转化有权利要求7所述的重组质粒。
  9. 权利要求1至3中任一项所述的IL-2突变体或权利要求4或5所述的IL-2突变体偶联物在制备用于治疗癌症的药物或制剂中的用途。
  10. 根据权利要求9所述的用途，其特征在于，所述癌症选自如下任意一种：肾癌、恶性黑色素瘤、胰腺癌、骨癌、前列腺癌、小细胞肺癌、非小细胞肺癌、间皮瘤、白血病、多发性骨髓瘤、淋巴瘤、肝癌、肉瘤、B细胞恶性肿瘤、乳腺癌、卵巢癌、结直肠癌、神经胶质瘤、多形性胶质母细胞瘤、脑膜瘤、垂体腺瘤、前庭神经鞘瘤、原发性中枢神经系统淋巴瘤、原始神经外胚层肿瘤、膀胱癌、食道癌、子宫癌、脑癌、头颈癌、宫颈癌、睾丸癌、甲状腺癌及胃癌。
PCT/CN2023/132133 2022-11-17 2023-11-16 Il-2突变体及其应用 WO2024104444A1 (zh)

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CN114651004A (zh) * 2019-06-14 2022-06-21 科优基因公司 用于癌症治疗的新型白介素-2变体
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CN101426916A (zh) * 2004-03-05 2009-05-06 诺华疫苗和诊断公司 改进的白介素-2突变蛋白
CN114651004A (zh) * 2019-06-14 2022-06-21 科优基因公司 用于癌症治疗的新型白介素-2变体
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