CN111893094A - 临床用tscm诱导培养和质量控制鉴定试剂盒及应用 - Google Patents
临床用tscm诱导培养和质量控制鉴定试剂盒及应用 Download PDFInfo
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Abstract
本发明涉及免疫细胞靶向治疗技术领域,特别涉及一种优化的从新鲜或低温冻存的人体外周血单个核细胞或脐血单个核细胞中分离纯化T细胞,T细胞诱导扩增干细胞样记忆性T细胞(TSCM)的试剂盒及产品质量控制鉴定的试剂盒体系和应用,TSCM具有更强的增殖、自我更新和存活能力,能分化成中央记忆性T细胞(TCM),效应记忆性T细胞(TEM)和效应T细胞,小鼠动物实验表明,TSCM和其他分化的T细胞亚群相比,在体内有更强的抗肿瘤效应。在临床应用中,TSCM细胞培养过程中采用的试剂较多,细胞质量标准难以控制,很难在短时间内培养出质量均一的TSCM细胞,迫切需要开发简单、格式化的试剂盒,对临床应用具有重要的意义。
Description
技术领域
本发明涉及免疫细胞靶向治疗技术领域,特别涉及一种优化的从新鲜或低温冻存的人体外周血单个核细胞或脐血单个核细胞中分离纯化T细胞,T细胞诱导扩增干细胞样记忆性T细胞(TSCM)的试剂盒及产品质量控制鉴定的试剂盒体系和应用。
背景技术
过继性细胞治疗技术为打破肿瘤患者中枢和外周免疫耐受,恢复或增强其抗肿瘤免疫功能提供了一种有效的方法。然而目前回输的免疫细胞都是基于终末分化的效应性T细胞,在体内难以长期存活,极大的影响了过继性细胞治疗的临床疗效。
Gattinoni等在小鼠中发现一类具有自我更新能力的抗原特异性CD8+T细胞,这些称为TSCM( T-memory stem cells )的表面标志CD62L+CD45RA+CD95+CD45RO-,TSCM具有更强的增殖、自我更新和存活能力,能分化成中央记忆性T细胞(TCM),效应记忆性T细胞(TEM)和效应T细胞,小鼠动物实验表明,TSCM和其他分化的T细胞亚群相比,在体内有更强的抗肿瘤效应。
本发明人2018年发明了《一种优化的临床用TCM分选、诱导培养和质量控制鉴定试剂盒及应用》(专利号:201811194036.2),TSCM和TCM表型不同,基因表达谱和功能差异很小,二者属于同一个记忆性干细胞池,TCM在体外培养过程中容易分化成TEM,体外培养20天后,TCM大部分失去增值能力,变成分化状态,TSCM保持了较高比例的表型,CD4TSCM占比83%,CD8TSCM占比100%,具有分化状态的记忆性T细胞亚群的存在会扰乱长期免疫的建立,因此纯化T细胞亚群对于成功的过继性免疫细胞治疗具有重要的意义。在临床应用中,TSCM细胞培养过程中采用的试剂较多,标准不一致,培养过程中工作量大,培养时间长,工艺繁琐,不稳定,染菌风险大,细胞质量标准难以控制,许多技术人员很难在短时间内培养出质量均一的TSCM细胞,迫切需要开发简单、格式化的试剂盒,同时本发明通过进一步优化诱导因子组合物TSCM-III,节省了原材料,降低了生产成本;诱导培养前用TSCM-II包被培养板TSCM细胞增殖倍数提高了2-3倍,凋亡指数降低,活性提高了10%-20%,有效细胞的绝对数量提高了2-3倍;TCR基因转染前,用TSCM-II包被培养板可提高转染效率。本发明改进了工艺,这对临床上提高肿瘤治疗疗效,清除残留病灶,防止肿瘤的复发和转移具有重要的意义。
发明内容
为了解决TCM在体外培养过程中容易分化成TEM的问题;本申请提供了一种从新鲜或低温冻存的人体外周血PBMC及脐血单个核细胞(UBMC)中分离纯化出T细胞,T细胞诱导TSCM,诱导活化率高的试剂盒及应用;纯化出T细胞相比PBMC直接诱导TSCM,纯化后诱导效率更高;然后将肿瘤特异或病毒特异性TCR基因转染自体TSCM,使免疫细胞治疗具有长效性和靶向性。
本发明是通过以下步骤得到的:
本发明提供一种TSCM诱导培养试剂盒,包括容器和装入容器中的临床用TSCM细胞无血清扩增完全培养液TSCM-I和TSCM细胞扩增包被液TSCM-II和诱导因子组合物TSCM-III:
本发明提供一种临床用无血清TSCM细胞诱导扩增完全培养液TSCM-I,含有人体细胞生长必须的碳水化合物、促生长因子和激素、维生素、人血白蛋白、氨基酸、无机盐离子、微量元素、水、人白介素7、人白介素21和人白介素15。本发明不排除可以采用上述无血清完全培养基之外的培养基实施培养。
本发明提供一种TSCM细胞扩增包被液TSCM-II,它含有重组人纤维蛋白片段和PBS,重组人纤维蛋白片段的工作浓度为12.5ug/ml-50ug/ml。
本发明提供一种TSCM细胞诱导因子组合物TSCM-III,无血清TSCM细胞扩增完全培养液TSCM-I中添加50-200ng/mLCD3单抗、50-200ng/mLCD28单抗、5-30ng/mL人白介素7、5-30ng /mL人白介素15和5-30ng /mL人白介素21。
优选步骤(7)中T细胞按照细胞密度1×106/mL用TSCM-III重悬,其中添加8ng /mLIL-7、8ng /mL人白介素15、8ng /mL IL-7、80ng/mL CD3单抗、80ng/mL CD28。第二天以后用TSCM-I培养液对T细胞细胞进行诱导扩增培养,经过14-22天的扩增培养,收获达到临床应用标准的TSCM细胞;对收获的TSCM细胞制剂进行内毒素、活性和表型检测,细胞鉴定结果表明,细胞活性达到95%以上,同时含有较高比例的CD62L+ PE-cy7/CD45RO-Percp-Cy5.5CD/45RA+FITC/CD95+PETSCM细胞;TSCM在体外活化过程中会获得CD45RO+的表达,同时保留CD45RA和CD62L的表达,这可能与CD3和CD28活化过程中下调CD45RA和CD62L的表达有关,这群细胞称为TSCM-like,二者抗肿瘤效率相同。
质量检测合格的 TSCM细胞装入50-100ml生理盐水中,制成免疫细胞制剂,配合肿瘤临床治疗需要静脉回输。
本发明提及的无血清培养基和诱导因子或者TSCM细胞诱导液的原料可以从市场上购买,并且长期冷冻保存细胞和小分子蛋白的技术已经非常成熟,本领域技术人员完全可以利用上述技术保存组成本发明试剂盒的培养基和组合物。
本发明还包括将上述TSCM细胞诱导试剂盒、无血清TSCM细胞扩增完全培养液TSCM-I和TSCM细胞扩增包被液体TSCM-II和TSCM-III装入专用洁净容器制备成试剂盒。
本发明通过包被TSCM-II工作液,TSCM细胞增殖倍数提高了2-3倍,凋亡指数降低,活性提高了10%-20%,这对临床上提高肿瘤治疗疗效,清除残留病灶,防止肿瘤的复发和转移具有重要的意义。
重组TCR逆转录病毒转染(培养后第3天)TSCM细胞。
根据目的基因序列,设计引物,通过PCR技术克隆目的基因,构建重组人逆转录病毒PLXPXSN-TCRa12-2-IRES-Vβ7.1,用HEK293细胞包装完整病毒颗粒后以MOI=100的比例转染TSCM。转染前TSCM-II 配成50ug/ml工作液,包被到六孔板中。
培养后5天用DC负载肝癌肿瘤抗原AFP进行肿瘤抗原再刺激。
台盼蓝染色法获取TSCM体外刺激活化后生长曲线(至体外培养至22天)。
本发明另一方面还提供一种TSCM诱导后鉴定试剂盒,所述临床用TSCM细胞流式表型和活性鉴定试剂盒包括五组双色抗体组合物,分别是CD3-BV421/CD4-APC-Cy/CD8APC/CD62L+PE-cy7/CD45RO-Percp-Cy5.5CD/45RA+FITC/CD95+PE和Annexin-FINKTC/PI,洗涤液50ml一管,固定液50ml一管;所述抗体按照体积1:1混合装于1支棕色EP管中,每20ul可检测一次,共计10-50套,所述洗涤液是磷酸盐缓冲液;所述固定液是1%多聚甲醛。
所述试剂盒制备中的质量控制鉴定方法,按照GMP要求,将相关物料密封装入试剂盒,按照《中国药典》进行无菌和内毒素检测;
所述制备的细胞制剂的质量控制鉴定方法,包括按照国家食品药品监督管理总局公布的《药品注册管理办法(修订稿)》意见中“生物制品注册分类和申报资料要求(试行)”进行质量控制,保证临床应用时的安全性。
上述方法中,在获得本发明TSCM之前,用生理盐水洗涤并重悬所述TSCM细胞3-4次,直至满足《中国药典》要求的无菌、内毒素的检测指标,保证本发明的TSCM细胞在回输给患者时残留的本发明所述的培养基组分和诱导因子组合物不会引起患者不适反应。所述TSCM细胞组合物包括1x105-3x107/ml本发明的TSCM,优选为1x106-2x107/ml。所述组合物还包括人血白蛋白;其中,以所述组合物为基础,所述人血白蛋白的质量体积含量为1-5%,优选为3%。
本发明还提供了一种本发明TSCM细胞组合物的临床应用和疗效评估方法。
本发明提供了一种TSCM在临床肿瘤综合治疗中的应用方法,其中包括让患者知晓本发明TSCM的治疗作用并签署知情同意书后,自由选择本发明TSCM细胞作为辅助手段在肿瘤综合治疗中的联合应用。
优选所述TSCM组合物使用的方式为滴注。具体地,使用所述TSCM细胞组合物的方式为采用6号针头或7号针头、输血器,静脉注射或静脉滴注。优选使用所述TSCM细胞组合物的方式为在手臂或者下肢采用7号针头输血器进行静脉滴注。
所述TSCM细胞组合物优选包含3x106-2x107/ml的本发明TSCM细胞,其有效量为50ml-100ml/个体的生理盐水组合物。
以上所述TSCM细胞组合物都是一次静脉滴注的剂量,一个疗程三-四次静脉滴注治疗,从第一次开始期间连续或每间隔一天再回输下一次。一个疗程结束后,适疗效情况可以间隔两到三个月后继续给药。如有必要,间隔时间为一年或两年后还可继续再治疗。
所述疗效评价的标准有:
1)起免疫增强作用的细胞因子如白介素2、白介素12、干扰素γ和肿瘤坏死因子a在内的Th1类细胞因子变化水平,一般升高为有效;
2)起免疫抑制作用的细胞因子如白介素6、白介素10、转化生长因子β和表皮细胞生长因子的变化水平,一般降低为有效;
3)T淋巴细胞亚群变化:CD3、CD4、CD8、CD62L+ PE-cy7/CD45RO-Percp-Cy5.5CD/45RA+FITC/CD95+PE阳性率的升高或改善,CD16/CD56阳性率的升高,以及CD4+FOX3+的降低;
4)其他疗效评价指标还有:患者肿瘤标志物检测,如甲胎蛋白、鳞癌细胞抗原、游离PSA与总PSA比值、癌胚抗原、糖链抗原125、糖链抗原CA242、细胞角蛋白19片段CYFRA21-1、游离绒毛膜促性腺激素β-HCG、神经炎特异性烯醇化酶(NSE)、糖链抗原199、总前列腺特异性抗原TPSA、游离前列腺特异性抗原TPSA、糖链抗原CA724在内的肿瘤特异性抗原的表达降低,主要通过流式荧光发光法检测;影像学检测,如核磁共振、CT和B超检测;以及患者生存期统计;
5)Karnofsky评分法和生活质量评分法。
优选本发明的TSCM细胞用于自体治疗,可最大限度的避免免疫排斥反应造成的副作用。
统计学分析
实验数据以均数士标准差来表示。应用SPSS16.0统计软件进行统计分析。各指标先进行正态性及方差齐性检验。两因素两水平数据比较采用2x2析因设计的方差分析(不同组细胞肿瘤杀伤活性比较,抗体抑制试验,各组细胞的细胞因子分泌。采用重复测量的方差分析完成各组细胞不同时间地点细胞数量分析。采用单因素方差分析对不同组细胞端粒长度进行分析。当P<0.05时,被认为差异具有统计学意义。
本发明所述疾病包括但不限于肾癌、恶性黑色素瘤、肺癌、肝癌、大肠癌、胃癌、淋巴瘤和病毒感染性疾病包括但不限病毒性肝炎、结核病和艾滋病等。
本发明的有益效果:
1)本发明提供了一种临床用TSCM细胞高效、快速制备方法,相关组合物和试剂盒及其应用方法。本发明对TSCM细胞的诱导、培养及产品质量控制进行了优化和完善,通过一套TSCM细胞诱导、培养及产品质量控制鉴定的试剂盒形式,规范TSCM细胞临床应用,为TSCM细胞高效快速制备和临床应用质量标准统一提供标准模式;
2)本发明中T复苏的细胞存活率93%±3%,复苏后TSCM诱导率为95%±3%;
3)T细胞诱导成TSCM的诱导率比PBMC直接诱导TSCM高57%±3%;
4)诱导培养前用TSCM-II包被培养板可提高TSCM的增殖能力;
5)TCR基因转染前,用TSCM-II包被培养板可提高转染效率。
附图说明
图1为T细胞诱导前形态特征A和诱导后TSCM形态特征B;
图2 为T细胞诱导15天后表型鉴定;
图3为 T细胞和PBMC诱导TSCM第15天时的表型对比;
图4为诱导培养22天时TSCM和TCM表型对比;
图5为不同诱导因子组合诱导后TSCM生长曲线。
具体实施方式
下面结合具体实施例对本发明进行进一步说明:
实施例1
一、外周血PBMC或脐血UBMC分离和T细胞纯化:
采集枸橼酸钠抗凝血100ml,通过淋巴细胞分离液分离其中的PBMC和UBMC,纯化T细胞,进行深低温冻存。
二、T细胞冷冻后复苏:取出冻存后的T细胞,迅速投入 37 ℃水浴中,并不断轻轻摇动,保证管内液体在1 min 内80%溶解,将细胞吸到一定量 DMEM 洗涤液中,并洗涤冻存管1 次,移入50 mL 离心管中,233 g 离心5 min。弃上清,加入适量培养基,轻轻混匀,留取500 微升做计数测细胞活性。
三、细胞计数和台盼蓝拒染法测细胞活性
Countstar检测细胞样本,读取屏幕上显示的细胞平均值数值。根据公式细胞数T=细胞数/ml*V计算细胞总数。
四、T细胞回收率
计算T细胞回收率,采用公式:T细胞细胞回收率=(分离后细胞数/分离前细胞数)×100%。
实施例2
在实施例1基础上,用TSCM诱导试剂盒进行TSCM的诱导:
本发明提供一种TSCM诱导培养试剂盒,包括容器和装入容器中的临床用TSCM细胞无血清扩增完全培养液TSCM-I和TSCM细胞扩增包被液TSCM-II和诱导因子组合物TSCM-III:
1)本发明提供一种临床用无血清TSCM细胞扩增完全培养液TSCM-I,含有人体细胞生长必须的碳水化合物、促生长因子和激素、维生素、人血白蛋白、氨基酸、无机盐离子、微量元素、水和人白介素7、人白介素15和人白介素21。本发明不排除可以采用上述无血清完全培养基之外的培养基实施培养。
2)本发明提供一种TSCM细胞扩增包被液TSCM-II,它含有重组人纤维蛋白片段和PBS,重组人纤维蛋白片段的工作浓度为12.5ug/ml-50ug/ml。
3)本发明提供一种TSCM细胞诱导因子组合物TSCM-III,无血清TSCM细胞扩增完全培养液TSCM-I中添加50-200ng/mLCD3单抗、50-200ng/mLCD28单抗、5-30ng/mL人白介素7、5-30ng /mL人白介素15和5-30ng /mL人白介素21。
D0天将TSCM-II 配成12.5ug/ml工作液,包被到培养瓶中,平放于37℃培养箱中大于2小时。T细胞按照细胞密度1×106/mL用TSCM-III重悬,其中添加8ng/mL人白介素7、8ng/mL人白介素15和8ng/mL人白介素21、80ng/mL CD3单抗、80ng/mL CD28。第二天以后用TSCM-I培养液对TSCM细胞进行扩增培养,经过12-15天的扩增培养,收获达到临床应用标准的TSCM细胞,并对细胞制剂进行内毒素、活性和表型检测,细胞鉴定结果表明,细胞活性达到95%以上,同时含有较高比例的CD3+-BV421/CD4+-APC-Cy/CD8+APC/CD62L+ PE-cy7/CD45RO-Percp-Cy5.5CD/45RA+FITC/CD95+PE细胞;见图1和图2。
实施例3 T细胞和PBMC诱导TSCM诱导效率对比
D0天将TSCM-II 配成12.5ug/ml工作液,包被到培养瓶中,平放于37℃培养箱中大于2小时。PBMC细胞按照细胞密度1×106/mL用TSCM-III重悬,其中添加8ng/mL人白介素7、8ng/mL人白介素15、80ng/mL CD3单抗、80ng/mL CD28。第二天以后用 TSCM-I培养液对TSCM细胞进行扩增培养,经过12-15天的扩增培养,收获达到临床应用标准的TSCM细胞,并对细胞制剂进行内毒素、活性和表型检测,细胞鉴定结果表明,PBMC细胞直接诱导TSCM的诱导率低于T细胞,见图3;同时,可以看出诱导培养22天后,CD8TCM占比0.92%,CDTCM占比3.65%,大部分失去增殖能力,变成分化状态,CD8TSCM占比99.58%,CDTSCM占比97.21%,始终保持较高的比例,见图4。
实施例4
重组TCR逆转录病毒转染(培养后第3天)TSCM细胞。
根据肝癌特异基因TCRa12-2和Vβ7.1序列,设计引物,通过PCR技术克隆目的基因,构建重组人逆转录病毒PLXPXSN-TCRa12-2-IRES-Vβ7.1,用HEK293细胞包装完整病毒颗粒,-80℃冰箱保存病毒颗粒,以MOI=100的比例转染TSCM。
1)将TSCM-II 配成50ug/ml工作液,包被到六孔板中,平放于37℃培养箱中大于2小时;
2)吸出RetroNectin溶液,每孔加入PBS终止液0.5 ml;
3)室温放置30分钟;
4)加入适量PBS清洗孔板;
5)吸出清洗液,得到RetroNectin包被的孔板;
6)逆转录病毒保存液 (-80℃) 于37℃水浴融解;
7)将病毒液加入包被后的六孔板,每孔1ml;
8)32℃放置4小时;
9)吸出病毒保存液;
10)每孔加入待转染的细胞悬液;
培养后5天用DC负载肝癌肿瘤抗原AFP肿瘤抗原再刺激;
台盼蓝染色法获取TSCM体外刺激活化后生长曲线(至体外培养至22天)。
上述细胞培养过程中及以下实施例中TSCM组合物制备都应该在GMP要求的百级洁净室或者操作台进行,严格保证细胞及其制剂的质量安全。
上述实施例包括培养袋培养,本发明的实施例中设计到的添加剂或者因子加入量只明示了其中某个比例,凡是试剂盒涉及的TSCM细胞培养鉴定,以及添加相关添加剂或者因子比例能满足在上述权利要求的浓度范围的都应属于本发明要求保护之列。
实施例5
诱导因子组合物TSCM-III配方进一步优化。
实验共分6组,培养15天时做流式,分析CD4TSCM和CD8TSCM的百分比和增殖倍数。
A组: 8ng/mL IL-7+8ng /ml IL-15+80ng/mL CD3+80ng/mL CD28;
B组:8ng/mL IL-7+8ng /ml IL-15+8ng /ml IL-21+80ng/mL CD3+80ng/mL CD28;
C组:4ng/mL IL-7+4ng /ml IL-15+40ng/mL CD3+40ng/mL CD28;
D组:4ng/mL IL-7+4ng /ml IL-15+4ng /ml IL-21+40ng/mL CD3+40ng/mL CD28;
E组:16ng/mL IL-7+16ng /ml IL-15+80ng/mL CD3+80ng/mL CD28;
F组:16ng/mL IL-7+16ng /ml IL-15+16ng /ml IL-21+80ng/mL CD3+80ng/mL CD28;
表1 不同诱导因子组合诱导后CD4TSCM和CD8TSCM的百分比和增殖倍数
Treatment Percentage of Growth multiple Percentage of Growth multiple
CD4+TSCM CD4+ TSCM CD8+TSCM CD8+TSCM
A组 99.33±0.6 493 100± 1.8 330
B组 99.19±0.3 1763 100± 1.68 1179
C组 97.22 ±0.25 43.9 100±1.5 29.3
D组 96.9±0.4 23.8 100±1.8 15.9
E组 98.65±0.2 383 100±1.3 256
F组 99.20±0.1 177 100±1.2 119
每天观察细胞生长状态,每隔1-2天添加TSCM-I培养基,应用Countstar计数仪进行细胞计数,绘制生长曲线,见图5。A-F组细胞间差异具有统计学意义(F=199.09,P<0.001)。
实施例6
TSCM组合物制备及临床应用:
(1)上述实施例中获得的TSCM细胞用0.9%医用生理盐水重悬,计数,在细胞重悬液中添加人血白蛋白3%,调整总体积100ml;
(2)事先准备临床用100ml装有0.9%生理盐水袋或瓶,将上述细胞组合物装入其中,贴上说明标签;
(3)待细胞组合物相应检测合格后,送临床实施自体TSCM细胞治疗回输。
(4)临床应用方法说明如下:
1)TSCM组合物给药的途径优选:在手臂或者下肢采用7号针头输血器进行静脉滴注;
2)所述TSCM细胞组合物临床有效剂量包括:组合物含3x106-2x107/ml细胞,途径80ml-100ml/个体,一个疗程连续回输3-4次;
3)从第一次开始期间连续或每间隔一天再回输下一次。一个疗程结束后,适疗效情况可以间隔两到三个月后继续给药。如有必要,间隔时间为一年或两年后还可继续再治疗;
4)TSCM细胞临床回输治疗后进行疗效评价:
经过TSCM细胞治疗1-2个疗程后,较治疗之前患者生活质量明显改善,KPS评分法评分提高20-30分,QOL评分法提高30-40分,患者免疫功能有不同程度改善,其中细胞因子检测显示能够使Th1类细胞因子水平提高3-6倍。
实施例7
TSCM分选和诱导后鉴定试剂盒制备及应用
在培养第15天,用本发明的TSCM诱导后鉴定试剂盒鉴定TSCM的表面marker CD3+-BV421/CD4+APCCy/CD8+APC/CD62L+PEcy7/CD45ROPercpCy5.5CD/45RA+FITC/CD95+PEL的表达,细胞培养第15d,CD4TSCM细胞比例占99.56%,CD8TSCM细胞比例占100%,结果见图2。
(1)单细胞悬液:细胞密度调整至5x105-5x106/ml;
(2)吸取样本100ul,加入CD3-BV421/CD4-APC-Cy/CD8APC/CD62L PE-cy7/CD45RO-Percp-Cy5.5CD/45RAFITC/CD95PE荧光标记抗体20ul,室温避光孵育15min;
(3)加入清洗液2ml,1000rpm离心5min;
(4)去上清,500ul PBS缓冲液重悬后上机检测。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受实施例的限制,其它任何未背离本发明的精神实质与原理下所做的改变、修饰、组合、替代、简化均应为等效替换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种临床用TSCM诱导培养和质量控制鉴定的试剂盒体系,其特征在于,包含从新鲜或低温冻存的人体外周血单个核细胞或脐血单个核细胞中分离纯化T细胞,T细胞诱导扩增干细胞样记忆性T细胞(TSCM)的试剂盒及产品质量控制鉴定的试剂盒体系和应用:
(1)TSCM诱导培养试剂盒,包括容器和装入容器中的临床用TSCM细胞无血清扩增完全培养液TSCM-I和TSCM细胞扩增包被液TSCM-II和诱导因子组合物TSCM-III:
本发明提供一种临床用无血清TSCM细胞诱导扩增完全培养液TSCM-I,含有人体细胞生长必须的碳水化合物、促生长因子和激素、维生素、人血白蛋白、氨基酸、无机盐离子、微量元素、水、人白介素7、人白介素21和人白介素15;本发明不排除可以采用上述无血清完全培养基之外的培养基实施培养;
本发明提供一种TSCM细胞扩增包被液TSCM-II,它含有重组人纤维蛋白片段和PBS,重组人纤维蛋白片段的工作浓度为12.5ug/ml-50ug/ml;
(2)本发明提供一种TSCM细胞诱导因子组合物TSCM-III,无血清TSCM细胞扩增完全培养液TSCM-I中添加50-200ng/mLCD3单抗、50-200ng/mLCD28单抗、5-30ng/mL人白介素7、5-30ng /mL人白介素15和5-30ng /mL人白介素21;
优选步骤(2)中T细胞按照细胞密度1×106/mL用TSCM-III重悬,其中添加8ng /mL IL-7、8ng /mL人白介素15、8ng /mL IL-7、80ng/mL CD3单抗、80ng/mL CD28,第二天以后用TSCM-I培养液对T细胞细胞进行诱导扩增培养,经过14-22天的扩增培养,收获达到临床应用标准的TSCM细胞;对收获的TSCM细胞制剂进行内毒素、活性和表型检测,细胞鉴定结果表明,细胞活性达到95%以上,同时含有较高比例的CD62L+ PE-cy7/CD45RO-Percp-Cy5.5CD/45RA+FITC/CD95+PETSCM细胞;TSCM在体外活化过程中会获得CD45RO+的表达,同时保留CD45RA和CD62L的表达,这可能与CD3和CD28活化过程中下调CD45RA和CD62L的表达有关,这群细胞称为TSCM-like,二者抗肿瘤效率相同;
(3)本发明另一方面还提供一种TSCM诱导后鉴定试剂盒,所述临床用TSCM细胞流式表型和活性鉴定试剂盒包括五组双色抗体组合物,分别是CD3-BV421/CD4-APC-Cy/CD8APC/CD62L+PE-cy7/CD45RO-Percp-Cy5.5CD/45RA+FITC/CD95+PE和Annexin-FINKTC/PI,洗涤液50ml一管,固定液50ml一管;所述抗体按照体积1:1混合装于1支棕色EP管中,每20ul可检测一次,共计10-50套,所述洗涤液是磷酸盐缓冲液,所述固定液是1%多聚甲醛;
(4)本发明还包括将上述TSCM细胞诱导试剂盒、无血清TSCM细胞扩增完全培养液TSCM-I和TSCM细胞扩增包被液体TSCM-II和TSCM-III装入专用洁净容器制备成试剂盒。
2.应用权利要求1所述的试剂盒体系制备TSCM的方法,其特征在于,包括以下步骤:
(1)外周血PBMC或脐血UBMC分离和T细胞纯化:
(2)TSCM诱导试剂盒使用:
1)D0天将TSCM-II 配成12.5ug/ml工作液,包被到培养瓶中,平放于37℃培养箱中大于2小时;
2)T细胞按照细胞密度1×106/mL用TSCM-III重悬,其中添加8ng/mL人白介素7、8ng /mL人白介素15和8ng/mL人白介素21、80ng/mL CD3单抗、80ng/mL CD28,第二天以后用 TSCM-I培养液对TSCM细胞进行扩增培养,经过12-15天的扩增培养,收获达到临床应用标准的TSCM细胞,并对细胞制剂进行内毒素、活性和表型检测,细胞鉴定结果表明,细胞活性达到95%以上,同时含有较高比例的CD3+-BV421/CD4+-APC-Cy/CD8+APC/CD62L+PE-cy7/CD45RO-Percp-Cy5.5CD/45RA+FITC/CD95+PE细胞;
(3)质量检测合格的TSCM细胞装入50-100ml生理盐水中,制成免疫细胞制剂,配合肿瘤临床治疗需要静脉回输。
3.根据权利要求2所述的干细胞样记忆性免疫细胞,其特征在于步骤(2)中T细胞按照密度1x106ml接种,采用TSCM-II包被培养瓶,采用T75、T175培养瓶和1L培养袋生产获得了临床应用TSCM细胞,培养时间为12-28天。
4.根据权利要求2所述的干细胞样记忆性免疫细胞,其特征在于诱导后使用重组TCR逆转录病毒转染(培养后第3天)TSCM细胞,转染前将TSCM-II 配成50ug/ml工作液,包被到六孔板中,平放于37℃培养箱中大于2小时。
5.根据权利要求2所述的干细胞样记忆性免疫细胞,其特征在于培养后5天用DC负载肝癌肿瘤抗原AFP进行肿瘤抗原再刺激。
6.根据权利要求2所述的干细胞样记忆性免疫细胞,其特征在于所述试剂盒制备中的质量控制鉴定方法,按照GMP要求,将相关物料密封装入试剂盒,按照《中国药典》进行无菌和内毒素检测;细胞制剂的质量控制鉴定方法,按照国家食品药品监督管理总局公布的《药品注册管理办法(修订稿)》意见中“生物制品注册分类和申报资料要求(试行)”进行质量控制,保证临床应用时的安全性。
7.根据权利要求2所述的干细胞样记忆性免疫细胞,其特征在于在获得本发明TSCM之前,用生理盐水洗涤并重悬所述TSCM细胞3-4次,直至满足《中国药典》要求的无菌、内毒素的检测指标,保证本发明的TSCM细胞在回输给患者是残留的本发明所述的培养基组分和诱导因子组合物不会引起患者不适反应。
8.所述TSCM细胞组合物包含3x106-2x107/ml的本发明TCM细胞,其有效量为50ml-100ml/个体的生理盐水组合物,密度为1x105-3x107/ml,优选为1x106-2x107/ml,还包括人血白蛋白;其中,以所述组合物为基础,所述人血白蛋白的质量体积含量为1-5%,优选为3%。
9.根据权利要求2所述的干细胞样记忆性免疫细胞,其特征在于上述步骤TSCM组合物使用的方式为滴注,具体地,使用所述TSCM细胞组合物的方式为采用6号针头或7号针头、输血器,静脉注射或静脉滴注,优选使用所述TSCM细胞组合物的方式为在手臂或者下肢采用7号针头输液器进行静脉滴注,以上所述TSCM细胞组合物都是一次静脉滴注的剂量,一个疗程三-四次静脉滴注治疗,从第一次开始期间连续或每间隔一天再回输下一次,一个疗程结束后,适疗效情况可以间隔两到三个月后继续给药,如有必要,间隔时间为一年或两年后还可继续再治疗。
10.一种权利要求1-9中任一项所述的干细胞样记忆性免疫细胞在临床肿瘤治疗、病毒感染性疾病和药物中的应用,包括但不限于肾癌、恶性黑色素瘤、肺癌、肝癌、大肠癌、胃癌、淋巴瘤和病毒感染性疾病包括但不限病毒性肝炎、结核病和艾滋病等。
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