CN114191430A - 青蒿素在靶向抑制髓系来源的抑制性细胞及其在制备肿瘤免疫治疗药物中的应用 - Google Patents
青蒿素在靶向抑制髓系来源的抑制性细胞及其在制备肿瘤免疫治疗药物中的应用 Download PDFInfo
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Abstract
本发明公开了青蒿素在靶向抑制髓系来源的抑制性细胞及其在制备肿瘤免疫治疗药物中的应用。本发明经过大量实验证明青蒿素可以抑制髓系来源的抑制性细胞的聚集，促进髓系来源的抑制性细胞的凋亡，靶向抑制髓系来源的抑制性细胞，解除髓系来源的抑制性细胞对效应T细胞的免疫抑制作用，逆转肿瘤微环境免疫抑制状态。青蒿素作为靶向髓系来源的抑制性细胞药物使用，具有广谱有效、毒副作用小、不宜耐药和成本经济等优势。青蒿素在肿瘤免疫治疗药物领域具有重要的应用前景，可以用于治疗肿瘤的联合免疫疗法，青蒿素和抗PD‑L1抗体免疫疗法联合应用，实现协同增效的抗肿瘤效果。
Description
技术领域
本发明属于生物医药技术领域，具体涉及青蒿素在靶向抑制髓系来源的抑制性细胞及其在制备肿瘤免疫治疗药物中的应用。
背景技术
肿瘤微环境中髓系来源的抑制性细胞(myeloid-derived suppressor cells，MDSCs)是一群异质性细胞，来源于骨髓祖细胞和未成熟髓细胞，是树突状细胞、巨噬细胞和粒细胞的前体。在荷瘤小鼠的血液、脾脏和肿瘤组织及肿瘤患者的外周血和肿瘤组织存在大量MDSCs的扩增。小鼠MDSCs被定义为共表达Gr-1和CD11b的细胞。根据Gr-1的抗原表位Ly6G和Ly6C两种分子表达的不同，小鼠MDSCs可分为粒细胞样MDSCs(G-MDSCs，CD11b+Ly6G+Ly6Clow)和单核细胞样MDSCs(M-MDSCs，CD11b+ Ly6G-Ly6Chigh)两种亚型。前者在形态学上与多形核粒细胞相似，后者则具有单核细胞的形态。人的MDSCs还没有统一的表面标记可用于鉴定，目前的研究表明也存在不同表型的人类MDSCs亚型，其中包括CD14+CD11b+HLA-DRlow单核细胞样MDSCs和CD15+粒细胞样MDSCs。M-MDSCs主要通过高表达精氨酸酶活性以抗原非特异性的方式抑制T细胞功能，而G-MDSCs则利用活性氧簇ROS作为免疫介质以抗原特异性地方式抑制T细胞反应。
基于MDSCs在肿瘤生长中发挥的作用，人们正在积极探索不同的治疗放法，目前治疗策略主要分为三种：第一类治疗策略是诱导MDSCs分化，如舒尼替尼和全反式维甲酸(ATRA)或维生素D3它们可以将MDSCs分化为例如树突状细胞(DC)和刺激性单核细胞等更成熟的细胞，显示出抗肿瘤疗效,多西他赛(docetaxel，DTX)是紫杉醇的类似物，主要通过抑制MDSCs的STAT3磷酸化和促使MDSCs向M1分化来减弱MDSC的抑制作用；第二类治疗策略是减少MDSCs聚集，临床前以及临床实验表明低剂量吉西他滨和5-氟尿嘧啶均可以减少体内MDSCs的含量。吉西他滨(gemcitabine，GEM)是一种嘧啶核苷类似物的抗代谢药，可以抑制核酸还原酶和DNA聚合酶α，阻止DNA的合成。临床上广泛用于胰腺癌、肺癌、乳腺癌等多种肿瘤的治疗。在多种肿瘤动物模型中，GEM通过降低MDSCs的数量来抵抗肿瘤免疫。同样地，另一种抗代谢药5-氟尿嘧啶(5-Fu)小剂量应用时也能诱导MDSCs凋亡，而对T细胞、B细胞及NK细胞的数量没有明显影响，机制可能是MDSCs低表达胸苷酸合成酶。因此，GEM对MDSCs的作用主要表现为诱导凋亡。阿霉素(adriamycin，ADM)是蒽环类抗生素，其代谢活性物嵌入DNA碱基中形成复合体，抑制DNA合成和转录。在多种荷瘤小鼠模型中发现ADM具有免疫调节效应，能选择性地清除和灭活MDSCs；趋化因子受体CXCR2拮抗剂已在多个临床前肿瘤模型中得到验证，通过阻断YAP-1-CXCL5抑制MDSCs积聚，可能成为MDSCs积聚的抑制剂；第三类治疗策略是抑制MDSCs免疫抑制功能，其中环氧化酶2(COX2)抑制剂、ROS抑制剂、5型磷酸二酯酶(PDE-5)抑制剂、硝基阿司匹林以及合成三菇类化合物均可以通过降低Arg-1、iNOS或NOX2等关键蛋白的表达起到抑制MDSCs的作用。阿扎胞苷(5-azacytidine，AZA)是一种去甲基化药物，在小鼠TC-1/A 9和TRAMP-C2肿瘤模型中可观察到，能减少MDSCs聚集和抑制MDSCs功能。
目前通过靶向MDSCs改善免疫治疗效果是该领域研究热点，但是已发表的相关研究中这些药物均具有靶点单一、容易产生耐药以及对机体毒副作用大等缺点。CXCR2除表达于MDSCs表面外，仍表达于成熟的中性粒细胞等；化疗药物引起MDSCs凋亡的同时诱导效应性细胞的凋亡；全反式维甲酸促进MDSCs向成熟细胞分化的同时诱导肿瘤相关的巨噬细胞和Treg细胞的产生。因此，上述手段只能部分恢复免疫治疗效果，进一步增强免疫治疗效应则迫切需要开发广谱性、多靶点、不易耐药和安全可靠的MDSCs靶向药物来改善免疫治疗效果，提高临床转化的安全性和有效性。
青蒿素(Artemisinin,ART)及其衍生物是一类含有过氧基团的倍半萜内酯类药物，现已成为国际社会上普遍认可并投入使用的抗疟疾首选药物，近年来，人们发现青蒿素及其衍生物不仅具有神奇的抗疟疾作用，而且具有抗肿瘤和免疫调节作用。虽然目前的研究已经试图开始尝试利用青蒿素来治疗肿瘤疾病，但目前尚无研究报道青蒿素靶向抑制髓系来源的抑制性细胞的疗法和抗PD-L1抗体组合的联合免疫疗法。
发明内容
为了解决现有技术中的不足，本发明旨在提供青蒿素在靶向抑制髓系来源的抑制性细胞及其在制备肿瘤免疫治疗药物中的应用。
本发明第一方面提供青蒿素在靶向抑制髓系来源的抑制性细胞中的应用。
进一步地，所述青蒿素抑制髓系来源的抑制性细胞的聚集，促进髓系来源的抑制性细胞的凋亡，抑制其免疫功能。
进一步地，所述青蒿素能够使MDSCs从M2型促肿瘤表型向M1型抗肿瘤表型极化。
本发明第二方面提供一种靶向抑制髓系来源的抑制性细胞的药剂，其活性成分为青蒿素。
本发明第三方面提供青蒿素在制备肿瘤免疫治疗药物中的应用。
本发明第四方面提供一种肿瘤免疫治疗药物组合物，其活性成分包括青蒿素。
进一步地，所述的肿瘤免疫治疗药物组合物，其活性成分还包括阻断免疫检查点的药剂。
优选地，所述阻断免疫检查点的药剂为抗PD-L1抗体。
本发明的有益效果为：
本发明经过大量实验证明青蒿素可以抑制髓系来源的抑制性细胞的聚集，促进髓系来源的抑制性细胞的凋亡，靶向抑制髓系来源的抑制性细胞，解除髓系来源的抑制性细胞对效应T细胞的免疫抑制作用，逆转肿瘤微环境免疫抑制状态。青蒿素作为靶向髓系来源的抑制性细胞药物使用，具有广谱有效、毒副作用小、不宜耐药和成本经济等优势。青蒿素在肿瘤免疫治疗药物领域具有重要的应用前景，可以用于治疗肿瘤的联合免疫疗法，该联合免疫疗法由靶向抑制髓系来源的抑制性细胞的疗法和抗PD-L1抗体免疫疗法组成，青蒿素和抗PD-L1抗体免疫疗法联合应用，实现协同增效的抗肿瘤效果。
附图说明
图1为青蒿素作用下抑制MDSCs的聚集，促进其凋亡及解除MDSCs对T细胞抑制作用的影响。图A-B：体外分离培养MDSCs三天后加入不同浓度(50μM、100μM、300μM、500μM)青蒿素以DMSO为对照组流式检测MDSCs的凋亡水平和比例变化。图C：体外分离培养MDSCs三天后加入100μM青蒿素处理12小时，与脾脏中CD3+T细胞以1:1、1:2、1：4、1：8的比例加入其中混合培养。各组混合培养3天，流式细胞术检测T细胞增殖情况。
图2为青蒿素能够使MDSCs从M2型促肿瘤表型向M1型抗肿瘤表型极化。图A-B：转录组测序结果。图C：qRT-PCR检测MDSCs的M1型巨噬细胞标志基因和M2型巨噬细胞标志基因的表达。图D：Western blot检测iNOS和ARG1蛋白表达。图E：利用商品化试剂盒检测MDSCs细胞内精氨酸酶活性。图F：流式检测一氧化氮水平。
图3为青蒿素能够在体内抑制肿瘤生长，并提高抗PD-L1免疫疗法。图A-B：取6-8周大小SPF级C57BL/6小鼠，皮下接种s.c.1×106B16F10细胞或1×107Hepa1-6细胞，待肿瘤长出后给予对照组(DMSO)处理、青蒿素药物(50mg/kg)处理，在肿瘤长出后第一周每隔一天给予anti-PD-L1(10mg/kg)，连续观察3周小鼠肿瘤生长情况，每天记录小鼠肿瘤长直径和短直径，按照公式肿瘤体积(mm3)＝长直径×短直径2×1/2统计小鼠肿瘤体积变化。图C-F：于肿瘤接种后第21天处死小鼠取肿瘤组织进行流式检测。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
(1)体外检测青蒿素处理对MDSCs聚集和凋亡的影响
新鲜分离的C57BL/6小鼠的骨髓细胞，用含10％FBS的RPMI 1640培养基将细胞密度调整至1×106/ml，依此加入40ng/ml GM-CSF和40ng/ml IL-6。充分混匀后，将细胞混合液铺板于24孔板中，培养于37℃、5％CO2培养箱中。在培养至第三天时加入ART(50μM、100μM、300μM、500μM)，以DMSO为对照组流式检测MDSCs比例和凋亡。
(2)体外检测青蒿素处理对解除MDSCs对T细胞的抑制作用的影响
取上述分离的MDSCs加入100μM青蒿素处理12小时，并用磁珠分离纯化Gr-1+细胞。无菌分离C57BL/6小鼠脾脏，流式分选纯化得到其脾脏CD3+T细胞、并以1μM CFSE对其染色，以1×106/ml细胞浓度铺板于96孔板中，实验组加ConA(5ug/ml)刺激，本底对照组不加ConA，每组设3个复孔。再将纯化的MDSCs以1:1、1:2、1：4、1：8的比例加入其中混合培养。各组混合培养3天，流式细胞术检测T细胞增殖情况。
图1为青蒿素作用下抑制MDSCs的聚集，促进其凋亡及解除MDSCs对T细胞抑制作用的影响。图A-B：体外分离培养MDSCs三天后加入不同浓度(50μM、100μM、300μM、500μM)青蒿素以DMSO为对照组流式检测MDSCs的凋亡水平和比例变化。结果显示与DMSO相比，随着青蒿素浓度越高MDSCs的凋亡水平逐渐增加，MDSCs比例越来越低，说明青蒿素可以在体外促进MDSCs凋亡并抑制MDSCs聚集，且成浓度依赖性。图C：体外分离培养MDSCs三天后加入100μM青蒿素处理12小时，与脾脏中CD3+T细胞以1:1、1:2、1：4、1：8的比例加入其中混合培养。各组混合培养3天，流式细胞术检测T细胞增殖情况。结果显示，青蒿素处理后的MDSCs细胞与对照组DMSO相比，T细胞的增殖水平明显升高，说明青蒿素处理的MDSCs解除了对T细胞的抑制作用。
实施例2
通过转录组测序结果分析显示，青蒿素作用下与对照组DMSO相比，MDSCs抑制M2型通路相关基因的表达。并用Western Blot和qRT-PCR检测MDSCs细胞内免疫抑制因子基因ARG1、M1型基因iNOS的表达，利用商品化试剂盒检测MDSCs细胞内精氨酸酶和一氧化氮水平。
图2：青蒿素能够使MDSCs从M2型促肿瘤表型向M1型抗肿瘤表型极化。
图A-B：转录组测序显示，青蒿素作用下与对照组DMSO相比，MDSCs的ARG1免疫抑制基因下调，且抑制M2型通路的相关基因表达。图C：qRT-PCR检测MDSCs的M1型巨噬细胞标志基因和M2型巨噬细胞标志基因的表达，结果显示与对照组DMSO相比，青蒿素可以抑制MDSCs的M2型基因表达并上调M1型基因的表达。图D：Westernblot检测iNOS和ARG1蛋白表达。结果显示在我们选用的100μM浓度时iNOS表达升高而ARG1表达降低。图E：利用商品化试剂盒检测MDSCs细胞内精氨酸酶活性。图F：流式检测一氧化氮水平。结果显示在青蒿素100μM的作用下MDSCs精氨酸含量降低而一氧化氮含量增加。说明青蒿素能够下调MDSCs的M2型基因和蛋白表达，上调M1型，使MDSCs从M2型向M1型极化。
实施例3
在C57BL/6小鼠体内分别建立B16F10、Hepa1-6两种肿瘤模型，验证体内青蒿素抑制肿瘤生长的效果，并与anti-PD-L1抗体联用增强anti-PD-L1免疫疗法。
取6-8周大小SPF级C57BL/6小鼠，皮下接种s.c.1×106B16F10细胞或1×107Hepa1-6细胞，待肿瘤长出后给予对照组(DMSO)处理、青蒿素药物(50mg/kg)处理，在肿瘤长出后第一周每隔一天给予anti-PD-L1(10mg/kg)，每天记录小鼠肿瘤长直径和短直径，按照公式肿瘤体积(mm3)＝长直径×短直径2×1/2统计小鼠肿瘤体积变化，于肿瘤接种后第21天，取小鼠骨髓、脾脏以及肿瘤组织进行流式检测。
图3：青蒿素能够在体内抑制肿瘤生长，并提高抗PD-L1免疫疗法。
图A-B：取6-8周大小SPF级C57BL/6小鼠，皮下接种s.c.1×106B16F10细胞或1×107Hepa1-6细胞，待肿瘤长出后给予对照组(DMSO)处理、青蒿素药物(50mg/kg)处理，在肿瘤长出后第一周每隔一天给予anti-PD-L1(10mg/kg)，连续观察3周小鼠肿瘤生长情况，每天记录小鼠肿瘤长直径和短直径，按照公式肿瘤体积(mm3)＝长直径×短直径2×1/2统计小鼠肿瘤体积变化。图C-F于肿瘤接种后第21天处死小鼠取肿瘤组织进行流式检测。结果显示与对照组DMSO相比，青蒿素和anti-PD-L1单独作用时均可抑制肿瘤生长，联用后效果更佳。在肿瘤组织中流式结果显示，青蒿素可以抑制肿瘤组织中MDSCs及其亚群的比例，增强CD3+T细胞、CD4+T细胞的比例，青蒿素与anti-PD-L1联用后可以提高anti-PD-L1对MDSCs及其亚群的抑制作用以及提高anti-PD-L1对CCD3+T细胞、CD4+T细胞免疫活力。说明青蒿素能够在体内抑制肿瘤生长，与抗PD-L1抗体联用后可以提高抗PD-L1免疫疗法。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (8)
1.青蒿素在靶向抑制髓系来源的抑制性细胞中的应用。
2.根据权利要求1所述的应用，其特征在于，所述青蒿素抑制髓系来源的抑制性细胞的聚集，促进髓系来源的抑制性细胞的凋亡。
3.根据权利要求1所述的应用，其特征在于，所述青蒿素使髓系来源的抑制性细胞从M2型促肿瘤表型向M1型抗肿瘤表型极化。
4.一种靶向抑制髓系来源的抑制性细胞的药剂，其特征在于，其活性成分为青蒿素。
5.青蒿素在制备肿瘤免疫治疗药物中的应用。
6.一种肿瘤免疫治疗药物组合物，其特征在于，其活性成分包括青蒿素。
7.根据权利要求6所述的肿瘤免疫治疗药物组合物，其特征在于，所述活性成分还包括阻断免疫检查点的药剂。
8.根据权利要求7所述的肿瘤免疫治疗药物组合物，其特征在于，所述阻断免疫检查点的药剂为抗PD-L1抗体。
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| CN112773788A (zh) * | 2020-09-29 | 2021-05-11 | 上海市肺科医院 | 双氢青蒿素在制备非小细胞肺癌药物中的应用 |
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2021
- 2021-12-01 CN CN202111458936.5A patent/CN114191430A/zh active Pending
- 2021-12-14 WO PCT/CN2021/137750 patent/WO2023097764A1/zh unknown
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|---|---|---|---|---|
| CN112891339A (zh) * | 2021-01-27 | 2021-06-04 | 中国药科大学 | 包封青蒿素的血红素纳米囊泡、制备方法和用途 |
| CN113304146A (zh) * | 2021-06-05 | 2021-08-27 | 澳门大学 | 双氢青蒿素类化合物在制备pd-l1表达抑制剂中的应用 |
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| Title |
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| 王盼等: "青蒿素及其衍生物在相关疾病中的免疫调节作用", 《中国热带医学》 * |
| 陈皖晴等: "髓源抑制细胞介导肿瘤免疫微环境重塑及靶向治疗的研究进展", 《上海中医药杂志》 * |
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