CN116999391A - 一种car-t细胞制剂及其制备方法和应用 - Google Patents
一种car-t细胞制剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种CAR‑T细胞制剂及其制备方法和应用,所述CAR‑T细胞制剂为微乳液;微乳液包括水、正丁酸乙酯、卵磷脂、免疫调节液、乙醇,其体积比为35‑45:4‑6:3‑8:2‑5:1‑3;免疫调节液按摩尔浓度包括0.01‑0.03mol/L淫羊藿苷、0.03‑0.06mol/L葛根素、0.02‑0.05mol/L大豆苷元、0.03‑0.08mol/L当归多糖。本发明通过的CAR‑T细胞制剂辅助修饰T细胞进行多发性骨髓瘤的对抗治疗。
Description
技术领域
本发明涉及细胞制剂领域,具体为一种CAR-T细胞制剂及其制备方法和应用。
背景技术
多发性骨髓瘤(multiplemyeloma,MM)是一种恶性浆细胞性肿瘤,可归为B细胞淋巴瘤范畴,多种机制参与疾病发生。特征为骨髓中恶性浆细胞浸润,常伴有单克隆免疫球蛋白或其片段(M蛋白)过度生成,导致靶器官损害。
目前普遍的治疗方法包括单克隆抗体、免疫调节类药物、激素类药物、蛋白酶体抑制剂联合自体造血干细胞移植治疗等,但由于遗传异质性和克隆演变等原因,多数患者短期内复发,复发/难治性患者RRMM及高危细胞遗传学患者预后尤其差。近年,随着生物治疗领域的飞速发展,RRMM患者有了更多的选择:靶向不同靶点的单克隆抗体、双特异性抗体、免疫检查点抑制剂、抗体药物偶联物、双特异性T细胞增殖剂、嵌合抗原受体T(chimericantigen receptor T,CAR-T)细胞等治疗方法。
CAR-T细胞在多种肿瘤治疗特别是血液系统肿瘤治疗中显示出良好疗效,是近年来抗肿瘤治疗领域最卓越的成果,然而,其在治疗多发性骨髓瘤时依然对肿瘤细胞杀伤力不足、增殖能力和表达能力仍有待提高。
发明内容
本发明为了解决现有技术中存在的缺陷,提供一种能对多发性骨髓瘤细胞有高杀伤力、对BCMA的CAR-T细胞有高表达力的CAR-T细胞制剂及其制备方法和应用。
本发明为了达到此目的,采用如下的技术方案:
一种CAR-T细胞制剂,所述CAR-T细胞制剂为微乳液;微乳液包括水、正丁酸乙酯、卵磷脂、免疫调节液、乙醇,其体积比为35-45:4-6:3-8:2-5:1-3;免疫调节液按摩尔浓度包括0.01-0.03mol/L淫羊藿苷、0.03-0.06mol/L葛根素、0.02-0.05mol/L大豆苷元、0.03-0.08mol/L当归多糖。
微乳液是粒径在10-100nm之间的一种液态溶液,主要是由水相、油相、表面活性剂组成的一种透明的混合溶液,是一种在热力学上相对稳定的体系,可以分为水包油型、油包水型、双连续相型。微乳液由于具有多相,能提高难溶于水的物质的溶解度,是一种新型的给药方法,其相对稳定性好,且药物分散性好,细胞对其的吸收率和增殖都具有良好的促进作用。本发明的CAR-T细胞制剂主要为O/W水包油型。
CAR-T疗法就是嵌合抗原受体T细胞免疫疗法,英文全称Chimeric AntigenReceptor T-Cell Immunotherapy。这是一种治疗肿瘤的新型精准靶向疗法,近几年通过优化改良在临床肿瘤治疗上取得很好的效果,是一种非常有前景的,能够精准、快速、高效,且有可能治愈癌症的新型肿瘤免疫治疗方法。
T细胞也叫T淋巴细胞,是人体白细胞的一种,来源于骨髓造血干细胞,在胸腺中成熟,然后移居到人体血液、淋巴和周围组织器官,发挥免疫功能。
CAR-T细胞治疗实体瘤,得找到特异性高表达的靶点,这样可以最大程度上避免损伤其他正常的细胞。因此合适的靶点是相对重要的,这样能比通用型靶点疗效更好,也减少对细胞的毒性作用。本申请的CAR-T细胞的抗原靶点主要为BCMA,这个靶点的高表达优于其他靶点。
本发明还公开一种上述的CAR-T细胞制剂的制备方法,包括如下步骤:
S1称取淫羊藿苷、葛根素、大豆苷元、当归多糖;
S2分别将淫羊藿苷、葛根素、大豆苷元、当归多糖溶于乙醇溶液中;
S3旋转蒸发以后离心;
S4冷冻干燥成纳米粉末;
S5将淫羊藿苷、葛根素、大豆苷元、当归多糖加入水溶液中加热溶解后冷却成免疫调节液;
S6将水、正丁酸乙酯、卵磷脂、免疫调节液、乙醇;按体积比为35-45:4-6:3-8:2-5:1-3的比例混合均匀制成CAR-T细胞制剂。
优选的,S2步骤中溶于乙醇溶液时加热。
优选的,S3步骤中旋转蒸发转速为40-80rpm。
优选的,S4步骤中为真空冷冻干燥。
优选的,S5步骤中加热温度为40-60℃。
优选的,S2步骤中还包括过滤步骤,乙醇溶液溶解后用微孔无菌器过滤。
优选的,S6步骤中混合均匀后避光保存。
本发明所述的CAR-T细胞制剂主要应用在制备细胞制剂和对多发性骨髓瘤的治疗中。
本发明的有益效果:
1.本发明通过CAR-T细胞制剂辅助修饰T细胞进行多发性骨髓瘤的治疗;
2.本发明的CAR-T细胞制剂在体外能增强CAR-T细胞的杀伤作用,提高CAR-T细胞疗法在多发性骨髓瘤治疗中的抗肿瘤作用;
3、本发明的CAR-T细胞制剂采用微乳化溶液,粒径小,稳定性高、活性物质细胞透过率高,能有效提高疗效;
4、本发明的CAR-T细胞制剂能辅助提高CAR-T细胞疗法的抑癌效果,有效提升了多发性骨髓瘤细胞的细胞凋亡率。
附图说明
图1为多发性骨髓瘤细胞活力实验检测中空白组细胞图;
图2为多发性骨髓瘤细胞活力实验检测中对照组细胞图;
图3为多发性骨髓瘤细胞活力实验检测中实施例四细胞图。
具体实施方式
为了使本领域的技术人员更好地理解发明的技术方案,下面结合具体实施方式对本发明作进一步的详细说明。
本发明公开了一种CAR-T细胞制剂,所述CAR-T细胞制剂为微乳液;微乳液包括水、正丁酸乙酯、卵磷脂、免疫调节液、乙醇,其体积比为35-45:4-6:3-8:2-5:1-3;免疫调节液按摩尔浓度包括0.01-0.03mol/L淫羊藿苷、0.03-0.06mol/L葛根素、0.02-0.05mol/L大豆苷元、0.03-0.08mol/L当归多糖。
更优选的,免疫调节液按摩尔浓度包括0.02mol/L淫羊藿苷、0.05mol/L葛根素、0.05mol/L大豆苷元、0.06mol/L当归多糖。
CAR-T细胞的抗原靶点为BCMA。
上述的CAR-T细胞制剂的制备方法,包括如下步骤:
S1称取淫羊藿苷、葛根素、大豆苷元、当归多糖;
S1步骤中称取淫羊藿苷、葛根素、大豆苷元、当归多糖并分别单独放置;
S2分别将淫羊藿苷、葛根素、大豆苷元、当归多糖溶于乙醇溶液中;
S2步骤中,淫羊藿苷溶解于乙醇溶液中,0.5-1mol淫羊藿苷配置到500-1000mL乙醇中,优选溶解温度为40-60摄氏度;葛根素溶解于乙醇溶液时,取0.2-0.4mol葛根素溶解到500-1000mL乙醇中,溶解温度优选的为45-55摄氏度;大豆苷元溶解到乙醇溶液中时,取0.2-0.5mol大豆苷元溶解到500-1000mL乙醇中,溶解温度优选为30-50摄氏度;当归多糖溶于乙醇时,取0.5-1mol溶解到500-1000mL乙醇中,溶解温度优选的为25-45摄氏度。乙醇溶液溶解后用微孔无菌器过滤。
S3旋转蒸发以后离心;
将淫羊藿苷、葛根素、大豆苷元、当归多糖的乙醇溶液分别进行旋转蒸发,控制旋转速度20-80rpm/min,优选的为真空旋转蒸发,蒸发温度控制在30-45℃。当乙醇量少于20mL时,将溶液倒出并进行离心,取下层沉淀物。
S4冷冻干燥成纳米粉末;
S4步骤中优选的采用真空冷冻干燥技术进行纳米粉末的制备,并单独放置;
S5将淫羊藿苷、葛根素、大豆苷元、当归多糖加入水溶液中加热溶解后冷却成免疫调节液;
称取0.01-0.03mol/L淫羊藿苷、0.03-0.06mol/L葛根素、0.02-0.05mol/L大豆苷元、0.03-0.08mol/L当归多糖混合均匀后,倒入1L的去离子水,加热至40-60℃溶解。未取用前避光保存。
S6将水、正丁酸乙酯、卵磷脂、免疫调节液、乙醇;按体积比为35-45:4-6:3-8:2-5:1-3的比例混合均匀制成CAR-T细胞制剂。
用水、正丁酸乙酯、卵磷脂、免疫调节液、乙醇制备水包油型微乳液。室温条件下进行制备,保持400rpm搅拌速度,先将水、正丁酸乙酯、卵磷脂、乙醇预混30min,再用滴水法滴加免疫调节液制备O/W型微乳液。待全部逐滴加入后,继续在搅拌器上衡速搅拌30min,最终获得澄清均匀分散体系。本发明的CAR-T细胞制剂为O/W型微乳液。
淫羊藿苷(Icariin)是淫羊藿的主要活性成分,为8-异戊烯基黄酮苷类化合物。可以从箭叶淫羊藿、柔毛淫羊藿、巫山淫羊藿,朝鲜淫羊藿等干燥茎叶中提取得到。为淡黄色针状结晶,溶于乙醇、乙酸乙酯,不溶于醚、苯、氯仿。淫羊藿地上部分主要含黄酮类化合物,地下部分主要含黄酮类化合物及生物碱。此外,淫羊藿属植物尚含木脂素类、蒽醌类、花青素、倍半萜类、苯乙醇苷类、多糖、葡萄糖、果糖、植物甾醇、棕榈酸、硬脂酸、亚麻酸、氯化钾等上百种化学成分,这些成分分布于淫羊藿属不同植物中。淫羊藿苷能增加心脑血管血流量、促进造血功能、免疫功能及骨代谢。
葛根素可以双向调节骨髓基质细胞的成骨和成脂分化,主要体现在两方面:一是通过促进成骨细胞生成直接促进骨的形成;二是通过抑制脂肪细胞的生成间接促进骨髓基质细胞成骨分化,并通过降低脂肪细胞对破骨细胞的促进作用,在整体上使骨量增加,从而起到预防骨质疏松的效果。葛根素能使大鼠血清中血清钙、血清磷含量及碱性磷酸酶水显著增加,而由成骨细胞分泌的骨钙素水平降低至正常水平,从而加快骨质合成、改善骨质疏松,其骨密度、骨矿物质含量、骨密度、肌肉含量水平及骨密质扫描结果均有改善,骨密质质地坚硬致密,骨松质内红骨髓增加,从而有效改善骨质疏松。
大豆苷元可通过调节ERs和PPARY等受体表达间接影响成骨细胞的药物反应.雌激素可明显影响大豆苷元的受体调节作用。大豆苷元对细胞受体表达的调节作用能对成骨细胞进行调节。
当归多糖(ASP)是从当归中分离得到的主要活性成分,具有较强的造血、免疫、抗氧化、抗肿瘤和保护肝脏的生物活性。当归多糖(ASP)对大鼠骨髓间充质干细胞成骨向分化有正向影响。
以上为本发明的CAR-T细胞制剂的说明,以下结合具体的实施例进行进一步的说明。
实验方法
一、CAR-T细胞制剂的制备
S6将水、正丁酸乙酯、卵磷脂、免疫调节液、乙醇;按体积比为35-45:4-6:3-8:2-5:1-3的比例混合均匀制成CAR-T细胞制剂。
用水、正丁酸乙酯、卵磷脂、免疫调节液、乙醇制备水包油型微乳液。室温条件下进行制备,保持400rpm搅拌速度,先将水、正丁酸乙酯、卵磷脂、乙醇预混30min,再用滴水法滴加免疫调节液制备O/W型微乳液。待全部逐滴加入后,继续在搅拌器上衡速搅拌30min,最终获得澄清均匀分散体系。
按下表1组分表进行配比后在透射电镜下检测微乳液样品形态
水 | 正丁酸乙酯 | 卵磷脂 | 免疫调节液 | 乙醇 | |
配比一 | 35 | 4 | 3 | 2 | 1 |
配比二 | 38 | 5 | 5 | 3 | 2 |
配比三 | 40 | 6 | 8 | 4 | 2 |
配比四 | 42 | 5 | 7 | 5 | 3 |
配比五 | 45 | 6 | 8 | 5 | 3 |
表1 CAR-T细胞制剂配置表
根据表1配置好的CAR-T细胞制剂的微乳液在透射电镜下的形态确认最佳配比为配比四。
二、多发性骨髓瘤细胞系的培养
本申请中的多发性骨髓瘤细胞为MM.1S,购买自中国(上海)科学院典型培养物保藏委员会细胞库。
1.细胞复苏
使用快速解冻法复苏细胞,将冻存的多发性骨髓瘤细胞从液氮中取出。在37℃的恒温水浴锅中快速解冻,预热融化多发性骨髓瘤细胞溶液,然后将多发性骨髓瘤细胞溶液转移到10%FBS培养基的离心管中,1200rpm离心5min后弃掉上清液,用培养基重悬多发性骨髓瘤细胞,并将其悬液均匀铺在细胞培养瓶中储存。储存环境为37℃,等融合后准备进行传代培养。
2.多发性骨髓瘤细胞的传代培养
取出多发性骨髓瘤细胞,转移到离心管中,1500rpm离心5min后去掉上清液,使用PBS缓冲液重悬,将重悬后的多发性骨髓瘤细胞混匀后再进行离心,离心后将离心管中的上清液去掉。接种培养到培养瓶中,然后放置于培养箱中进行传代培养,培养箱的培养环境为37℃,5%CO2。
3.多发性骨髓瘤细胞的冻存
待细胞长至80%-90%融合度时,去掉培养液并加入PBS,洗涤1-2次,再收集细胞悬液,1200rpm离心5-8min。去掉上清液。按DMSO:FBS体积比1:9配置冻存液,保存于4℃冰箱待用。如果非直接取用时,储存于-20℃,以便长期保存。利用冻存液重悬细胞,待细胞充分悬起后,每个试管中加入1-2mL细胞冻存液进行保存。
三、BCMACAR-T细胞的制备
取多发性骨髓瘤细胞,分选CD3阳性细胞后,体外培养并活化后,将BCMA CAR-T载体转导到T细胞中,并持续观察检测,直到T细胞稳定表达靶向BCMA抗体的CAR受体稳定扩增为1ⅹ106/L。
四、细胞增殖能力测定(CCK-8法)
1.CCK-8法基本原理
CCK-8法可以通过比色检测方法测定细胞增殖或毒性。其利用水溶性四唑盐
_WST-8,在电子载体1-MethoxyPMSF存在的条件下能够被还原成水溶性的甲臜染料。生成的甲臜量与活细胞的数量成正比。利用酶标仪检测450nm处培养液吸光值,可间接反应出活细胞的数量。CCK-8溶液可直接加入到细胞样品中,与MTT法比较,CCK-8法显色时间短,不需要预配。置各种成分,操作简便、灵敏度高、重复性好。
2.制作CCK-8最佳孵育时间曲线
将细胞以3x103个/孔接种于96孔板中,平行5个孔,待细胞贴壁后,加入CCK-8溶液,分别孵育1h、2h、3h、4h后测定吸光值,以孵育时间为横坐标(X轴),吸光值为纵坐标(Y轴)的曲线,绘制标准曲线。
五、不同浓度对多发性骨髓瘤细胞活力的影响
(1)取正常生长的多发性骨髓瘤细胞,收集细胞计数,重悬细胞,按照3x103个/孔,接种100uL细胞悬液于96孔板并滴加培养基,过夜培养使细胞贴壁。
(2)取2mL细胞悬液,去掉旧培养基,加入CCK-8溶液和培养基。加药组加入细胞量数为多发性骨髓瘤细胞四分之一的BCMACAR-T细胞和1mg CAR-T细胞制剂,对照组加入细胞量数为多发性骨髓瘤细胞四分之一的BCMACAR-T细胞和1mg含有等体积DMSO载体溶液,空白组不再加入溶液。培养24h,36h,48h,每孔加入10μLCCK-8溶液,细胞培养箱孵育2h后于450nm处检测吸光度。
(3)细胞活力计算公式:细胞活力(%)=[A(加药)-A(空白)]/[A(对照组)-A(空白)]X 100%.
A(加药):含有BCMACAR-T细胞、CCK-8溶液和CAR-T细胞制剂溶液
A(空白):不含BCMA CAR-T细胞,只含培养基和CCK-8溶液
A(对照组):含有BCMACAR-T细胞、CCK-8溶液、等体积DMSO溶液
(4)根据活力计算公式得出的结果找到最佳作用浓度和作用时间。
按表1中的配比四配置微乳液,并按下表2的免疫调节液的摩尔浓度配比制备加药组的CAR-T细胞制剂。
表2CAR-T细胞制剂浓度配比表
将细胞接种于96孔板中,浓度分别按实施例1-6的浓度配比为,对照组为DMSO培养基,分别作用24h、36h、48h后利用CCK-8法检测其吸光值,按照细胞活力计算公式计算细胞活力。作用24h后,以空白组的多发性骨髓瘤细胞增值数为基础值计算细胞增值量。对照组和加药组的各实施例对多发性骨髓瘤细胞的细胞凋亡均有显著影响。对照组和加药组对多发性骨髓瘤细胞的细胞凋亡率减少量分别为:对照组11.4%、实施例一15.2%、实施例二17.5%、实施例三18.3%、实施例四20.8%、实施例五18.7%、实施例六19.2%。图1为空白组细胞图,图2为对照组细胞图,图3为实施例四细胞图,从图中可以看出,对照组和加药组对细胞的凋亡率均有一定程度的增高,与对照组相比,加药组的作用效果有极显著性差异(P<0.01),各实施例组之间略有差异。并且实施例六效果最为明显,促凋亡效果最强。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种CAR-T细胞制剂,其特征在于:所述CAR-T细胞制剂为微乳液;微乳液包括水、正丁酸乙酯、卵磷脂、免疫调节液、乙醇,其体积比为35-45:4-6:3-8:2-5:1-3;免疫调节液按摩尔浓度包括0.01-0.03mol/L淫羊藿苷、0.03-0.06mol/L葛根素、0.02-0.05mol/L大豆苷元、0.03-0.08mol/L当归多糖。
2.根据权利要求1所述的CAR-T细胞制剂,其特征在于:CAR-T细胞的靶点为BCMA。
3.一种权利要求1所述的CAR-T细胞制剂的制备方法,其特征在于:包括如下步骤:
S1称取淫羊藿苷、葛根素、大豆苷元、当归多糖;
S2分别将淫羊藿苷、葛根素、大豆苷元、当归多糖溶于乙醇溶液中;
S3旋转蒸发以后离心;
S4冷冻干燥成纳米粉末;
S5将淫羊藿苷、葛根素、大豆苷元、当归多糖加入水溶液中加热溶解后冷却成免疫调节液;
S6将水、正丁酸乙酯、卵磷脂、免疫调节液、乙醇;按体积比为35-45:4-6:3-8:2-5:1-3的比例混合均匀制成CAR-T细胞制剂。
4.根据权利要求3所述的CAR-T细胞制剂的制备方法,其特征在于:S2步骤中溶于乙醇溶液时加热。
5.根据权利要求3所述的CAR-T细胞制剂的制备方法,其特征在于:S3步骤中旋转蒸发转速为40-80rpm。
6.根据权利要求3所述的CAR-T细胞制剂的制备方法,其特征在于:S4步骤中为真空冷冻干燥。
7.根据权利要求3所述的CAR-T细胞制剂的制备方法,其特征在于:S5步骤中加热温度为40-60℃。
8.根据权利要求3所述的CAR-T细胞制剂的制备方法,其特征在于:S2步骤中还包括过滤步骤,乙醇溶液溶解后用微孔无菌器过滤。
9.根据权利要求3所述的CAR-T细胞制剂的制备方法,其特征在于:S6步骤中混合均匀后避光保存。
10.根据权利要求1所述的CAR-T细胞制剂在制备细胞制剂中的应用。
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