WO2022088555A1 - 用于白血病的car t细胞疗法的t细胞增效剂及获得增效t细胞的方法 - Google Patents

用于白血病的car t细胞疗法的t细胞增效剂及获得增效t细胞的方法 Download PDF

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WO2022088555A1
WO2022088555A1 PCT/CN2021/076819 CN2021076819W WO2022088555A1 WO 2022088555 A1 WO2022088555 A1 WO 2022088555A1 CN 2021076819 W CN2021076819 W CN 2021076819W WO 2022088555 A1 WO2022088555 A1 WO 2022088555A1
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cells
car
rapamycin
cell
pi3k
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魏海明
粘志刚
郑小虎
孙汭
田志刚
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中国科学技术大学
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Definitions

  • the present invention relates to chimeric antigen receptor (CAR) T cell therapy for tumors, in particular to a T cell potentiator in CAR T cell therapy for hematological tumors such as acute myeloid leukemia, and use of the same to enhance CAR T cells method of therapeutic effect.
  • CAR chimeric antigen receptor
  • Acute myeloid leukemia is a tumor derived from the bone marrow, and although chemotherapy can induce a remission rate of up to 70%, the vast majority of patients relapse.
  • intramedullary relapse occurs when the leukemia cells in the bone marrow exceed 5%, and extramedullary relapse occurs in the extramedullary part (usually the central nervous system and testis), childhood leukemia is intramedullary and extramedullary Recurrence is about 50%, and 95% of adults have intramedullary recurrence.
  • the leukemia stem cells in the bone marrow can be eliminated is the key factor for the success or failure of the treatment of acute myeloid leukemia.
  • CD19-targeting chimeric antigen receptor T cells in the treatment of acute lymphoblastic leukemia (ALL) has achieved surprising results (Maude et al., 2014). It shows the great potential of CAR T cell therapy for the treatment of hematological diseases, especially hematological tumors. So far, targets such as CD123, CD33, and CLL1 have been validated preclinically as potential targets for acute myeloid leukemia (Kenderian et al., 2015; Mardiros et al., 2013; Wang et al., 2018). However, cell therapy using CAR T cells designed against these sites has not achieved satisfactory results in the treatment of AML, that is, there is no report of significant effects of CAR T cell therapy in the treatment of AML.
  • CAR T cells are difficult to take effect in AML
  • the inventor's point of view after the research is that the important reasons affecting the effect of CAR T are that it is difficult for CAR T cells to enter the tumor site, and it is difficult for CAR T cells to enter the body in vivo. continue to exist.
  • Several clinical studies have suggested that the migration of CAR T cells to the bone marrow is the basis for affecting the therapeutic effect of CAR T cells. It has been reported that the more CAR T cells used for treatment migrate to the bone marrow, the better the patient's treatment effect will be (Ritchie et al., 2013; Wang et al., 2015). Therefore, improving the ability of CAR T cells to migrate to the tumor site during treatment has become a research direction to improve the therapeutic effect of CAR T cells.
  • CAR T cells Another key factor that determines the efficacy of CAR T cells is whether CAR T cells can persist in the body. Short-term survival of CAR T cells in vivo often leads to poor therapeutic effects. Therefore, there is a need for a method to improve the survival of CAR T cells in vivo.
  • CAR T cells The in vitro construction of CAR T cells involves the proliferation and differentiation of T cells.
  • the PI3K-Akt/mTOR signaling pathway plays a key role in T cell proliferation, survival, migration, and differentiation of effector/memory subsets.
  • CD3/CD28 antibody magnetic beads, IL2 cytokines, and CAR structures and other signals transmitted to T cells can lead to the activation of the PI3K-Akt/mTOR signaling pathway.
  • Rapamycin is produced by Streptomyces hygroscopicus and was first isolated from Easter Island samples of Streptomyces hygroscopicus by Surandranath and colleagues in 1972.
  • the compound was originally named rapamycin (Sirolimus) after the island's original name, Rapa Nui. Sirolimus was originally developed as an antifungal agent. However, this use was abandoned due to its ability to inhibit mTOR, with potent immunosuppressive and antiproliferative properties. Approved by the U.S. Food and Drug Administration and launched in September 1999, it is marketed by Pfizer (formerly Wyeth) under the trade name Rapamune.
  • Rapamycin is a macrolide compound that is used clinically as an immunosuppressant to prevent organ transplant rejection, and it is also used to treat a rare lung disease called lymphangioleiomyoma sick. It has immunosuppressive function in humans when used at therapeutic concentrations (10-30 ⁇ g ⁇ L -1 ), and is particularly useful in preventing renal transplant rejection. Regarding its mechanism of action, it is generally believed to reduce the sensitivity of T cells and B cells to interleukin 2 (IL-2) through mTOR inhibition, thereby inhibiting the activation of T cells and B cells.
  • IL-2 interleukin 2
  • the inventor's previous research found that during the in vitro construction of CAR T cells, the expression of CXCR4 was reduced, and more short-lived CAR T cells were generated.
  • the present invention relates to a CAR T cell therapy for targeting tumors, preferably hematological tumors (leukemia), including the use of PI3K-AKT/mTOR signaling pathway inhibitors as T cell potentiators, thereby enhancing the CAR T/NK cells Bone marrow migration and prolongation of survival time in vivo, in particular, the PI3K-AKT/mTOR signaling pathway inhibitor is rapamycin.
  • the overactivation of PI3K-Akt/mTOR during the in vitro production of CAR T cells can be attenuated, the expression of CXCR4 can be up-regulated, and the effect of CAR T on bone marrow can be enhanced. lethality of AML.
  • the in vivo survival time of potent CAR T cells can be increased, and the anticancer activity of CAR T cells in AML treatment can be further enhanced.
  • the present invention relates to the application of rapamycin as a T cell potentiator in the preparation of a medicine for the treatment of hematological tumors by being used in combination with CAR T cell therapy, and the cells used for treatment in the preparation of the CAR T cell therapy, containing Use of the cells in pharmaceutical compositions.
  • rapamycin By using rapamycin, the expression of CXCR4 on CAR T cells can be enhanced, the ability of CAR T cells to migrate to the bone marrow can be improved, the removal of tumor cells in the bone marrow can be accelerated, and the efficacy of CAR T cells can be improved to generate more cells with longer lifespan CAR T cells.
  • the present invention specifically includes the following contents.
  • PI3K-AKT/mTOR signaling pathway inhibitor as a T cell potentiator for treating CAR T cells or cell populations containing CAR T cells, wherein,
  • the cell therapy is chimeric antigen receptor T/NK cell therapy, and the PI3K-AKT/mTOR signaling pathway inhibitor is, for example, LY294002 or rapamycin, preferably rapamycin.
  • the CAR T cells or the cell population containing the CAR T cells is preferably activated by a cytokine, wherein the cytokine and the PI3K-AKT/mTOR signaling pathway inhibitor simultaneously treat the T cells, or T cells were treated before or after treatment with PI3K-AKT/mTOR signaling pathway inhibitors.
  • a method of enhancing CAR T cells or a cell population containing CAR T cells comprising culturing T cells or a cell population containing T cells with a medium supplemented with a PI3K-AKT/mTOR signaling pathway inhibitor, the T cells or containing
  • the cell population of T cells is preferably activated by cytokines, wherein the cytokines treat T cells concurrently with the PI3K-AKT/mTOR signaling pathway inhibitor, or treat T cells before or after the PI3K-AKT/mTOR signaling pathway inhibitor treats T cells cells, the PI3K-AKT/mTOR signaling pathway inhibitor is, for example, LY294002 or rapamycin, preferably rapamycin.
  • the cytokines are IL-2, IL7 and IL15, preferably IL-2.
  • a potentiated CAR T cell prepared by the method of any one of items 3-5.
  • the tumor is preferably the use in the medicament of leukemia.
  • a pharmaceutical composition comprising a CAR T cell obtained by the method of any one of items 3-5, or the method of item 7.
  • Figure 1 Flow cytometry plot of CXCR4 expression in initially isolated T cells and CAR T cells.
  • Figure 2 Flow cytometry plot of phosphorylation of Mtor signaling pathway in CAR T cells.
  • FIG. 1 Flow cytometry of the expression level of CXCR4 in rapamycin-treated CAR T cells.
  • Figure 5 Flow cytometry plot of bone marrow migration of rapamycin-treated CAR T cells.
  • FIG. 7 Depletion of myeloid AML by rapamycin-treated EpCAM CAR T cells.
  • Fluorescence imaging detects tumor changes.
  • (b) Representative plot of tumor burden by fluorescence imaging in mice (n 8).
  • FIG. 1 Rapamycin treatment enhances the antitumor ability of CD33 CAR T cells.
  • (b) Representative plot of tumor burden by fluorescence imaging in mice (n 6).
  • rapamycin is used in the following examples, other drugs that inhibit the PI3K-AKT/mTOR signaling pathway, such as LY294002, can also be used. When such a drug is used, it is administered at a concentration equivalent to the effect of 5 to 40 nM of rapamycin to increase CXCR4 in T cells.
  • concentration of rapamycin used, although a final concentration of 20 nM was used to incubate the cells in the following examples, it may be 5, 10, 20, 30, 40 nM or a concentration range therebetween.
  • PI3K-AKT/mTOR signaling pathway inhibitor for the timing of adding PI3K-AKT/mTOR signaling pathway inhibitor in cell culture, it can be added at any stage of in vitro culture of T cells or a population containing T cells, and it can be added at the same time as cytokines such as IL-2. IL-2 can also be added to the medium after a period of time as described in the Examples.
  • the cytokines that can be used in the present invention may be, for example, IL7 and IL15, preferably IL-2, or a combination of IL-2 and these.
  • EpCAM CAR T cells and CD33 CAR T cells were prepared. Except for the different CAR chimeric sequences, other operations and reagents were the same for the two cells.
  • CAR chimeric sequence of EpCAM CAR T cells connect the mouse-derived anti-human EpCAM scFv to the CD8 transmembrane region, 41bb costimulatory domain and CD3 ⁇ to obtain the mouse-derived anti-human EpCAM chimeric sequence (AE4 scFV-CD8-CD28- CD3 ⁇ , sequence shown in SEQ ID No. 1), was inserted into PCDH-MSCV-MCS-EF1-copGFP (purchased from Addgene Company).
  • CAR chimeric sequence of CD33 CAR T cells connect the humanized anti-human CD33 scFv to the CD8 transmembrane region, 41bb costimulatory domain and CD3 ⁇ to obtain the humanized anti-human CD33 chimeric sequence (such as SEQ ID No. 3), inserted into PCDH-MSCV-MCS-EF1-copGFP (purchased from Addgene)
  • the three plasmids in the following table were respectively transfected into 293T cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) using PEI (Polyscience, 23966) according to the instructions, and the cell culture supernatants of 48h and 72h were collected to transduce T cells. .
  • Mononuclear cells were isolated from fresh human peripheral blood (from Anhui Provincial Blood Center) by Ficoll density gradient centrifugation, and T cells were isolated using a CD3 T cell positive selection kit (Miltenyi, 30-097-043). The initially isolated T cells were divided into two, one for flow cytometry and the other for CAR T cell construction. Unless otherwise specified below, the day of initial separation will be taken as day 0.
  • the X-VIVO 15 medium ( lonza, BE02-060F) T cells were resuspended at a concentration of 5 x 105/ml, and anti-CD3/CD28 Dynabeads (thermo, 11161D) were added in a 1:1 ratio by number.
  • IL-2 was added at the same time as the anti-CD3/CD28 Dynabeads were added to make the final concentration of IL-2 at 100 U/ml, and IL-2 was supplemented every two days.
  • the concentrated lentiviruses obtained by centrifugation at 50,000g, 4°C for 2h from the cell culture supernatant collected above were aggregated with MOI 50 and polybrene (Sigma, H9268) with a final concentration of 8ng/ml.
  • Instructions for the operation of proteomine Add it to the activated T cells, centrifuge at 720g at 32°C for 1h, change the medium after 6-8h, and add rapamycin (Sigma, V900930-1MG) with a final concentration of 20nM, and incubate at 37°C In the medium culture, rapamycin was supplemented every two days, and a control group without rapamycin treatment was established at the same time.
  • T cells were monitored daily during transduction and complete X-VIVO 15 medium was added (to maintain a cell concentration of 0.5–1 x 10 cells/mL. On day 4 post-transduction, Dynabeads were removed. Harvest activation after day 5) The T cells, namely EpCAM CAR T cells and CD33 CAR T cells, were used for subsequent analysis and in vivo experiments, respectively.
  • Measurement group T cells (represented by T) initially isolated in Example 1, and EpCAM CAR T cells (represented by CAR T) after 5 days of in vitro culture in Example 1.
  • the cells to be assayed were detected using a BD LSRII flow cytometer and analyzed using a Flowjo V10.
  • the anti-human antibody EpCAM was purchased from biolengend, (324208), the anti-human CXCR4 antibody was purchased from eBioscience (12-9999-42), the anti-human mTOR antibody was purchased from BD (583489), and the anti-human S6 antibody was purchased from BD (583489).
  • Antibody was purchased from CST (14733S), anti-human CD62L antibody was purchased from BD (555544), and anti-human CD45RO antibody was purchased from BD (560607).
  • EpCAM T cell line harvested above was washed once in phosphate buffer supplemented with 2% fetal bovine serum, and incubated with mouse serum for 30 min. After blocking Fc receptors, the cells were stained at 4°C in the dark. , PBS was washed twice and then detected on the machine (Figure 1, 2, T cells from the same source and CAR T cells are connected with a straight line).
  • ebiscience's fixative transmembrane solution (Invitrogen, cat. No. 00-5521-00)
  • intracellular antibodies in the amount recommended by the manufacturer's instructions, and stain in the dark for 1 h.
  • flow cytometry (BD LSRII) was performed.
  • Ribosomal S6 protein is a downstream protein of mTORC1 (mTOR complex 1). Ribosomal S6 protein includes multiple isoforms, among which p70S6K is activated by phosphorylation to form p-p70S6K under the action of upstream regulators such as mammalian mTOR.
  • Assay group EpCAM CAR T cells cultured in vitro and treated with rapamycin from 24 h (represented by +Rapa), EpCAM CAR T cells cultured in vitro but not treated with rapamycin (represented by -RaPa). Sample collection time points: Days 3, 6, 9, and 12.
  • T cells were isolated and transfected with lentivirus as in Example 1, and CAR T cells were treated with rapamycin of 0 nM, 5 nM, 10 nM, 20 nM, and 40 nM respectively, and the expression of CXCR4 was detected by flow cytometry after 6 days of culture. The results are shown in FIG. 3 .
  • T cells were isolated as in Example 1, and subjected to lentiviral transfection and rapamycin (20 nM) treatment to obtain rapamycin-treated CAR T cells (hereinafter also referred to as enhanced CAR T cells) and CAR T cells not treated with rapamycin (control). Among them, the cells were collected on the 3rd, 6th, 9th, and 12th days of culture after lentivirus transfection, and the CXCR4 antibody (same as Example 1) was used to treat the CAR without rapamycin and rapamycin-treated CAR. T cells were detected by flow cytometry. The preparation of the sample and the flow detection method are the same as those in Example 1, and the flow detection results of the expression levels of CXCR4 in the CAR T cells not treated with rapamycin and the CAR T cells treated with rapamycin are shown in Figure 3.
  • CXCR4 CXCR4MFI
  • T cells in the process of preparing CAR T cells, the ability of T cells to migrate to the bone marrow is significantly reduced after activation of T cells by antibodies to CD3 and CD28 and infection with virus, while treatment with rapamycin can improve the ability of T cells to migrate to the bone marrow.
  • T cells with an improved ability to migrate to the bone marrow can even obtain excellent booster T cells that have a higher ability to migrate to the bone marrow than the initially isolated T cells. It is presumed that such T cells can enter the tumor site more efficiently, and an increased therapeutic effect can be expected.
  • the chemotactic response of the enhanced CAR T cells to CXCL12 was detected by transwell (Costar Company) with a 5 ⁇ m pore size.
  • EpCAM CAR T cells (denoted as +Rapa) that were isolated, cultured in vitro and treated with rapamycin as in Example 1, EpCAM CAR T cells that were not treated with rapamycin (denoted as -Rapa), Each group was divided into a group in which CXCL12 (SDF-1) was added to the lower chamber (denoted as +CXCL12), and a group in which CXCL12 (SDF-1) was not added (indicated as -CXCL12), resulting in a total of 4 groups.
  • the cells in the lower chamber were collected, centrifuged at 300g for 5 min at 4°C, the supernatant was discarded, and resuspended in 200ul of pre-cooled PBS buffer.
  • the samples were detected by flow cytometry in the same manner as in Example 1 (FIG. 4).
  • CXCL12 (SDF-1) is a ligand for CXCR4.
  • SDF-1 cytokine CXCL12
  • Effective CAR T cells have enhanced bone marrow migratory capacity and can be expected to have an increased therapeutic effect.
  • Measurement group EpCAM CAR T cells (+Rapa) isolated, cultured in vitro and treated with rapamycin as in Example 1, EpCAM CAR T cells (-Rapa) not treated with rapamycin.
  • mice Using immunodeficient female NCG mice (6-10 weeks old, purchased from JiCuiYaoKang), they were divided into two groups, each group of 4 mice, and 5 ⁇ 10 6 rapamycin-treated CAR T cells were transfused into the tail vein respectively. , or CAR T cells not treated with rapamycin. Mice were harvested 7 days after infusion, peripheral blood and bone marrow were collected, and erythrocytes were lysed using RBC lysis buffer (Biolegend, 420301) according to the instructions, and flow cytometry was performed as above ( FIG. 5 ).
  • RBC lysis buffer Biolegend, 420301
  • T cells can be divided into four subgroups Tem, Tcm, Tscm, and Teff by CD62L and CD45RO, of which Tcm and Tscm represent long-lived subgroups, especially Tscm subgroups.
  • the in vivo survival time of CAR T cells and the decline in their potency can be measured by detecting the expression of their exhaustion markers. When the level of expression depletion marker is high, it indicates that such CAR T cells have a short in vivo survival time and poor therapeutic activity. Therefore, in this example, the enhanced EpCAM CAR T cell subsets Tem, Tcm, Tscm, and Teff were divided, and the expression of typical exhaustion markers PD-1 and Tim3 were detected.
  • Measurement group EpCAM CAR T cells (+Rapa) isolated, cultured in vitro and treated with rapamycin as in Example 1, EpCAM CAR T cells (-Rapa) not treated with rapamycin.
  • CAR T cells Cell separation and in vitro culture of CAR T cells were carried out in the same manner as in Example 1, and two groups were tested after 12 days of in vitro expansion.
  • the ratio of each CAR T subpopulation (Tem, Tcm, Tscm, Teff) was measured using flow assay, and the above CAR T cell subpopulations were divided using CD45RO and CD62L (the antibodies used were described in Example 1).
  • the proportions of CAR T cells expressing the exhaustion marker PD-1 and Tim3 positive were detected by flow cytometry ( Figure 6, CAR T cells from the same source are connected by straight lines).
  • Assay group PBS (negative control group), EpCAM CAR T cells not treated with rapamycin, EpCAM CAR T cells treated with rapamycin.
  • NCG mice Use female NCG mice (6-10 weeks, purchased from JiCuiYaoKang) to establish a tail vein transfusion leukemia xenograft model (Kenderian et al., 2015), and determine tumor formation and grouping on the 5th day to ensure the average fluorescence of each group. The strength is the same. Then, EpCAM CAR T (infusion volume: 1 ⁇ 10 6 cells/time) was infused into tail vein once for treatment, and 15 mg/ml of fluorescein potassium salt (Gold biotechnology, lock) was injected intraperitoneally every week for fluorescence imaging detection. Tumor burden, treatment schedule and assay results are shown in Figure 7.
  • HL60 purchased from Shanghai Cell Bank, Chinese Academy of Sciences
  • EpCAM CAR T cells treated with rapamycin can more effectively clear tumors in an intravenously transfused leukemia xenograft model.
  • Assay group PBS (negative control group), CD33 CAR T cells not treated with rapamycin, CD33 CAR T cells treated with rapamycin
  • the T cell potentiator of the present invention for use in CAR T cell therapy against hematological tumors such as acute myeloid leukemia, and the method for enhancing the effect of CAR T cell therapy using the same are effective, and the potentiation obtained by such a method is effective T cells can be used for the preparation of antitumor drugs based on multiple sites, and have wide applicability.
  • T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model.Blood 113,6392-6402.
  • CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity again human acute myeloid leukemia. Leukemia 29, 1637-1647.

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Abstract

提供用于针对血液肿瘤如急性髓系白血病的CAR T细胞疗法中的T细胞增效剂，及使用其增强CAR T细胞疗法效果的方法。该方法包括使用PI3K-AKT/mTOR信号通路抑制剂作为T细胞增效剂在体外培养过程中处理在CAR T疗法中使用的T细胞。该T细胞增效剂能够增强CAR T/NK细胞向骨髓的迁移，提高CAR T细胞疗法清除肿瘤的效果。

Description

用于白血病的CAR T细胞疗法的T细胞增效剂及获得增效T细胞的方法 技术领域
本发明涉及针对肿瘤的嵌合抗原受体(CAR)T细胞疗法，具体涉及用于针对血液肿瘤如急性髓系白血病的CAR T细胞疗法中的T细胞增效剂，及使用其增强CAR T细胞疗法效果的方法。
背景技术
急性髓系白血病(Acute myeloid leukemia,AML)是一种来源于骨髓的肿瘤，尽管化疗可以诱导高达70％的缓解率，但绝大部分患者都会复发。((Bishop,1997))，当骨髓中白血病细胞超过5％则为髓内复发，如果出现在骨髓外的部分则成为髓外复发(通常为中枢神经系统和睾丸)，儿童白血病髓内髓外复发大约各占一半，而成人95％髓内复发。能否清除骨髓内的白血病干细胞是治疗急性髓系白血病成败的关键因素。
目前已报道使用靶向CD19嵌合抗原受体T细胞治疗急性淋巴细胞白血病(Acute lymphocytic leukemia,ALL)取得惊人的效果(Maude et al.,2014)。显示CAR T细胞治疗对于治疗血液病尤其是血液肿瘤的巨大潜力。至今，CD123，CD33，CLL1等靶点已经在临床前被验证为急性髓细胞白血病的潜在靶点(Kenderian et al.,2015；Mardiros et al.,2013；Wang et al.,2018)。然而，使用了针对这些位点设计的CAR T细胞的细胞疗法在治疗AML中没有达到满意的效果，即，没有在治疗AML中CAR T细胞疗法取得显著效果的报道。
对于CAR T细胞为何在AML中难以起效，发明人在研究后提出的观点是，影响CAR T起效的重要原因为CAR T细胞很难进入肿瘤部位，以及进入体内的CAR T在体内很难持续地存在。目前已有的几项临床研究提示，CAR T细胞向骨髓的迁移性是影响CAR T细胞治疗效果的基础。据报道，用于治疗的CAR T细胞向骨髓迁移越多，则患者的治疗效果越佳(Ritchie et al.,2013；Wang et al.,2015)。因此，提高CAR T细胞在治疗过程中向肿瘤部位迁移的能力成为改善CAR T细胞的治疗效果的一个研究方向。
决定CAR T细胞疗效的另一关键因素是CAR T细胞能否在体内持续存在，短期的CAR T细胞体内存活往往导致了治疗效果不佳。因此，需要一种改善CAR T细胞体内存活的方法。
发明内容
发明概述
CAR T细胞在体外的构建过程涉及到T细胞的增殖分化。在该过程中，PI3K-Akt/mTOR信号通路对于T细胞的增殖，存活，迁移以及效应/记忆亚群的分化扮演关键角色。其中，CD3/CD28抗体磁珠和IL2细胞因子以及CAR结构等向T细胞传递的信号都会导致PI3K-Akt/mTOR信号通路的活化。而过度的PI3K-Akt/mTOR信号通路的激活会促进短寿命的终末效应T细胞的形成，且会下调CD62L，CCR7及CXCR4等趋化因子受体的表达(Arojo et al.,2018；Sinclair et al.,2008)。而目前已有文章报道，通过使CAR T细胞过表达趋化因子受体从而促进了CAR T细胞向肿瘤内的迁移(Di Stasi et al.,2009；Moon et al.,2011)。
雷帕霉素是由吸水链霉菌产生，在1972年首次由苏兰德拉纳特和同事从复活节岛的吸水链霉菌样品中分离发现。该化合物最初以该岛拉帕努伊的原名命名为雷帕霉素(Sirolimus)。Sirolimus最初被开发为抗真菌剂。但是，由于发现其具有抑制mTOR的能力，具有强大的免疫抑制和抗增殖特性，因此放弃了该用途。经美国食品药品监督管理局批准于1999年9月上 市，由辉瑞公司(以前由惠氏)以商品名Rapamune销售。
雷帕霉素是一种大环内酯化合物，其在临床上使用作为免疫抑制剂，用于防止器官移植排斥，其还用于治疗一种罕见的肺部疾病，称为淋巴管平滑肌瘤病。它在以治疗浓度(10～30μg·L -1)使用时在人体中具有免疫抑制功能，尤其是在防止肾脏移植排斥中特别有用。关于其作用机理，它通常被认为通过mTOR抑制作用而降低T细胞和B细胞对白细胞介素2(IL-2)的敏感性，从而能够抑制T细胞和B细胞的活化。
发明人的前期研究发现，在CAR T细胞体外构建过程中，CXCR4的表达会降低，且产生更多的是短寿命的CAR T细胞。
发明人进行了深入研究，通过转录组分析发现，在CAR T细胞中PI3K-AKT/mTOR信号通路被明显活化。最终发现，在CAR T细胞构建过程中，通过向培养体系中添加mTOR的经典抑制剂雷帕霉素，可以有效上调CXCR4的表达，并且生成更长寿命的CAR T细胞。这样获得的CAR T细胞可以更为有效地向骨髓迁移，并且在体内存活时间更长，从而达到更好地治疗白血病的疗效。
本发明涉及一种用于针对肿瘤，优选为血液肿瘤(白血病)的CAR T细胞疗法，包括使用作为T细胞增效剂的PI3K-AKT/mTOR信号通路抑制剂，从而增强CAR T/NK细胞的骨髓迁移性及延长体内存活时间，特别地，所述PI3K-AKT/mTOR信号通路抑制剂为雷帕霉素。
在一个实施方式中，通过在培养过程中添加适当浓度例如20nM的雷帕霉素，可以减弱CAR T细胞体外生产过程中PI3K-Akt/mTOR的过度活化，上调CXCR4的表达，增强CAR T对于骨髓AML的杀伤性。此外，还可以通过增加Tscm细胞的比例，下调耗竭标记的表达，从而提高具有效能的CAR T细胞的体内存活时间，进一步增强CAR T细胞在AML治疗中的抗癌活性。
本发明涉及雷帕霉素作为T细胞增效剂通过与CAR T细胞疗法联合使用而在制备治疗血液肿瘤的药物中的应用，及在制备所述CAR T细胞疗法中用于治疗的细胞，含有该细胞的药物组合物中的应用。通过使用雷帕霉素，可以增强CAR T细胞上CXCR4的表达，提高CAR T细胞向骨髓迁移的能力，加快清除骨髓中的肿瘤细胞，同时改善CAR T细胞的效能，生成更多具有更长寿命的CAR T细胞。
本发明具体包括以下内容。
1.PI3K-AKT/mTOR信号通路抑制剂作为T细胞增效剂用于处理CAR T细胞或含有CAR T细胞的细胞群体中的应用，其中，
所述细胞疗法为嵌合抗原受体T/NK细胞疗法，所述PI3K-AKT/mTOR信号通路抑制剂例如为LY294002或雷帕霉素，优选为雷帕霉素。
2.根据项1的应用，其中，所述CAR T细胞或含有CAR T细胞的细胞群体优选经过细胞因子激活，其中所述细胞因子与PI3K-AKT/mTOR信号通路抑制剂同时处理T细胞，或在PI3K-AKT/mTOR信号通路抑制剂处理T细胞之前或之后处理T细胞。
3.增效CAR T细胞或含有CAR T细胞的细胞群体的方法，包括用添加PI3K-AKT/mTOR信号通路抑制剂的培养基培养T细胞或含有T细胞的细胞群体，所述T细胞或含有T细胞的细胞群体优选经过细胞因子激活，其中所述细胞因子与PI3K-AKT/mTOR信号通路抑制剂同时处理T细胞，或在PI3K-AKT/mTOR信号通路抑制剂处理T细胞之前或之后处理T细胞，所述PI3K-AKT/mTOR信号通路抑制剂例如为LY294002或雷帕霉素，优选为雷帕霉素。
4.根据项3的方法，所述细胞因子为IL-2，IL7和IL15，优选为IL-2。
5.根据项3或4的方法，其中雷帕霉素在培养基中的终浓度为5、10、20、30、40nM，优选为20nM，优选雷帕霉素处理时间不少于3天，更优选不少于5天。
6.增效的CAR T细胞，通过项3-5中任一项所述的方法制备。
7.根据项3-5中任一项所述的方法或项6所述的CAR T细胞，其中，所述CAR为人EpCAM嵌合抗原受体，氨基酸序列如SEQ ID No.2所示。
8.根据项1或2所述的应用、或项3-5中任一项所述的方法，或项6所述的CAR T细胞，或项7所述的方法或CAR T细胞在制备治疗肿瘤优选为白血病的药物中的用途。
9.药物组合物，其包含通过项3-5中任一项所述的方法，或项7所述的方法获得的CAR T细胞。
附图简述
图1.初始分离T细胞和CAR T细胞中CXCR4的表达的流式检测图。
图2.CAR T细胞中Mtor信号通路的磷酸化的流式检测图。
图3.雷帕霉素处理的CAR T细胞的CXCR4的表达水平的流式检测图。
图4.雷帕霉素处理增强CAR T细胞响应CXCL12的趋化的transwell迁移实验。
图5.雷帕霉素处理的CAR T细胞的骨髓迁移的流式检测图。
图6.CAR T细胞各亚群比例及耗竭标记的表达的流式检测图
图7.雷帕霉素处理的EpCAM CAR T细胞的骨髓AML的清除。(a)荧光成像检测肿瘤变化。(b)小鼠荧光成像的肿瘤负荷代表图(n＝8)。(c)总荧光值统计图。(d)骨髓部位总荧光值统计。(e)Kaplan-Meier生存分析。(f)抗人EpCAM抗体骨髓横切面组化染色。
图8.雷帕霉素处理增强CD33 CAR T细胞的抗肿瘤能力。(a)荧光成像检测肿瘤变化。(b)小鼠荧光成像的肿瘤负荷代表图(n＝6)。(c)总荧光值统计图。
具体实施方式
虽然在下述实施例中使用了雷帕霉素，但也可以使用其他对PI3K-AKT/mTOR信号通路具有抑制作用的药物，例如LY294002。在使用这样的药物时，其施用浓度为与施用5～40nM的雷帕霉素使T细胞的CXCR4升高的效果相同的浓度。
对于雷帕霉素的使用浓度而言，虽然在下述实施例中使用了20nM的终浓度孵育细胞，但也可以为5、10、20、30、40nM或这之间的浓度范围。
对于PI3K-AKT/mTOR信号通路抑制剂在细胞培养中加入的时机而言，可以在T细胞或含有T细胞的群体在体外培养的任一阶段加入，可以与细胞因子如IL-2同时加入，也可以如实施例中所述那样在加入IL-2一段时间后加入到培养基中。
能够用于本发明的细胞因子除了IL-2以外，也可以为例如：IL7和IL15，优选为IL-2，或IL-2与这些的组合。
实施例1 EpCAM CAR T细胞和CD33 CAR T细胞的构建和制备
在本实施例中制备了EpCAM CAR T细胞和CD33 CAR T细胞，两种细胞除了CAR嵌合序列不同外，其他操作及试剂均相同。
EpCAM CAR T细胞的CAR嵌合序列：将鼠源抗人EpCAM的scFv连上CD8跨膜区，41bb共刺激域及CD3ζ，得到鼠源抗人EpCAM的嵌合序列(AE4 scFV-CD8-CD28-CD3ζ，序列如SEQ ID No.1所示)，插入到PCDH-MSCV-MCS-EF1-copGFP(购自Addgene公司)中。
CD33 CAR T细胞的CAR嵌合序列：将人源化抗人CD33的scFv连上CD8跨膜区，41bb 共刺激域及CD3ζ，得到人源化抗人CD33的嵌合序列(如SEQ ID No.3所示)，插入到PCDH-MSCV-MCS-EF1-copGFP(购自Addgene公司)中
接着分别将下表的3种质粒利用PEI(Polyscience,23966)按照说明书操作转染到293T细胞(购自中国科学院上海细胞库)中，收集48h和72h的细胞培养液上清以转导T细胞。
Figure PCTCN2021076819-appb-000001
通过Ficoll密度梯度离心从新鲜人外周血(来自安徽省血液中心)分离单个核细胞，使用CD3T细胞阳选分离试剂盒(Miltenyi，30-097-043)分离T细胞。将初始分离的T细胞分成两份，一份用于流式检测，另一份用于构建CAR T细胞。以下如无特殊说明，均将进行初始分离之日作为第0天。
对用于构建各组CAR T细胞的分离的T细胞，在第0天用补充了5％人AB型血清(GEMINI,100-512)，2mmol/L谷氨酰胺的X-VIVO 15培养基(lonza，BE02-060F)以5×10 5/ml的浓度重悬T细胞，并按数目比为1:1比例加入抗CD3/CD28 Dynabeads(thermo，11161D)。在加入抗CD3/CD28 Dynabeads的时候同时加入IL-2，使IL-2的终浓度为100U/ml，每两天补充一次IL-2。
刺激24小时后，将从上述收集的细胞培养液上清中利用50000g，4℃，离心2h得到的浓缩慢病毒以MOI 50与终浓度为8ng/ml的聚凝胺(Sigma,H9268)按聚凝胺的操作说明加入活化的T细胞中，720g，32℃离心1h，6-8h后换液，并加入终浓度为20nM的雷帕霉素(Sigma,V900930-1MG)，在37℃培养箱中培养，每两天补充一次雷帕霉素，同时设立未添加雷帕霉素处理的对照组。在转导过程中每天监测细胞培养，并添加完全X-VIVO 15培养基(以保持细胞浓度为0.5–1×10 6细胞/mL。转导后第4天，去除Dynabeads。第5天后收获激活的T细胞，即EpCAM CAR T细胞和CD33 CAR T细胞，分别用于后续分析及体内实验。
实施例2.体外培养后的CAR T细胞的趋化因子下调、mTOR激活
测定组别：实施例1中初始分离的T细胞(以T表示)，及实施例1中体外培养5天后的EpCAM CAR T细胞(以CAR T表示)。
流式细胞的测定
针对待测定的细胞采用BD LSRII流式细胞仪进行检测，使用Flowjo V10进行分析。
在本实施例的测定中使用了抗人抗体EpCAM购自biolengend,(324208)，抗人CXCR4抗体购自eBioscience(12-9999-42)，抗人mTOR抗体购自BD(583489)，抗人S6抗体购自CST(14733S)，抗人CD62L抗体购自BD(555544)，抗人CD45RO抗体购自BD(560607)。
把上述收获的体外培养的EpCAM T细胞系在补充有2％胎牛血清的磷酸盐缓冲液中洗涤一次，并用小鼠血清与细胞共孵育30min，阻断Fc受体后于4℃避光染色，PBS洗涤两次后上机检测(图1，2，来自同一来源的T细胞与CAR T细胞用直线连接)。
具体而言，对于胞内染色，先用ebiscience的固定穿膜液(Invitrogen，货号00-5521-00) 固定透化后按厂家说明书建议用量加入胞内抗体，避光染色1h，穿膜液和PBS各洗一次后进行流式细胞仪(BD LSRII)检测。
结果显示，相比于初始分离的T细胞，体外培养5天后的CAR T细胞的CXCR4的表达明显下调；相比于初始分离T细胞，体外培养5天后CAR T细胞的mTOR，核糖体S6蛋白的磷酸化明显上调。核糖体S6蛋白为mTORC1(mTOR复合体1)下游蛋白。核糖体S6蛋白包括多种亚型，其中p70S6K在哺乳动物mTOR等上游调节因子的作用下被磷酸化激活形成p-p70S6K。
结果提示，在制备为CAR T细胞后，T细胞向骨髓迁移的能力下降，并且存在mTOR途径的激活，提示这可能是造成CAR T细胞对AML的治疗效果不佳的原因之一。
实施例3.雷帕霉素处理对EpCAM CAR T细胞的趋化因子的影响
测定组别：体外培养并在24h起利用雷帕霉素处理的EpCAM CAR T细胞(以+Rapa表示)，体外培养但未使用雷帕霉素处理的EpCAM CAR T细胞(以-RaPa表示)。样品收集时间点：第3，6，9，12天。
浓度筛选：同实施例1地分离了T细胞并进行慢病毒转染后分别用0nM,5nM,10nM,20nM,40nM的雷帕霉素处理CAR T细胞，培养6天后流式检测CXCR4的表达。结果示于图3。
结果显示：5nM,10nM,20nM,40nM均可用于处理CAR T细胞，20nM为最优浓度，可以最为有效上调CXCR4的表达。
同实施例1地分离了T细胞并进行慢病毒转染和雷帕霉素(20nM)的处理，得到雷帕霉素处理的CAR T细胞(下文中也称为增效的CAR T细胞)和未用雷帕霉素处理的CAR T细胞(对照)。其中，分别在慢病毒转染后培养的第3，6，9，12天收集细胞，使用CXCR4抗体(同实施例1)，对未用雷帕霉素处理和经雷帕霉素处理的CAR T细胞进行流式检测。样品的准备及流式检测方法同实施例1，并将未用雷帕霉素处理的CAR T细胞和雷帕霉素处理CAR T细胞的CXCR4的表达水平流式检测结果示于图3。
结果显示：在第3，6，9，12天的时间点中，雷帕霉素处理组的CXCR4的表达(CXCR4MFI)均显著高于未处理组，在第3，6，9，12天均超过其一倍。
结果提示，在制备为CAR T细胞的过程中，T细胞向骨髓迁移的能力在通过CD3和CD28的抗体活化T细胞并用病毒感染后出现明显下降，而通过利用雷帕霉素处理，能够获得向骨髓迁移的能力发生提高的T细胞，甚至可以得到向骨髓迁移的能力高于初始分离T细胞的优秀增效T细胞。推测这样的T细胞能够更有效地进入肿瘤部位，可以期待其增加的治疗效果。
实施例4增效的T细胞在CXCL12的趋化下的Transwell迁移实验
为了进一步探讨利用本发明的T细胞增效剂得到的增效EpCAM CAR T细胞的迁移能力，采用5μm孔径的transwell(Costar公司)检测了增效的CAR T细胞对CXCL12的趋化的响应。
测定组别：同实施例1分离、体外培养并利用雷帕霉素处理的EpCAM CAR T细胞(记作+Rapa)，未使用雷帕霉素处理的EpCAM CAR T细胞(记作-Rapa)，对于每组各分为在下室中加入CXCL12(SDF-1)的组(记作+CXCL12)，和未加入CXCL12(SDF-1)的组(记作-CXCL12)，得到共4个组别。
试验时，在24孔板中加入500ul含100ng/ml(CXCL12)SDF-1(爱必信，abs01114)的RPMI 1640完全培养基；然后将上室放入孔内，接着将200ul用cellTrace violet(thermo，C34571)按说明书进行标记的增效的CAR T细胞(1×10 5)加入transwell上室。各组细胞在 37℃，5％CO2条件下孵育4h。
收集下室的细胞，300g，4℃离心5min，弃上清，并重悬于200ul预冷的PBS缓冲液中，每个样品中加入1×10 5的未用cellTrace violet标记的CAR T细胞，以实施例1同样的方法利用流式细胞仪检测样品(图4)。
结果显示：在未加入趋化因子CXCL12(SDF-1)的两组之间，未用雷帕霉素处理组的CAR T细胞和雷帕霉素处理组向下室迁移的能力没有显著性差异；而在加入CXCL12(SDF-1)的两组之间，相比于未用雷帕霉素处理组的CAR T细胞，雷帕霉素处理组中能够迁移到下室的细胞更多，有显著性差异(**，表示p<0.01)。
CXCL12(SDF-1)是CXCR4的配体。以上结果提示，通过雷帕霉素处理增效的CAR T细胞对于作为CXCR4的配体的细胞因子CXCL12(SDF-1)的趋化能够更好地响应，进而进行迁移，进一步证明本发明的增效CAR T细胞具有增强的骨髓迁移能力，可期待具有增加的治疗效果。
实施例5增效的CAR T细胞在免疫缺陷小鼠体内的迁移追踪
为了在体内证实经本发明的T细胞增效剂处理的增效CAR T细胞的骨髓迁移情况，进行了增效EpCAM CAR T细胞在免疫缺陷小鼠体内的迁移追踪。
测定组别：同实施例1分离、体外培养并利用雷帕霉素处理的EpCAM CAR T细胞(+Rapa)，未使用雷帕霉素处理的EpCAM CAR T细胞(-Rapa)。
使用免疫缺陷小鼠雌性NCG鼠(6-10周，购自集萃药康)，分为两组，每组4只，分别尾静脉转输5×10 6的雷帕霉素处理的CAR T细胞，或未使用雷帕霉素处理的CAR T细胞。输注7天后收获小鼠，收集外周血和骨髓，利用RBC裂解液(Biolegend，420301)按说明书裂解红细胞后同上地进行了流式检测(图5)。
结果显示：从图5可知，在转输CAR T细胞后第7天收获的小鼠中，在外周血(PB)中，未用雷帕霉素处理的CAR T细胞和雷帕霉素处理组的数量没有显著性差异(ns)。但在骨髓(BM)中，雷帕霉素处理的CAR T细胞的比例为(0.38)，明显高于未用雷帕霉素处理组(0.10)，接近其4倍(**，p<0.01)；以数量进行统计时，雷帕霉素处理的CAR T细胞的数量为175043(数值)，明显高于未用雷帕霉素处理组的数量77790。通过在免疫缺陷小鼠中的动物体内试验，进一步证实，使用雷帕霉素处理增效的CAR T细胞的骨髓迁移能力确实得到了增强。
实施例6增效的CAR T细胞中耗竭标记的表达降低
T细胞可以通过CD62L和CD45RO划分为Tem，Tcm，Tscm，Teff四个亚群，其中Tcm，Tscm代表长寿命的亚群，尤其是Tscm亚群。CAR T细胞的体内存活时间长短及其效能的减退能够通过检测其耗竭标记的表达来衡量。当表达耗竭标记的水平较高时，表示这样的CAR T细胞的体内存活时间短，治疗活性较差。因此在本实施例中对增效的EpCAM CAR T细胞亚群Tem，Tcm，Tscm，Teff进行划分，并检测了典型耗竭标记PD-1和Tim3的表达。
测定组别：同实施例1分离、体外培养并利用雷帕霉素处理的EpCAM CAR T细胞(+Rapa)，未使用雷帕霉素处理的EpCAM CAR T细胞(-Rapa)。
同实施例1进行了细胞分离、CAR T细胞的体外培养，其中，在体外扩增12天后对两组进行试验。使用流式检测测量了CAR T各亚群(Tem，Tcm，Tscm，Teff)的比例，以上CAR T细胞亚群使用CD45RO和CD62L(使用的抗体见实施例1的描述)来划分。并用流式分别检测表达耗竭标记PD-1，Tim3阳性的CAR T细胞的比例(图6，来自同一来源的CAR T细胞用直线连接)。
结果显示，相比于未用雷帕霉素处理的CAR T细胞，在雷帕霉素处理组中，Tscm亚群的比例明显增加。Tscm亚群的增加，表示在增效的细胞中生成了更多长寿命的细胞。相比于未用雷帕霉素处理的CAR T细胞，在雷帕霉素处理组中，CAR T耗竭标记PD-1和Tim3的表达明显降低，PD-1有统计差差异(P＝0.0628)，Tim3中有统计差异(*表示p<0.05)。上述结果证明，经过本发明的增效剂雷帕霉素的处理，获得的CAR T细胞的质量更高寿命更长。
实施例7增效的EpCAM CAR T细胞的体内抗肿瘤试验
首先，使用免疫缺陷小鼠进行了增效的EpCAM CAR T细胞体内抗肿瘤能力的试验。
测定组别：PBS(阴性对照组)，未用雷帕霉素处理的EpCAM CAR T细胞，雷帕霉素处理的EpCAM CAR T细胞。
使用雌性NCG鼠(6-10周,购自集萃药康)建立尾静脉转输白血病异种移植模型(Kenderian et al.,2015)，在第5天确定成瘤并分组，保证每组的平均荧光强度一致。然后输注尾静脉输注EpCAM CAR T(输注量：1×10 6细胞/次)一次进行治疗，每周通过腹腔注射15mg/ml的荧光素钾盐(Gold biotechnology,lock)进行荧光成像检测肿瘤负荷，处理日程表及检测结果示于图7中。
具体而言，包括给NCG鼠进行尾静脉转输1×10 6的稳定表达荧光素酶白血病细胞的HL60(购自中国科学院上海细胞库)诱导成瘤，在第5天确定成瘤后，将鼠分为三组，每组8只，使其平均荧光保持一致。分别尾静脉转输1×10 6的未用雷帕霉素处理的EpCAM CAR T和1×10 6数量的雷帕霉素处理的EpCAM CAR T，等体积PBS。然后第5、9、16、23天进行荧光成像检测肿瘤变化。将处理日程表示于图7a。小鼠荧光成像的肿瘤负荷代表图(n＝8)和总荧光值的统计图示于图7b。
进一步，在注射诱导的第23天对(图7d)为(图7c)的骨髓部位总荧光值统计，进行Kaplan-Meier生存分析(图7e)。并将小鼠的骨髓组织在4％多聚甲醛溶液中固定过夜，脱钙后进行组织包埋切片，使用抗人EpCAM抗体(abcam，ab223582)和抗人CD3抗体(CST，#85061)进行组化染色(图7f)。
结果显示，根据肿瘤负荷荧光图及相应的总荧光值统计，可知与未用雷帕霉素处理CAR T细胞相比，在第9天，雷帕霉素处理组小鼠中的肿瘤明显更低，在第23天，可观察到几乎已不存在肿瘤，可知本发明的增效CAR T细胞的消除效果更好。对总荧光值的统计显示了肿瘤残存量的定量结果，可知注射了HL60的小鼠中，两种CAR T细胞组在各时间点的荧光值均低于PBS组，其中雷帕霉素处理组的总荧光值均低于未用雷帕霉素处理组，到第23天，雷帕霉素处理组中的总荧光值明显更低。
以上结果表明，在体内，使用雷帕霉素处理的EpCAM CAR T细胞能够更为有效地清除静脉转输白血病异种移植模型中的肿瘤。
进一步，骨髓部位(BM)的总荧光值统计显示，在第23天，未用雷帕霉素处理的CAR T细胞组中，骨髓部位的荧光值与PBS组相比没有明显降低，而雷帕霉素处理的CAR T细胞组中，骨髓部位的荧光值明显降低，提示其实现了对骨髓部位的肿瘤清除。抗人EpCAM抗体骨髓横切面组化染色显示，雷帕霉素处理的EpCAM CAR T治疗组的骨髓中，几乎没有EpCAM阳性AML的存在，而未用雷帕霉素处理组中则仍有EpCAM阳性的肿瘤细胞残留。
Kaplan-Meier生存分析结果显示，在第42天，未雷帕霉素处理的EpCAM CAR T细胞治疗组的生存百分比为75％，雷帕霉素处理的EpCAM CAR T细胞治疗组的生存百分比为100％， 在第60天在第40天，未雷帕霉素处理的EpCAM CAR T细胞治疗组的生存百分比为25％，雷帕霉素处理的EpCAM CAR T细胞治疗组的生存百分比为75％。由此可知，与PBS组、未雷帕霉素处理的EpCAM CAR T细胞相比，雷帕霉素处理的EpCAM CAR T细胞显著延长了小鼠的生存时间(分别为***,p<0.001和*,p<0.05)。上述结果证明雷帕霉素处理的EpCAM CAR T细胞可以更好地清除骨髓AML，更为有效地治疗AML。
实施例8增效的CD33 CAR T细胞的体内抗肿瘤试验
然后，以与实施例7相同的方法和操作，使用免疫缺陷小鼠对雷帕霉素处理的CD33 CAR T细胞的体内抗肿瘤能力进行了检测。
测定组别：PBS(阴性对照组)，未用雷帕霉素处理的CD33 CAR T细胞，雷帕霉素处理的CD33 CAR T细胞
同实施例7地给NCG鼠尾静脉转输1×10 6的HL60，在第5天确定成瘤而将鼠分为三组，每组6只，保证平均荧光强度一致。然后分别尾静脉转输1×10 6的未用雷帕霉素处理的CD33 CAR T和1×10 6雷帕霉素处理的CD33 CAR T细胞，等体积PBS。然后第5、16、23、30天使用小动物成像仪(PerkinElmer)进行荧光成像检测肿瘤变化。将处理日程表示于图8a。小鼠荧光成像的肿瘤负荷代表图(n＝6)示于图8b，总荧光值的统计图示于图8c。
结果显示，根据肿瘤负荷荧光图及相应的总荧光值统计，可知与未用雷帕霉素处理的CAR T细胞组相比，在第16天，雷帕霉素处理组小鼠中的肿瘤荧光明显更低，在第30天，可观察到几乎已不存在肿瘤，可知本发明的增效CAR T细胞的消除效果更好。对总荧光值的统计显示了肿瘤残存量的定量结果，可知注射了HL60的小鼠中，两种CAR T细胞组在各时间点的荧光值均低于PBS组，其中雷帕霉素处理组的总荧光值均低于未用雷帕霉素处理组，到第30天，雷帕霉素处理组中的总荧光值接近于0。
以上结果表明，在体内，使用雷帕霉素处理的CD33 CAR T细胞也能够比处理之前更为有效地清除静脉转输白血病异种移植模型中的肿瘤。
上述结果证明，通过使用雷帕霉素作为T细胞增效剂处理用于抗肿瘤的细胞疗法中使用的细胞，能够获得增强抗肿瘤特别是AML的效应的CAR T细胞，这样的方法能够适用于EpCAM靶点，也可以适用于其他治疗白血病的靶点如CD33。
因此，本发明的用于针对血液肿瘤如急性髓系白血病的CAR T细胞疗法中的T细胞增效剂，及使用其增强CAR T细胞疗法效果的方法有效，并通过这样的方法获得的增效T细胞能够用于基于多种位点的抗肿瘤药物的制备，具有广泛的适用性。
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Claims (9)

  1. PI3K-AKT/mTOR信号通路抑制剂作为T细胞增效剂用于处理CAR T细胞或含有CAR T细胞的细胞群体中的应用，其中，
    所述细胞疗法为嵌合抗原受体T/NK细胞疗法，所述PI3K-AKT/mTOR信号通路抑制剂例如为LY294002或雷帕霉素，优选为雷帕霉素。
  2. 根据权利要求1的应用，其中，所述CAR T细胞或含有CAR T细胞的细胞群体优选经过细胞因子激活，其中所述细胞因子与PI3K-AKT/mTOR信号通路抑制剂同时处理T细胞，或在PI3K-AKT/mTOR信号通路抑制剂处理T细胞之前或之后处理T细胞。
  3. 增效CAR T细胞或含有CAR T细胞的细胞群体的方法，包括用添加PI3K-AKT/mTOR信号通路抑制剂的培养基培养T细胞或含有T细胞的细胞群体，所述T细胞或含有T细胞的细胞群体优选经过细胞因子激活，其中所述细胞因子与PI3K-AKT/mTOR信号通路抑制剂同时处理T细胞，或在PI3K-AKT/mTOR信号通路抑制剂处理T细胞之前或之后处理T细胞，所述PI3K-AKT/mTOR信号通路抑制剂例如为LY294002或雷帕霉素，优选为雷帕霉素。
  4. 根据权利要求3的方法，所述细胞因子为IL-2，IL7和IL15，优选为IL-2。
  5. 根据权利要求3或4的方法，其中雷帕霉素在培养基中的终浓度为5、10、20、30、40nM，优选为20nM，优选雷帕霉素处理时间不少于3天，更优选不少于5天。
  6. 增效的CAR T细胞，通过权利要求3-5中任一项所述的方法制备。
  7. 根据权利要求3-5中任一项所述的方法或权利要求6所述的CAR T细胞，其中，所述CAR为人EpCAM嵌合抗原受体，氨基酸序列如SEQ ID No.2所示。
  8. 根据权利要求1或2所述的应用、或权利要求3-5中任一项所述的方法，或权利要求6所述的CAR T细胞，或权利要求7所述的方法或CAR T细胞在制备治疗肿瘤优选为白血病的药物中的用途。
  9. 药物组合物，其包含通过权利要求3-5中任一项所述的方法，或权利要求7所述的方法获得的CAR T细胞。
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