WO2024104444A1 - Mutant d'il-2 et son utilisation - Google Patents

Mutant d'il-2 et son utilisation 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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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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Publication of WO2024104444A1 publication Critical patent/WO2024104444A1/fr

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19Cytokines; Lymphokines; Interferons
    • A61K38/20Interleukins [IL]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/04Immunostimulants
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/54Interleukins [IL]
    • C07K14/55IL-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

La présente invention concerne un mutant d'IL-2 et son utilisation. Le mutant d'IL-2 comprend une mutation d'acide aminé en position 125 d'une protéine représentée par SEQ ID NO : 1 et une mutation d'acide aminé dans au moins und des sites suivants : F42 ou R38, la protéine ayant la séquence d'acides aminés représentée par SEQ ID NO : 1 étant IL-2 de type sauvage, la capacité d'activation du mutant d'IL-2 en IL-2Rαβγ est inférieure à la capacité d'activation d'IL-2 de type sauvage en IL-2Rαβγ, et il n'y a pas de différence évidente entre la capacité d'activation du mutant d'IL-2 en IL-2Rαβγ et la capacité d'activation d'IL-2 de type sauvage en IL-2Rαβγ. Le mutant d'IL-2 améliore la polarisation cellulaire, peut activer sélectivement des cellules effectrices T CD8 + et des cellules NK, évite une immunosuppression provoquée par l'activation des cellules Treg, et résout efficacement le problème de faible polarisation cellulaire de l'état de la technique.
PCT/CN2023/132133 2022-11-17 2023-11-16 Mutant d'il-2 et son utilisation WO2024104444A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN111018961A (zh) * 2019-12-17 2020-04-17 北京志道生物科技有限公司 白介素-2衍生物
CN112724259A (zh) * 2020-11-16 2021-04-30 天津林达生物科技有限公司 人血清白蛋白与白介素2的融合蛋白及其用途
CN113321722A (zh) * 2021-04-13 2021-08-31 苏州复融生物技术有限公司 白介素2突变体及其应用
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
US20220289806A1 (en) * 2019-08-15 2022-09-15 Cytimm Therapeutics, Inc. Modified Interleukin 2 (IL-2) Polypeptides, Conjugates and Uses Thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US20220289806A1 (en) * 2019-08-15 2022-09-15 Cytimm Therapeutics, Inc. Modified Interleukin 2 (IL-2) Polypeptides, Conjugates and Uses Thereof
CN111018961A (zh) * 2019-12-17 2020-04-17 北京志道生物科技有限公司 白介素-2衍生物
CN112724259A (zh) * 2020-11-16 2021-04-30 天津林达生物科技有限公司 人血清白蛋白与白介素2的融合蛋白及其用途
CN113321722A (zh) * 2021-04-13 2021-08-31 苏州复融生物技术有限公司 白介素2突变体及其应用

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