WO2023246820A1 - 体外诱导干细胞样记忆细胞的化合物及其应用 - Google Patents

体外诱导干细胞样记忆细胞的化合物及其应用 Download PDF

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WO2023246820A1
WO2023246820A1 PCT/CN2023/101528 CN2023101528W WO2023246820A1 WO 2023246820 A1 WO2023246820 A1 WO 2023246820A1 CN 2023101528 W CN2023101528 W CN 2023101528W WO 2023246820 A1 WO2023246820 A1 WO 2023246820A1
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cells
stem cell
compound
butyl
formula
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PCT/CN2023/101528
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French (fr)
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赵阳兵
刘晓军
宋晓东
周雨凡
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上海优替济生生物医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • This application belongs to the technical field of immune cell expansion in vitro, and specifically relates to a compound for inducing stem cell-like memory cells in vitro and its application.
  • Cellular adoptive immunotherapy is a treatment method that activates, transforms and amplifies autologous or allogeneic immune cells in vitro and then infuses them back into the patient's body.
  • Genetically modified T cells chimeric antigen receptor (CAR-T) ) and T cell receptor (TCR-T) therapy are considered to be the most promising adoptive cellular immunotherapy methods at present.
  • adoptive reinfusion of T cells still faces some difficulties. For example, after highly differentiated terminal-stage T cells are reinfused into the body, their persistence is significantly reduced, which is one of the important reasons for poor therapeutic effects.
  • Tscm stem cell-like memory T cells
  • Tscm stem cell-like memory T cells
  • Tscm stem cell-like memory T cells
  • This group of cells develops from initial T cells and subsequently develops into central memory T cells and effector memory T cells.
  • Tscm has the characteristics of memory T cells that produce cytokines and rapidly proliferates, and has the characteristics of stem cells with strong survival and multipotential differentiation capabilities. Due to its potential for sustained expansion in vivo, Tscm is expected to become an important source of cells for adoptive immunotherapy of tumors.
  • Tscm in adoptive immunotherapy, it is particularly critical to explore methods for preparing Tscm cells in vitro. Therefore, it is of great practical significance to solve the contradiction between promoting Tscm self-renewal and inhibiting proliferation in a targeted manner, and to find effective and safe drugs for in vitro co-culture.
  • the technical problem to be solved by the present invention is to overcome the lack of therapeutic potential in the prior art for improving adoptive cell-based immunotherapy, especially the expansion induced by stem cell-like memory T cells. Therefore, the present invention provides a compound for inducing stem cell-like memory cells in vitro and its application.
  • the present invention solves the above technical problems through the following technical solutions.
  • the first aspect of the present invention provides the use of a compound represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in inducing stem cell-like memory cells in vitro:
  • R 1 , R 3 , R 4 and R 6 are independently C 1 -C 4 alkyl
  • R 2 is fluorine, chlorine, bromine or iodine
  • the compound represented by formula (I) or the compound represented by formula (II) satisfies one or more of the following conditions:
  • the R 1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, such as methyl;
  • the R 2 is fluorine
  • the R 3 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, such as methyl;
  • the R 4 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, such as methyl;
  • the R 6 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, such as ethyl;
  • n 1, 2, 3 or 4, preferably 2.
  • the compound has glycogen synthase kinase 3 inhibitory activity.
  • the compound has a structural formula as shown in formula (III) and has glycogen synthase kinase 3 ⁇ inhibitory activity:
  • the compound has a structural formula as shown in formula (IV) and has glycogen synthase kinase 3 ⁇ / ⁇ inhibitory activity:
  • the compound has a structural formula as shown in formula (V) and has glycogen synthase kinase 3 ⁇ inhibitory activity:
  • the compound represented by formula (III) is IM-12(3-(4-Fluorophenylethylamino)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole- 2,5-dione), which has glycogen synthase kinase 3 ⁇ inhibitory activity.
  • cytokines When combined with cytokines, it can effectively increase the absolute number and number of stem cell-like memory cells such as CD8+Tscm (stem cell-like memory CD8+T cells). Forming a ratio to promote the in vitro expansion of stem cell-like memory cells, such as stem cell-like memory T cells, with good safety.
  • the compound represented by formula (IV) is BRD0705((S)-4-ethyl-7,7-dimethyl-4-phenyl-2,4,6,7,8,9-hexahydro-5H- pyrazolo[3,4-b]quinolin-5-one), which has glycogen synthase kinase 3 ⁇ / ⁇ inhibitory activity.
  • stem cell-like memory cells such as CD8+Tscm (stem cell-like memory).
  • the absolute number and formation ratio of CD8+ T cells promotes the in vitro expansion of stem cell-like memory cells, such as stem cell-like memory T cells, and has good safety.
  • the compound represented by formula (V) is BRD5648 ((4R)-4-ethyl-7,7-dimethyl-4-phenyl-1,6,8,9-tetrahydropyrazolo[3,4-b] quinolin-5-one), which has glycogen synthase kinase 3 ⁇ inhibitory activity, can effectively increase the absolute number and number of stem cell-like memory cells such as CD8+Tscm (stem cell-like memory CD8+T cells) when combined with cytokines Forming a ratio to promote the in vitro expansion of stem cell-like memory cells, such as stem cell-like memory T cells, with good safety.
  • BRD5648 ((4R)-4-ethyl-7,7-dimethyl-4-phenyl-1,6,8,9-tetrahydropyrazolo[3,4-b] quinolin-5-one
  • CD8+Tscm stem cell-like memory CD8+T cells
  • cytokines Forming a ratio to promote the in vitro expansion
  • the compound with glycogen synthase kinase 3 inhibitory activity may also be:
  • Cromolyn (sodium) [5-[3-(2-carboxylato-4-oxochromen-5-yl)oxy-2-hydroxypropoxy]-4-oxochromene-2-carboxylate], etc., such as the compounds shown in Table 1, their targets
  • the points and structural formulas are shown in Table 1.
  • the stem cell-like memory cells are selected from the group consisting of stem cell-like memory T cells, stem cell-like memory NK cells and stem cell-like memory macrophages.
  • the stem cell-like memory cells are stem cell-like memory T cells.
  • the source of the stem cell-like memory cells is immune cells or tumor-associated lymphocytes.
  • the compound or a pharmaceutically acceptable salt thereof can reduce the differentiation of terminal cells of NK and macrophages.
  • the immune cells are selected from T cells, NK cells, NKT cells, macrophages, monocytes and granulocytes.
  • the T cells can be any kind of T lymphocytes, including tumor infiltrating lymphocytes (TIL), cytokine-induced killer cells (CIK), lymphokine-activated killer cells (LAK) and bone marrow. Infiltrating lymphocytes (MIL).
  • TIL tumor infiltrating lymphocytes
  • CIK cytokine-induced killer cells
  • LAK lymphokine-activated killer cells
  • MIL Infiltrating lymphocytes
  • the T cells are naive T cells.
  • the initial T cells are selected from the group consisting of initial CD3+ T cells, CD4+ T cells, CD8+ T cells, CD4+/CD8+ double-positive T cells, CD4/CD8 double-negative T cells, ⁇ T cells and helper One or more types of T cells.
  • the initial T cells are CD3+ T cells.
  • the T cells are genetically engineered T cells; the genetically engineered T cells are selected from the group consisting of recombinantly expressed chimeric antigen receptor (CAR), T cell receptor (TCR) and Bispecific T cell engager (BiTE) T cells, the CAR, TCR and BiTE bind tumor-associated antigens or viral antigens.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • BiTE Bispecific T cell engager
  • the tumor-related antigen is selected from Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, B7H3, GD2, PSCA, EBV16-E7 , H3.3, EGFRvIII, BCMA, 5T4, AGS-16, ALK1, ANG-2, B7-H4, c-fms, CA6, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b , CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR- ⁇ , GCC, GPNMB, HER3, HLA-DR, ICAM -1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b,
  • the viral antigen is selected from one or more of HPV, EBV, HBV, VZV, CMV, HSV and HIV.
  • the chimeric antigen receptor is well known to those skilled in the art. It is usually composed of a scFv single chain antibody (which is formed by linking the amino acid sequence of the VL region and the amino acid sequence of the VH region of the antibody through a Linker).
  • the hinge structure is connected to transmembrane and intracellular signaling structures derived from TCR complexes or IgE high-affinity receptors.
  • Those skilled in the art can introduce CAR-encoding nucleic acid into T cells through conventional transduction techniques in the art, such as mRNA electrotransduction, viral transduction, etc., to obtain engineered T cells modified by genetic engineering.
  • the TCR may be a T cell receptor expressed on heterologous cells that targets a specific antigen and is well known to those skilled in the art.
  • the T cell receptor can be genetically engineered to activate or deactivate specific genes, thereby affecting The role of T cell receptors in adoptive cell therapy.
  • the bispecific T cell engager (BiTE) is an antibody that contains two antibody variable domains capable of binding two independent antigens on a single polypeptide chain, which is well known to those skilled in the art.
  • the CAR, TCR or BiTE is a polynucleotide.
  • the vector encoding the polynucleotide may be an RNA vector, a retroviral vector, a lentiviral vector, an adenovirus vector or an adeno-associated virus vector.
  • the genetic engineering transformation is achieved by introducing a recombinant expression vector into T cells; the recombinant expression vector is an expression vector carrying nucleic acid encoding scFv, CAR, TCR or BiTE.
  • the scFv, CAR, TCR or BiTE targets MSLN M12 or BCMA31.BBZ.
  • the scFv, CAR, TCR or BiTE targeting BCMA31.BBZ includes a heavy chain variable region or a light chain variable region, and the heavy chain variable region includes an amino acid sequence such as SEQ ID CDR1, CDR2 and CDR3 shown in NO: 1, 2 and 3, the light chain variable region includes the CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO: 4, 5 and 6 respectively; or, the The scFv, CAR, TCR or BiTE targeting MSLN M12 includes a heavy chain variable region or a light chain variable region comprising CDR1 whose amino acid sequences are set forth in SEQ ID NO: 7, 8 and 9, respectively. , CDR2 and CDR3, the light chain variable region includes CDR1, CDR2 and CDR3 whose amino acid sequences are shown in SEQ ID NO: 10, 11 and 12 respectively.
  • the heavy chain variable region of the scFv, CAR, TCR or BiTE targeting BCMA31.BBZ includes the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region Comprising the amino acid sequence shown in SEQ ID NO: 14; alternatively, the heavy chain variable region of the scFv, CAR, TCR or BiTE targeting MSLN M12 comprises the amino acid sequence shown in SEQ ID NO: 15, The light chain variable region includes the amino acid sequence shown in SEQ ID NO:16.
  • the amino acid sequence of the scFv targeting BCMA31.BBZ is shown in SEQ ID NO:17, and the amino acid sequence of the CAR is shown in SEQ ID NO:18; or, the amino acid sequence of the CAR targeting MSLN M12
  • the amino acid sequence is shown in SEQ ID NO:19, and the nucleotide sequence is shown in SEQ ID NO:20.
  • the expression vector can be conventional in the art, preferably an RNA vector, a retroviral vector, a lentiviral vector, an adenovirus vector or an adeno-associated virus vector.
  • Those skilled in the art can introduce nucleic acids encoding CAR, TCR or BiTE into T cells through conventional transduction techniques in the art, such as mRNA electrotransduction, viral transduction, etc., to obtain engineered T cells modified by genetic engineering.
  • a second aspect of the present invention provides the use of a composition for inducing stem cell-like memory cells in vitro, wherein the composition includes at least one of the compounds used in the application as described in the first aspect.
  • the composition further includes cytokines.
  • the cytokine is IL-2, IL-7 and/or IL-15.
  • the stem cell-like memory cells are as described in the first aspect.
  • a third aspect of the present invention provides a compound for in vitro induction of stem cell-like memory cells or a pharmaceutically acceptable salt thereof.
  • the compound is represented by the following formula (I) or (II):
  • R 1 , R 3 , R 4 and R 6 are independently C 1 -C 4 alkyl
  • R 2 is fluorine, chlorine, bromine or iodine
  • the compound represented by formula (I) or the compound represented by formula (II) satisfies one or more of the following conditions:
  • the R 1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, such as methyl;
  • the R 2 is fluorine
  • the R 3 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, such as methyl;
  • the R 4 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, such as methyl;
  • the R 6 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, such as ethyl;
  • n 1, 2, 3 or 4, preferably 2.
  • the compound has glycogen synthase kinase 3 inhibitory activity.
  • the compound has a structural formula as shown in formula (III) and has glycogen synthase kinase 3 ⁇ inhibitory activity:
  • the compound has a structural formula as shown in formula (IV) and has glycogen synthase kinase 3 ⁇ / ⁇ inhibitory activity:
  • the compound has a structural formula as shown in formula (V) and has glycogen synthase kinase 3 ⁇ inhibitory activity:
  • the stem cell-like memory cells are as described in the first aspect.
  • the stem cell-like memory cells are stem cell-like memory T cells.
  • a fourth aspect of the present invention provides a composition for in vitro induction of stem cell-like memory cells, which composition includes at least one of the compounds described in the third aspect or a pharmaceutically acceptable salt thereof.
  • the composition further includes cytokines.
  • the cytokine is IL-2, IL-7 and/or IL-15.
  • the stem cell-like memory cells are as described in the first aspect.
  • the stem cell-like memory cells are stem cell-like memory T cells.
  • a fifth aspect of the present invention provides the use of a compound as described in the third aspect or a composition as described in the fourth aspect in inducing stem cell-like memory cells in vitro or in preparing a preparation for inducing stem cell-like memory cells in vitro.
  • the stem cell-like memory cells are as described in the first aspect.
  • the stem cell-like memory cells are stem cell-like memory T cells.
  • the content of the compound is 0.3-21 ⁇ M and does not include endpoint values.
  • the content of the compound when the structural formula of the compound is as shown in formula (I), the content of the compound is greater than 0.313 ⁇ M and less than 10 ⁇ M; when the structure of the compound is as shown in formula (II) When indicated, the content of the compound is greater than 0.313 ⁇ M and less than 20 ⁇ M.
  • the content of the compound is 0.625-5 ⁇ M; for example, the content of the compound is 5 ⁇ M.
  • the content of the cytokine is 30 IU to 100 IU or 2 ng to 15 ng.
  • the cytokine includes IL-2, and the content of IL-2 is preferably 30 to 50 IU.
  • the cytokine includes IL-7, and the content of IL-7 is preferably 5 to 10 ng.
  • the cytokine includes IL-15, and the content of IL-15 is preferably 5 to 10 ng.
  • a sixth aspect of the present invention provides a method for in vitro induction of stem cell-like memory cells.
  • the in vitro induction method includes the following steps:
  • the inducer is a compound as described in the third aspect or a pharmaceutically acceptable salt thereof, or a composition as described in the fourth aspect.
  • the stem cell-like memory cells are as described in the first aspect.
  • the stem cell-like memory cells are stem cell-like memory T cells.
  • the initial immune cells are CD3+ T cells.
  • the working concentration of the inducer is 0.313-20 ⁇ M; preferably 5 ⁇ M.
  • the medium for induction culture is R10 medium
  • the R10 medium is RPMI-1640 medium containing 10% (v/v) FBS.
  • the induction culture medium and inducer are supplemented every 2 days 4 days after adding the inducer.
  • the induction culture medium and the induction agent are supplemented every 2 days thereafter.
  • the activation refers to culturing initial CD3+ T cells in an activation medium; the activation medium is a T cell culture medium containing 0.75% TransAct (v/v%).
  • a seventh aspect of the present invention provides a kit for in vitro induction of stem cell-like memory cells, which kit includes the compound described in the third aspect or a pharmaceutically acceptable salt thereof, or the combination described in the fourth aspect things.
  • the stem cell-like memory cells are as described in the first aspect.
  • the stem cell-like memory cells are stem cell-like memory T cells
  • the kit further includes a T cell culture medium.
  • the present invention screens out compounds IM-12 with glycogen synthase kinase 3 ⁇ inhibitory activity and compounds with glycogen synthase kinase 3 ⁇ inhibitory activity that can promote the in vitro expansion of stem cell-like memory cells, such as stem cell-like memory T cells, from a compound library.
  • BRD5648 and compound BRD0705 with glycogen synthase kinase 3 ⁇ / ⁇ inhibitory activity This type of compound is an activator of the Wnt signaling pathway and can dephosphorylate ⁇ -catenin, promote its nuclear transfer and induce the expression of downstream genes.
  • glycogen synthase kinase 3 ⁇ inhibitor IM-12 When the glycogen synthase kinase 3 ⁇ inhibitor IM-12 is combined with cytokines, it can effectively increase the absolute number and formation ratio of CD8+Tscm (stem cell-like memory CD8+T cells), and its induction effect is better than that of the control without drug. group, and has no significant impact on T cell expansion within a certain concentration range, such as 5 ⁇ M culture system, and has good safety.
  • Figure 1A is a graphical representation of a relative measure of the absolute expanded number of stem cell memory CD8+ T cells (expressing CD45RO - CCR7 + CD8 + ).
  • Figure 1B is a graph showing the fold change of T cell expansion on day 12.
  • Figure 2 is a flow chart of high-throughput T cell expansion screening and phenotypic analysis.
  • Figure 3 shows the expansion fold of T cells on day 12 after treatment with different concentrations of IM-12 (from 3 donors).
  • Figures 4A-4B show the phenotypic ratio of T cells on day 12 after treatment with different concentrations of IM-12 (from 3 donors).
  • Figure 5 shows the expansion fold of CD45RO-CCR7 + CD8 + T cells on day 12 after treatment with different concentrations of IM-12.
  • Figure 6 shows the expansion fold of T cells on day 12 after treatment with different concentrations of BRD0705 (from 2 donors).
  • Figure 7A-7B shows the phenotypic ratio of T cells on day 12 after treatment with different concentrations of BRD0705 (from 2 donors)
  • Figure 8 shows the expansion times of CD45RO-CCR7 + CD8 + T cells on day 12 after treatment with different concentrations of BRD0705. number.
  • Figure 9 shows the cell expansion fold on D13 after the drug was combined with interleukins IL-2, IL-7, and IL-15.
  • drugs 5 ⁇ M IM-12, 5 ⁇ M BRD0705; IL7/IL15: 5ng/ml IL-7, 10ng/ml IL-15.
  • Figure 10 shows the cell phenotype of CD8 + CAR + T cells from D5 to D13 under the combined culture of drugs and interleukins.
  • Figure 11 shows the cell phenotype of CD8 + CAR - T cells from D5 to D13 under combined culture with drugs and interleukins.
  • Figures 12A-12B show the proportion of stem cell-like memory CD8 + CAR + T cells.
  • Figure 13 shows the killing and inhibition rates of CAR T cells on target cells under different efficacy-to-target ratios.
  • Figure 14 shows the use of ELISA to detect the secretion of IL-2 and IFN- ⁇ released by CAR T cells and target cells after co-culture.
  • Figures 15A-15B show the results of the mouse model, tumor volume ( Figure 15A) and survival rate (Figure 15B) at different time points.
  • Example 1 In vitro high-throughput screening of drugs that can improve the enrichment and expansion of Tscm CD8 T cells
  • Primary human lymphocytes Primary human lymphocytes were isolated from healthy donors using the CD4/CD8 Magnetic Bead Isolation Kit (Miltenyi Biotech) T.
  • T cells were seeded in 96-well flat-bottom plates at 5e4 cells/well, stimulated with 0.75% TransAct (v/v%, Miltenyi Biotech), and cultured in R10 medium (RPMI with 10% FBS) containing 30 IU interleukin-2. -1640; Invitrogen) culture.
  • R10 medium RPMI with 10% FBS
  • DMSO 0.05%
  • the flow cytometry fluorescent antibodies used are from: anti-human CD3 BV421 (Biolegend), anti-human CD8 AF700 (Biolegend), anti-human CCR7 APC (Biolegend), anti-human CD45RO FITC (Biolegend), anti-human CD62L BV605 (Biolegend), anti-human CD28 BV785 (Biolegend) and anti-human CD95PE/Cy7 (Biolegend).
  • Example 2 Using IM-12 to increase the enrichment and expansion of Tscm CD8 T cells
  • Cells were stimulated with 0.75% TransAct (v/v%, Miltenyi Biotech) and cultured in R10 medium (RPMI-1640 with 10% FBS; Invitrogen) containing 30 IU interleukin-2.
  • Different dosing groups were added with 0.313 ⁇ M, 0.625 ⁇ M, 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M or 20 ⁇ M IM-12.
  • the control group was added with an equal volume of DMSO to make the final concentration of DMSO 0.2% (v/v).
  • Each group Set up 3 donors. From the 4th day onwards, fresh culture medium and corresponding concentrations of drugs were added every 2 days. On day 12 of cell culture, cells were counted.
  • Figure 4A-4B results show that when the concentration of IM-12 is 0.313 ⁇ M, 0.625 ⁇ M, 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M or 10 ⁇ M, the proportion of stem cell-like memory cells (Tscm) population in CD8+ T cells increases with the concentration of IM-12. high and proportionally increased.
  • the IM-12 concentration ranges from 0.625 ⁇ M to 5 ⁇ M.
  • the expansion fold of CD45RO - CCR7 + CD8 + T cell stem cell-like memory cells increases.
  • the amplification fold was 1.5 times that of the control group.
  • Example 3 Using BRD0705 to increase the enrichment and expansion of Tscm CD8 T cells
  • Cells were stimulated with 0.75% TransAct (v/v%, Miltenyi Biotech) and cultured in R10 medium (RPMI-1640 with 10% FBS; Invitrogen) containing 30 IU interleukin-2.
  • Different dosing groups were added with 0.313 ⁇ M, 0.625 ⁇ M, 1.25 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M or 20 ⁇ M BRD0705.
  • the control group was added with an equal volume of DMSO to make the final concentration of DMSO 0.2% (v/v). Each group was set to 2 donor. From the 4th day onwards, fresh medium and corresponding concentrations of drugs were replenished every 2 days. On day 12 of cell culture, cells were counted.
  • the BRD0705 concentration ranges from 0.313 ⁇ M to 20 ⁇ M.
  • the expansion fold of CD45RO - CCR7 + CD8 + T cell stem cell-like memory cells increases gradually.
  • the concentration of BRD0705 was 20 ⁇ M, the amplification fold was 2.1 times that of the control group.
  • Example 4 Combination of IM-12, BRD0705 and different interleukins can improve the enrichment of Tscm cells
  • CD8 + anti-mesothelin CAR + T cells and CAR - T were as follows: R10-IL-2 was 22% vs. R10-IL-2-added group was 50.8%; R10-IL-7/IL-15 was 11.9% compared to 30.9% in the R10-IL-7/IL-15-added group.
  • the CD8 + anti-mesothelin CAR - T cell subpopulation classification on D13 was as follows: R10-IL-2 was 28% vs. R10-IL-2-added group was 55%; R10-IL-7/IL-15 was 12.4% compared to 32.4% in the R10-IL-7/IL-15-added group.
  • Example 5 Using IM-12 to improve the function of lentiviral anti-mesothelin CAR T cells in vitro
  • CD3-positive T cells isolated from PBMC were activated with anti-CD3/CD28 dynabeads (CTS Dynabeads CD3/CD28, Gibco, Cat#402031) and cultured in R10 medium (RPMI-1640 with 10% FBS; Invitrogen) containing 100 IU interleukin-2. )nourish.
  • the drug-added group added 5 ⁇ M glycogen synthase kinase 3 ⁇ inhibitor (IM-12), and the control group added an equal volume of DMSO to make the final concentration of DMSO 0.05% (v/v).
  • Lentivirus LV.A40C2828-2A-M12.BBZ
  • IM-12 can significantly increase the proportion of Tscm population in CD8 + anti-mesothelin CAR + T cells.
  • Figure 12A shows phenotypic data from three donors (ND022, ND023 and ND025).
  • Figures 12A-12B show the phenotype of ND022 anti-mesothelin CD8+CAR+ cells.
  • the lentivirus LV.A40C2828-2A-M12.BBZ CAR-T cell drug group (IM-12 treatment) and control group (DMSO treatment) were tested in the IncuCyte S3 live cell analysis system against the mesothelin-positive tumor cell line SKOV3- The killing ability of MSLN/CD40.
  • Example 6 Anti-tumor efficacy of IM-12-treated anti-mesothelin CAR T in SKOV3-MSLN/CD40 xenogeneic NOG tumor model
  • mice 5E6 SKOV3-MSLN/CD40 tumor cells were injected into mice (subcutaneous inoculation) after clicking chitin green (SKOV3-MSLN/CD40-CBG). After 2 weeks, mice were infused with 0.1E6 CAR-positive transduced T cells. (tail vein injection). Tumor volumes were measured at different time points and the survival status of mice was observed. The results showed that the drug-added group (IM-12 treatment) significantly inhibited tumor growth (Figure 15A) and improved the survival rate of mice (Figure 15B) compared with the control group (DMSO treatment) and other negative control groups.
  • IM-12 treatment significantly inhibited tumor growth
  • Figure 15B improved the survival rate of mice

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Abstract

提供一种体外诱导干细胞样记忆细胞的化合物及其应用。所述化合物如下式(I)或(II)所示并具有糖原合成酶激酶3抑制活性。所述化合物与细胞因子联用后，有效提高干细胞样记忆性CD8+T细胞的绝对数和形成比例，促进干细胞样记忆细胞体外扩增，并且安全性好。

Description

体外诱导干细胞样记忆细胞的化合物及其应用 技术领域
本申请属于免疫细胞体外扩增技术领域，具体涉及一种体外诱导干细胞样记忆细胞的化合物及其应用。
背景技术
细胞过继免疫治疗是通过体外活化、改造和扩增自体或异体免疫细胞后回输到患者体内的一种治疗方法，经基因修饰改造的T细胞嵌合抗原受体(chimeric antigen receptor，CAR-T)和T细胞受体(T cell receptor，TCR-T)疗法被认为是当前最具潜力的过继性细胞免疫治疗手段。目前，T细胞过继性回输仍面临一些难题，如，高度分化的终末阶段的T细胞回输体内后，持久性明显降低，是导致治疗效果不佳的重要原因之一。
近年来，干细胞样记忆性T细胞(T Stem Memory Cell，Tscm)陆续在小鼠、猕猴等模式生物及人体中发现。这群细胞由初始T细胞发育而来，随后发育为中枢性记忆性T细胞和效应性记忆性T细胞。Tscm既具有产生细胞因子、快速增殖的记忆性T细胞的特征，又具有较强的存活和多潜能分化的能力的干细胞特征。由于其具有在体内持久扩增的潜力，Tscm将有望成为过继性免疫***的重要细胞来源。
基于Tscm在过继性免疫治疗中的广阔应用前景，探索体外制备Tscm细胞的方法尤为关键。因此，有针对性的解决其中促进Tscm自我更新与增殖抑制之间的矛盾，找到有效、安全性高的体外共培养的药物非常有现实意义。
发明内容
本发明所要解决的技术问题是为了克服现有技术中缺少用来改进过继性的基于细胞的免疫疗法的治疗潜力，尤其是干细胞样记忆T细胞诱导的扩增。因此，本发明提供了一种体外诱导干细胞样记忆细胞的化合物及其应用。
本发明通过以下技术方案解决上述技术问题。
本发明第一方面提供了一种如式(I)或式(II)所示的化合物或其药学上可接受的盐在体外诱导干细胞样记忆细胞中的应用：
其中，
R1、R3、R4和R6独立地为C1-C4烷基；
R2为氟、氯、溴或碘；
较佳地，所述式(I)所示化合物或式(II)所示化合物满足如下条件中的一个或多个：
所述R1为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如甲基；
所述R2为氟；
所述R3为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如甲基；
所述R4为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如甲基；
所述R6为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如乙基；
m为1、2、3或4，优选为2。
本发明一些实施方案中，所述化合物具有糖原合成酶激酶3抑制活性。本发明一些实施方案中，所述化合物的结构式如式(III)所示且具有糖原合成酶激酶3β抑制活性：
本发明另一些实施方案中，所述化合物的结构式如式(IV)所示且具有糖原合成酶激酶3α/β抑制活性：
本发明另一些实施方案中，所述化合物的结构式如式(V)所示且具有糖原合成酶激酶3α抑制活性：
本发明中，如式(III)所示的化合物为IM-12(3-(4-Fluorophenylethylamino)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione)，其具有糖原合成酶激酶3β抑制活性，与细胞因子联用后，能够有效提高干细胞样记忆细胞例如CD8+Tscm(干细胞样记忆性CD8+T细胞)的绝对数和形成比例，促进干细胞样记忆细胞例如干细胞样记忆T细胞的体外扩增，并且安全性好。
本发明中，如式(IV)所示的化合物为BRD0705((S)-4-ethyl-7,7-dimethyl-4-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazolo[3,4-b]quinolin-5-one)，其具有糖原合成酶激酶3α/β抑制活性，与细胞因子联用后，能够有效提高干细胞样记忆细胞例如CD8+Tscm(干细胞样记忆性CD8+T细胞)的绝对数和形成比例，促进干细胞样记忆细胞例如干细胞样记忆T细胞的体外扩增，并且安全性好。
本发明中，如式(V)所示的化合物为BRD5648((4R)-4-ethyl-7,7-dimethyl-4-phenyl-1,6,8,9-tetrahydropyrazolo[3,4-b]quinolin-5-one)，其具有糖原合成酶激酶3α抑制活性，与细胞因子联用后，能够有效提高干细胞样记忆细胞例如CD8+Tscm(干细胞样记忆性CD8+T细胞)的绝对数和形成比例，促进干细胞样记忆细胞例如干细胞样记忆T细胞的体外扩增，并且安全性好。
本发明中，所述具有糖原合成酶激酶3抑制活性的化合物还可为:
BRD3731[(4S)-3-(2,2-dimethylpropyl)-4,7,7-trimethyl-4-phenyl-2,6,8,9-tetrahydropyrazolo[3,4-b]quinolin-5-one]；
'(R)-BRD3731[(4R)-3-(2,2-dimethylpropyl)-4,7,7-trimethyl-4-phenyl-2,6,8,9-tetrahydropyrazolo[3,4-b]quinolin-5-one]；
Cromolyn(sodium)[5-[3-(2-carboxylato-4-oxochromen-5-yl)oxy-2-hydroxypropoxy]-4-oxochromene-2-carboxylate]等，例如表1所示的化合物，其靶点和结构式如表1所示。
表1化合物及其靶点与结构式



本发明一些实施方案中，所述干细胞样记忆细胞选自干细胞样记忆T细胞、干细胞样记忆NK细胞和干细胞样记忆巨噬细胞。
本发明一些具体实施方案中，所述干细胞样记忆细胞为干细胞样记忆T细胞。
本发明一些实施方案中，所述干细胞样记忆细胞的来源为免疫细胞或肿瘤相关淋巴细胞。
本发明中，所述化合物或其药学上可接受的盐可以减少NK、巨噬细胞的终末细胞的分化。
本发明一些实施方案中，所述免疫细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞、单核细胞和粒细胞。
本发明一些实施方案中，所述T细胞可以是任何一种T淋巴细胞，包括肿瘤浸润淋巴细胞(TIL)、细胞因子诱导的杀伤细胞(CIK)、淋巴因子激活的杀伤细胞(LAK)和骨髓浸润淋巴细胞(MIL)。
本发明一些较佳实施方案中，所述T细胞为初始T细胞。
本发明一些实施方案中，所述初始T细胞选自初始CD3+T细胞、CD4+T细胞、CD8+T细胞、CD4+/CD8+双阳性T细胞、CD4/CD8双阴性T细胞、γδT细胞和辅助性T细胞中的一种或多种。
本发明一些具体实施方案中，所述初始T细胞为CD3+T细胞。
本发明一些实施方案中，所述T细胞为经基因工程改造的T细胞；所述经基因工程改造的T细胞选自重组表达嵌合抗原受体(CAR)、T细胞受体(TCR)和双特异性T细胞接合子(BiTE)的T细胞，所述CAR、TCR和BiTE结合肿瘤相关抗原或病毒抗原。
本发明中，所述肿瘤相关抗原选自Her2、NY-ESO-1、CD19、CD20、CD22、PSMA、c-Met、GPC3、IL13ra2、EGFR、CD123、CD7、B7H3、GD2、PSCA、EBV16-E7、H3.3、EGFRvIII、BCMA、5T4、AGS-16、ALK1、ANG-2、B7-H4、c-fms、CA6、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD74、CD79b、CD98、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CXCR4、DLL-4、EGP-1、ENPP3、EphA3、ETBR、FGFR2、FN、FR-α、GCC、GPNMB、HER3、HLA-DR、ICAM-1、IGF-1R、IL-3R、LIV-1、MSLN、MUC16、MUC1、NaPi2b、结合素-4、Notch 2、Notch 1、PD-L1、PD-L2、PDGFR-α、PS、SLTRK6、STEAP1、TEM1、VEGFR、CD25、CD27L、DKK-1、CSF-1R、CD138和间皮素中的一种或多种。
本发明中，所述病毒抗原选自HPV、EBV、HBV、VZV、CMV、HSV和HIV中的一种或多种。
本发明中，所述嵌合抗原受体(CAR)已为本领域技术人员熟知，其通常由一个scFv单链抗体(由抗体VL区氨基酸序列和VH区氨基酸序列经Linker连接而成)，通过铰链结构与源于TCR复合体或者IgE高亲和受体的跨膜和胞内信号结构连接构成。本领域技术人员可通过本领域常规的转导技术例如mRNA电转导、病毒转导等将编码CAR的核酸导入T细胞，以获得经基因工程改造修饰的工程化T细胞。
本发明中，所述TCR可为本领域技术人员熟知的表达在异源细胞上的靶向特定抗原的T细胞受体。所述T细胞受体可进行基因工程改造来激活或失活特定基因，从而影响 T细胞受体在过继性细胞疗法中的作用。
本发明中，所述双特异性T细胞接合子(BiTE)为本领域技术人员熟知的在单条多肽链上包含能够结合两种独立抗原的两个抗体可变域的抗体。
本发明一些实施方案中，所述CAR、TCR或BiTE是一种多核苷酸。编码多核苷酸的载体可为RNA载体、逆转录病毒载体、慢病毒载体、腺病毒载体或腺相关病毒载体。
本发明一些实施方案中，所述基因工程改造通过向T细胞中导入重组表达载体实现；所述重组表达载体为携带编码scFv、CAR、TCR或BiTE的核酸的表达载体。
本发明一些实施方案中，所述scFv、CAR、TCR或BiTE靶向MSLN M12或BCMA31.BBZ。
本发明一些具体实施方案中，所述靶向BCMA31.BBZ的scFv、CAR、TCR或BiTE包括重链可变区或轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、2和3所示的CDR1、CDR2和CDR3，所述轻链可变区包含氨基酸序列分别如SEQ ID NO:4、5和6所示的CDR1、CDR2和CDR3；或者，所述靶向MSLN M12的scFv、CAR、TCR或BiTE包括重链可变区或轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO:7、8和9所示的CDR1、CDR2和CDR3，所述轻链可变区包含氨基酸序列分别如SEQ ID NO:10、11和12所示的CDR1、CDR2和CDR3。
本发明一些较佳实施方案中，靶向BCMA31.BBZ的scFv、CAR、TCR或BiTE的所述重链可变区包含如SEQ ID NO:13所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:14所示的氨基酸序列；或者，靶向MSLN M12的scFv、CAR、TCR或BiTE的所述重链可变区包含如SEQ ID NO:15所示的氨基酸序列，所述轻链可变区包含如SEQ ID NO:16所示的氨基酸序列。
本发明一些更佳实施方案中，靶向BCMA31.BBZ的scFv的氨基酸序列如SEQ ID NO:17所示，CAR的氨基酸序列如SEQ ID NO:18所示；或者，靶向MSLN M12的CAR的氨基酸序列如SEQ ID NO:19所示，核苷酸序列如SEQ ID NO:20所示。
本发明一些实施方案中，所述表达载体可为本领域常规，优选为RNA载体、逆转录病毒载体、慢病毒载体、腺病毒载体或腺相关病毒载体。
本领域技术人员可通过本领域常规的转导技术例如mRNA电转导、病毒转导等将编码CAR、TCR或BiTE的核酸导入T细胞，以获得经基因工程改造修饰的工程化T细胞。
本发明第二方面提供了一种组合物在体外诱导干细胞样记忆细胞中的应用，其中，所述组合物包括如第一方面所述的应用中的所述化合物中的至少一种。
本发明一些实施方案中，所述组合物还包括细胞因子。
本发明一些具体实施方案中，所述细胞因子为IL-2、IL-7和/或IL-15。
本发明一些实施方案中，所述干细胞样记忆细胞如第一方面所述。
本发明第三方面提供一种用于干细胞样记忆细胞体外诱导的化合物或其药学上可接受的盐，所述化合物如下式(I)或(II)所示：
其中，
R1、R3、R4和R6独立地为C1-C4烷基；
R2为氟、氯、溴或碘；
较佳地，所述式(I)所示化合物或式(II)所示化合物满足如下条件中的一个或多个：
所述R1为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如甲基；
所述R2为氟；
所述R3为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如甲基；
所述R4为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如甲基；
所述R6为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如乙基；
m为1、2、3或4，优选为2。
本发明一些实施方案中，所述化合物具有糖原合成酶激酶3抑制活性。
本发明一些实施方案中，所述化合物的结构式如式(III)所示且具有糖原合成酶激酶3β抑制活性：
所述化合物的结构式如式(IV)所示且具有糖原合成酶激酶3α/β抑制活性：
所述化合物的结构式如式(V)所示且具有糖原合成酶激酶3α抑制活性：
本发明一些实施方案中，所述干细胞样记忆细胞如第一方面所述。
本发明一些具体实施方案中，所述干细胞样记忆细胞为干细胞样记忆T细胞。
本发明第四方面提供一种用于干细胞样记忆细胞体外诱导的组合物，所述组合物包括如第三方面所述的化合物或其药学上可接受的盐中的至少一种。
本发明一些实施方案中，所述组合物还包括细胞因子。
本发明一些具体实施方案中，所述细胞因子为IL-2、IL-7和/或IL-15。
本发明一些实施方案中，所述干细胞样记忆细胞如第一方面所述。
本发明一些具体实施方案中，所述干细胞样记忆细胞为干细胞样记忆T细胞。
本发明第五方面提供一种如第三方面所述的化合物或第四方面所述的组合物在体外诱导干细胞样记忆细胞或制备体外诱导干细胞样记忆细胞的制剂中的应用。
本发明一些实施方案中，所述干细胞样记忆细胞如第一方面所述。
本发明一些具体实施方案中，所述干细胞样记忆细胞为干细胞样记忆T细胞。
本发明一些实施方案中，所述化合物的含量为0.3～21μM且不含端点值。
本发明一些较佳的实施方案中，当所述化合物的结构式如式(I)所示时，所述化合物的含量为大于0.313μM且小于10μM；当所述化合物的结构如式(II)所示时，所述化合物的含量为大于0.313μM且小于20μM。
本发明一些更佳的实施方案中，所述化合物的结构式为式(I)时，所述化合物的含量为0.625～5μM；例如，所述化合物的含量为5μM。
本发明一些实施方案中，所述细胞因子的含量为30IU～100IU或2ng～15ng。
本发明一些较佳的实施方案中，所述细胞因子包括IL-2，所述IL-2的含量优选为30～50IU。
本发明另一些较佳的实施方案中，所述细胞因子包括IL-7，所述IL-7的含量优选为5～10ng。
本发明另一些较佳的实施方案中，所述细胞因子包括IL-15，所述IL-15的含量优选为5～10ng。
本发明第六方面提供一种干细胞样记忆细胞的体外诱导方法，所述体外诱导方法包括以下步骤：
向活化的初始免疫细胞中加入诱导剂进行诱导培养，即得；
其中，所述诱导剂为如第三方面所述的化合物或其药学上可接受的盐，或者如第四方面所述的组合物。
本发明一些实施方案中，所述干细胞样记忆细胞如第一方面所述。
本发明一些具体实施方案中，所述干细胞样记忆细胞为干细胞样记忆T细胞。
本发明一些实施方案中，所述初始免疫细胞为CD3+T细胞。
本发明一些实施方案中，所述的诱导剂的工作浓度为0.313～20μM；优选为5μM。
本发明一些实施方案中，所述诱导培养的培养基为R10培养基，所述R10培养基为含有10％(v/v)FBS的RPMI-1640培养基。
本发明一些较佳实施方案中，在加入所述诱导剂4天后每2天补充所述诱导培养的培养基和诱导剂。
本发明一些较佳实施方案中，在加入诱导剂3天后，之后每2天补充所述诱导培养的培养基和诱导剂。
本发明中，所述活化是指将初始CD3+T细胞在激活培养基中进行培养；所述激活培养基为含有0.75％TransAct(v/v％)的T细胞培养液。
本发明第七方面提供一种用于体外诱导干细胞样记忆细胞的试剂盒，所述试剂盒包括第三方面所述的化合物或其药学上可接受的盐，或者如第四方面所述的组合物。
本发明一些实施方案中，所述干细胞样记忆细胞如第一方面所述。
本发明一些具体实施方案中，所述干细胞样记忆细胞为干细胞样记忆T细胞，所述试剂盒还包括T细胞培养液。
本发明的有益效果是：
本发明从化合物库中筛选到能促进干细胞样记忆细胞例如干细胞样记忆T细胞体外扩增的具有糖原合成酶激酶3β抑制活性的化合物IM-12、具有糖原合成酶激酶3α抑制活性的化合物BRD5648和具有糖原合成酶激酶3α/β抑制活性的化合物BRD0705。该类化合物是Wnt信号通路的激活剂，可使β-catenin去磷酸化，促进其核转移并诱导下游基因表达。糖原合成酶激酶3β抑制剂IM-12与细胞因子联用后，有效提高CD8+Tscm(干细胞样记忆性CD8+T细胞)的绝对数和形成比例，其诱导效果优于不加药的对照组，并且在一定浓度范围内例如5μM培养体系下对T细胞扩增没有显著影响，安全性好。
附图说明
图1A为干细胞记忆性CD8+T细胞(表达CD45RO-CCR7+CD8+)绝对扩增数量的相对量度的图示。
图1B为T细胞在第12天的扩增倍数变化图，数据显示为平均值±标准误差(s.e.m.)(n＝6，来自3个供体)；其中：A：培养基中含有30IU IL-2，0.05％DMSO；B：培养基中含有30IU IL-2，5μM IM-12，0.05％DMSO；C：培养基中含有30IU IL-2，5μM TWS119，0.05％DMSO。
图2为高通量T细胞扩增筛选以及表型分析流程图。
图3为不同浓度的IM-12处理后T细胞第12天的扩增倍数(来自3个供体)。
图4A-4B为不同浓度的IM-12处理后T细胞第12天的表型比例(来自3个供体)。
图5为不同浓度的IM-12处理后第12天CD45RO-CCR7+CD8+T细胞的扩增倍数。
图6为不同浓度的BRD0705处理后T细胞第12天的扩增倍数(来自2个供体)。
图7A-7B为不同浓度的BRD0705处理后T细胞第12天的表型比例(来自2个供体)
图8为不同浓度的BRD0705处理后第12天CD45RO-CCR7+CD8+T细胞的扩增倍 数。
图9为药物与介素IL-2、IL-7、IL-15联用后D13天细胞的扩增倍数。其中：药物：5μM IM-12，5μM BRD0705；IL7/IL15：5ng/ml IL-7，10ng/ml IL-15。
图10为药物和白介素组合培养下，CD8+CAR+T的D5-D13天的细胞表型。
图11为药物和白介素组合培养下，CD8+CAR-T的D5-D13天的细胞表型。
图12A-12B为干细胞样记忆性CD8+CAR+T细胞的比例。
图13为不同效靶比下CAR T细胞对靶细胞的杀伤抑制率。
图14为使用ELISA检测CAR T细胞与靶细胞共培养后释放的IL-2和IFN-γ的分泌情况。
图15A-15B为小鼠模型的结果，不同时间点的肿瘤体积(图15A)和生存率(图15B)。
具体实施方式
实施例1：体外高通量筛选可提高Tscm CD8T细胞的富集和扩增的药物
原代人淋巴细胞：使用CD4/CD8磁珠分选试剂盒(Miltenyi Biotech)从健康供体中分离原代人淋巴细胞T。
细胞培养： T细胞以5e4细胞数/孔接种在96孔平底板中，使用0.75％TransAct(v/v％，Miltenyi Biotech)刺激细胞，并在含有30IU白介素-2的R10培养基(含有10％FBS的RPMI-1640；Invitrogen)培养。加药组添加5μM表1中的药物，对照组加入等体积DMSO，使DMSO终浓度为0.05％(v/v)。每组处理设置6个重复，即3个供体，每个供体2个复孔，并在细胞培养第4、6、8、10、12天转移细胞到新的孔板，并添加新鲜培养基和相应浓度的药物和DMSO。
细胞计数：细胞培养第4和第12天，通过Incucyte荧光成像***(Satorius Inc.)对使用1ng/ul AO(Acridine Orange，丫啶橙，Solarbio)染色的T细胞进行计数。当前细胞的浓度与初始细胞浓度比较后，可计算出细胞扩增的倍数。筛选流程如图2所示。
细胞亚群分析：
使用的流式荧光抗体来自于：anti-human CD3 BV421(Biolegend)、anti-human CD8 AF700(Biolegend)、anti-human CCR7 APC(Biolegend)、anti-human CD45RO FITC(Biolegend)、anti-human CD62L BV605(Biolegend)、anti-human CD28 BV785(Biolegend)和anti-human CD95PE/Cy7(Biolegend)。
在细胞培养第14天，使用流式细胞仪检测T细胞亚群分型。采用CD3、CD8、CD45RO、 CCR7、CD28、CD62L、CD95等表面分子对CD8+T细胞中的Tscm进行细胞群确定。
综合细胞扩增和T细胞亚群分型数据，计算出细胞相对扩增系数(药物组扩增倍数/DMSO组扩增倍数)和CD45RO-CCR7+CD8+干细胞样记忆细胞相对富集系数(药物组CD45RO-CCR7+CD8+干细胞样记忆细胞比例/DMSO组CD45RO-CCR7+CD8+干细胞样记忆细胞比例)。使用相对扩增系数乘以CD45RO-CCR7+CD8+干细胞样记忆细胞相对富集系数得出CD45RO-CCR7+CD8+T细胞扩增系数，结果如图1A所示，其中IM-12、BRD5648、BRD0705可提高CD45RO-CCR7+CD8+干细胞样记忆细胞的扩增能力。同时，与使用已公开的具有糖原合成酶激酶3β抑制活性的TWS119相比，在5μM IM-12对细胞扩增没有明显抑制，如图1B所示。
实施例2：使用IM-12提高Tscm CD8T细胞的富集和扩增数量
使用0.75％TransAct(v/v％，Miltenyi Biotech)刺激细胞，并在含有30IU白介素-2的R10培养基(含有10％FBS的RPMI-1640；Invitrogen)培养。不同加药组分别添加0.313μM、0.625μM、1.25μM、2.5μM、5μM、10μM或20μM的IM-12，对照组添加等体积DMSO，使DMSO终浓度为0.2％(v/v)，每组设置3个供体。从第4天起，每2天补充新鲜培养基和对应浓度的药物。在细胞培养第12天，对细胞进行计数。如图3所示IM-12浓度为0.313μM、0.625μM、1.25μM、2.5μM或5μM时，细胞扩增没有受到明显抑制，IM-12浓度10μM或20μM时，细胞扩增受到抑制。使用流式细胞仪检测T细胞亚群分型。采用CD3、CD8、CD45RO、CCR7等表面抗体对CD8+T细胞中的干细胞样记忆细胞(Tscm)进行细胞群确定。图4A-4B结果显示IM-12浓度为0.313μM、0.625μM、1.25μM、2.5μM、5μM或10μM时，CD8+T细胞中的干细胞样记忆细胞(Tscm)群体比例随着IM-12浓度升高而比例增加。综合扩增和表型结果，如图5所示，IM-12浓度在0.625μM～5μM范围内，随着IM-12浓度提高，CD45RO-CCR7+CD8+T细胞干细胞样记忆细胞的扩增倍数呈现梯度提高。当IM-12浓度为5μM时，扩增倍数为对照组的1.5倍。
实施例3：使用BRD0705提高Tscm CD8T细胞的富集和扩增数量
使用0.75％TransAct(v/v％，Miltenyi Biotech)刺激细胞，并在含有30IU白介素-2的R10培养基(含有10％FBS的RPMI-1640；Invitrogen)培养。不同加药组分别添加0.313μM、0.625μM、1.25μM、2.5μM、5μM、10μM或20μM的BRD0705，对照组添加等体积DMSO，使DMSO终浓度为0.2％(v/v)，每组设置2个供体。从第4天起，每2天补充新鲜培养基和对应浓度的药物。在细胞培养第12天，对细胞进行计数。如图6所 示，BRD0705浓度为0.313μM、0.625μM、1.25μM、2.5μM、5μM、10μM或20μM时，细胞扩增没有受到明显抑制。使用流式细胞仪检测T细胞亚群分型。采用CD3、CD8、CD45RO、CCR7等表面抗体对CD8+T细胞中的干细胞样记忆细胞(Tscm)进行细胞群确定。如图7A-7B所示，CD8+T细胞中的干细胞样记忆细胞(Tscm)群体比例随着BRD0705浓度升高而比例增加。综合扩增和表型结果，如图8所示，BRD0705浓度在0.313μM～20μM范围内，随着BRD0705浓度提高，CD45RO-CCR7+CD8+T细胞干细胞样记忆细胞的扩增倍数呈现梯度提高。当BRD0705浓度为20μM时，扩增倍数为对照组2.1倍。
实施例4：IM-12、BRD0705与不同白介素组合可提高Tscm细胞的富集
使用0.75％TransAct(v/v％，Miltenyi Biotech)刺激细胞，在R10(含有10％FBS的RPMI-1640；Invitrogen)的基础培养基中培养。不同白介素分组为IL-2(100IU/ml)、IL-7/IL-15(5ng/ml IL-7和10ng/ml IL-15)。加药组为5μM/ml的IM-12和5μM/ml BRD0705，对照组添加等体积DMSO，使DMSO终浓度为0.4％(v/v)。激活后24小时，在T细胞中加入慢病毒(LV.A40C2828-2A-M12.BBZ)，MOI＝3。从第4天起，每2天补充新鲜培养基和对应浓度的药物和白介素。如图9所示，药物与IL-7/IL-15的组合表现出较好的细胞扩增，相比IL-7/IL15不加药物组。药物与IL-2的组合相比IL-2不加药物组细胞扩增相当。在细胞培养过程中，在第5、8、11、13天使用流式细胞仪检测T细胞亚群分型。采用anti-Meso-FC、CD3、CD8、CD45RO、CCR7等表面抗体对CD8+CAR+T细胞中的Tscm进行细胞群确定。结果显示药物组可以显著提高CD8+anti-mesothelin CAR+T细胞和CAR-T中Tscm群体(CCR7+/CD45RO-)的比例(如图10、图11所示)。其中D13天CD8+anti-mesothelin CAR+T细胞亚群分型为：R10-IL-2为22％对比R10-IL-2-加药组为50.8％；R10-IL-7/IL-15为11.9％对比R10-IL-7/IL-15-加药组为30.9％。其中D13天CD8+anti-mesothelin CAR-T细胞亚群分型为：R10-IL-2为28％对比R10-IL-2-加药组为55％；R10-IL-7/IL-15为12.4％对比R10-IL-7/IL-15-加药组为32.4％。
实施例5：使用IM-12提高慢病毒anti-mesothelin CAR T细胞体外功能
用抗CD3/CD28dynabeads(CTS Dynabeads CD3/CD28，Gibco，Cat#402031)激活从PBMC分离的CD3阳性T细胞，并在含有100IU白介素-2的R10培养基(含有10％FBS的RPMI-1640；Invitrogen)培养。加药组添加5μM的糖原合成酶激酶3β抑制剂(IM-12)，对照组添加等体积DMSO，使DMSO终浓度为0.05％(v/v)。激活后24小时，在 T细胞中加入慢病毒(LV.A40C2828-2A-M12.BBZ)，MOI＝3。激活后5天，用磁选法将Dynabeads从T细胞中清除掉。在细胞培养第12天，使用流式细胞仪检测T细胞亚群分型。采用anti-Meso-FC、CD3、CD8、CD45RO、CCR7等表面抗体对CD8+CAR+T细胞中的Tscm进行细胞群确定。结果显示IM-12可以显著提高CD8+anti-mesothelin CAR+T细胞中Tscm群体的比例，图12A示出了来自三个供体(ND022、ND023和ND025)的表型数据。图12A-12B示出了ND022 anti-mesothelin CD8+CAR+细胞的表型。
在IncuCyte S3活细胞分析***中测试慢病毒LV.A40C2828-2A-M12.BBZ CAR-T细胞加药组(IM-12处理)和对照组(DMSO处理)对间皮素阳性肿瘤细胞株SKOV3-MSLN/CD40的杀伤能力。结果显示，在不同效靶比(E:T)下，IM-12处理的细胞和对照组细胞都能有效地杀死SKOV3-MSLN/CD40肿瘤靶细胞(图13所示)，其中IM-12处理组在E:T＝0.1下，对肿瘤细胞的杀伤抑制率显著高于对照组。同时通过ELISA检测CAR-T细胞与SKOV3-MSLN/CD40共培养过程中的IFN-γ和IL2释放水平，结果表明在共培养过程中IM-12处理组的IFN-γ和IL2释放水平显著高于对照组(图14)。表明IM-12处理组的CAR-T细胞对抗原阳性的肿瘤细胞具有更好的体外杀伤功能。
实施例6：IM-12处理的anti-mesothelin CAR T在SKOV3-MSLN/CD40异种NOG肿瘤模型中的抗肿瘤疗效
将5E6 SKOV3-MSLN/CD40肿瘤细胞经点击甲壳素绿(SKOV3-MSLN/CD40-CBG)后注射给小鼠(皮下接种)，2周后给小鼠输注0.1E6 CAR阳性转导的T细胞(尾静脉注射)。在不同的时间点测量肿瘤体积并观察小鼠的生存状态。结果表明，加药组(IM-12处理)相比对照组(DMSO处理)和其它阴性对照组，显著抑制了肿瘤生长(图15A)，提高了小鼠生存率(图15B)。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (15)

  1. 一种如式(I)和/或式(II)所示的化合物或其药学上可接受的盐在体外诱导干细胞样记忆细胞中的应用：
    其中，
    R1、R3、R4和R6独立地为C1-C4烷基；
    R2为氟、氯、溴或碘；
    较佳地，所述化合物具有糖原合成酶激酶3抑制活性；和/或，所述式(I)所示化合物或式(II)所示化合物满足如下条件中的一个或多个：
    所述R1为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如甲基；
    所述R2为氟；
    所述R3为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如甲基；
    所述R4为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如甲基；
    所述R6为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如乙基；
    m为1、2、3或4，优选为2。
  2. 如权利要求1所述的应用，其中，所述化合物如式(III)所示且具有糖原合成酶激酶3β抑制活性：
    或，
    所述化合物如式(IV)所示且具有糖原合成酶激酶3α/β抑制活性：
    或，
    所述化合物如式(V)所示且具有糖原合成酶激酶3α抑制活性：
  3. 如权利要求1所述的应用，其特征在于，所述干细胞样记忆细胞为干细胞样记忆T细胞；和/或，所述干细胞样记忆细胞的来源为免疫细胞或肿瘤相关淋巴细胞；
    较佳地，所述免疫细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞、单核细胞和粒细胞；
    更佳地，所述T细胞为初始T细胞；
    进一步更佳地，所述初始T细胞选自初始CD3+T细胞、CD4+T细胞、CD8+T细胞、CD4+/CD8+双阳性T细胞、CD4/CD8双阴性T细胞、γδT细胞和辅助性T细胞中的一种或多种；
    例如，所述初始T细胞为CD3+T细胞。
  4. 如权利要求3所述的应用，其特征在于，所述T细胞为经基因工程改造的T细胞；所述经基因工程改造的T细胞选自重组表达嵌合抗原受体(CAR)、T细胞受体(TCR)和双特异性T细胞接合子(BiTE)的T细胞，所述CAR、TCR和BiTE结合肿瘤相关抗原或病毒抗原；
    较佳地，所述肿瘤相关抗原选自Her2、NY-ESO-1、CD19、CD20、CD22、PSMA、c-Met、GPC3、IL13ra2、EGFR、CD123、CD7、B7H3、GD2、PSCA、EBV16-E7、H3.3、EGFRvIII、BCMA、5T4、AGS-16、ALK1、ANG-2、B7-H4、c-fms、CA6、EpCAM、CD30、CD32b、CD37、CD38、CD40、CD52、CD70、CD74、CD79b、CD98、CEA、CEACAM5、CLDN18.2、CLDN6、CS1、CXCR4、DLL-4、EGP-1、ENPP3、EphA3、ETBR、FGFR2、FN、FR-α、GCC、GPNMB、HER3、HLA-DR、ICAM-1、IGF-1R、IL-3R、LIV-1、MSLN、MUC16、MUC1、NaPi2b、结合素-4、Notch 2、Notch 1、PD-L1、PD-L2、PDGFR-α、PS、SLTRK6、STEAP1、TEM1、VEGFR、CD25、CD27L、DKK-1、CSF-1R、CD138和间皮素中的一种或多种；和/或，
    所述病毒抗原选自HPV、EBV、HBV、VZV、CMV、HSV和HIV中的一种或多种；和/或，
    所述基因工程改造通过向T细胞中导入重组表达载体实现；所述重组表达载体为携带编码scFv、CAR、TCR或BiTE的核酸的表达载体；和/或
    所述表达载体为RNA载体、逆转录病毒载体、慢病毒载体、腺病毒载体或腺相关病毒载体。
  5. 一种组合物在体外诱导干细胞样记忆细胞中的应用，其特征在于，所述组合物包括如权利要求1所述的应用中的所述化合物中的至少一种；
    较佳地，所述组合物还包括细胞因子；
    更佳地，所述细胞因子为IL-2、IL-7和/或IL-15。
  6. 如权利要求5所述的应用，其中，所述干细胞样记忆细胞为干细胞样记忆T细胞。
  7. 一种用于干细胞样记忆细胞体外诱导的化合物或其药学上可接受的盐，其特征在于，所述化合物如下式(I)或(II)所示：

    其中，
    R1、R3、R4和R6独立地为C1-C4烷基；
    R2为氟、氯、溴或碘；
    较佳地，所述化合物具有糖原合成酶激酶3抑制活性；和/或，所述式(I)所示化合物或式(II)所示化合物满足如下条件中的一个或多个：
    所述R1为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如甲基；
    所述R2为氟；
    所述R3为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如甲基；
    所述R4为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如甲基；
    所述R6为甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基，例如乙基；
    m为1、2、3或4，优选为2；
    更佳地，所述化合物的结构式如式(III)所示且具有糖原合成酶激酶3β抑制活性：
    或，
    所述化合物的结构式如式(IV)所示且具有糖原合成酶激酶3α/β抑制活性：
    或，
    所述化合物的结构式如式(V)所示且具有糖原合成酶激酶3α抑制活性：
  8. 如权利要求7所述的化合物，其特征在于，所述干细胞样记忆细胞为干细胞样记忆T细胞。
  9. 一种用于干细胞样记忆细胞体外诱导的组合物，其特征在于，所述组合物包括如权利要求7所述的化合物或其药学上可接受的盐中的至少一种；
    较佳地，所述组合物还包括细胞因子；
    更佳地，所述细胞因子为IL-2、IL-7和/或IL-15。
  10. 如权利要求9所述的组合物，其特征在于，所述干细胞样记忆细胞为干细胞样记忆T细胞。
  11. 一种如权利要求7所述的化合物或权利要求9所述的组合物在体外诱导干细胞样记忆细胞或制备体外诱导干细胞样记忆细胞的制剂中的应用；
    较佳地，所述干细胞样记忆细胞为干细胞样记忆T细胞。
  12. 如权利要求11所述的应用，其特征在于，所述化合物的含量为0.3～21μM且不含端点值；和/或，所述细胞因子的含量为30IU～100IU或2ng～15ng；
    较佳地，当所述化合物的结构式如式(I)所示时，所述化合物的含量为大于0.313μM且小于10μM；当所述化合物的结构如式(II)所示时，所述化合物的含量为大于0.313μM且小于20μM；和/或，当所述细胞因子包括IL-2时，所述IL-2的含量为30～50IU；当所述细胞因子包括IL-7时，所述IL-7的含量为5～10ng；当所述细胞因子包括IL-15时，所述IL-15的含量为5～10ng；
    更佳地，所述化合物的结构式为式(I)时，所述化合物的含量为0.625～5μM；例如， 所述化合物的含量为5μM。
  13. 一种干细胞样记忆细胞的体外诱导方法，其特征在于，所述体外诱导方法包括以下步骤：
    向活化的初始免疫细胞中加入诱导剂进行诱导培养，即得；
    其中，所述诱导剂为如权利要求7所述的化合物或其药学上可接受的盐，或者如权利要求9所述的组合物。
  14. 如权利要求13所述的体外诱导方法，其特征在于，所述干细胞样记忆细胞为干细胞样记忆T细胞；和/或，所述初始免疫细胞为CD3+T细胞；
    较佳地，所述诱导剂的工作浓度为0.313～20μM，优选为0.625～5μM；和/或，所述诱导培养的培养基为R10培养基，优选为含有10％(v/v)FBS的RPMI-1640培养基；
    更佳地，在加入所述诱导剂3天或4天后，每2天补充所述诱导培养的培养基和所述诱导剂。
  15. 一种用于体外诱导干细胞样记忆细胞的试剂盒，其特征在于，所述试剂盒包括如权利要求7所述的化合物或者如权利要求9所述的组合物；
    较佳地，所述干细胞样记忆细胞为干细胞样记忆T细胞；
    更佳地，所述试剂盒还包括T细胞培养液。
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