CN112725281A - 一种个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法及应用 - Google Patents
一种个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法及应用 Download PDFInfo
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Abstract
本发明是一种个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法，步骤包括，A、肿瘤原代细胞培养；B、可冻存的肿瘤抗原负载的DC细胞的获得；C、肿瘤新抗原特异性T细胞获得；D、新抗原特异性T细胞功能检测。本方法与传统相比，具有创新性，能够在短时间内分析鉴定数百个突变多肽序列，筛选出能产生特异性抗肿瘤免疫应答的突变多肽制备疫苗，提高疗效。
Description
技术领域
本发明涉及生物技术领域，具体来说是一种个体化肿瘤新抗原介导细胞免疫应答的体外预测方法及应用。
背景技术
癌细胞突变基因编码的氨基酸序列称为 “新抗原”（neoantigen)，主要由点突变、缺失突变、融合突变等产生的、正常细胞没有的、新的异常蛋白/多肽。新生抗原用作疫苗，经DC细胞摄取、改造和提呈，产生具识别肿瘤新抗原的特异性T淋巴细胞，发挥抗肿瘤细胞免疫应答。然而，很多研究结果显示，虽然依靠遗传信息推算得出的肿瘤新生抗原数量很多，但能被自体T细胞识别的却只有大约1-2%，即使是浸润在肿瘤内的T细胞也只能识别1/11的新抗原。因此，如何鉴定突变多肽的免疫原性，是确保新抗原多肽疫苗疗效的关键。
发明内容
本发明的目的是提供一种个体化肿瘤新抗原介导的细胞免疫应答的体外预测方法。
为实现上述目的，本发明采用的技术方案是：一种个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法，步骤包括：
A、肿瘤原代细胞培养；
B、可冻存的肿瘤抗原负载的DC细胞的获得；
C、肿瘤新抗原特异性T细胞获得；
D、新抗原特异性T细胞功能检测。
进一步的，所述肿瘤原代细胞培养步骤包括：
A1.在1-2 h内取下肿瘤块并用生理盐水或者PBS清洗，然后物理剪切成小块并置于高糖DMEM细胞培养基中；
A2.将组织块转移到加有HBSS的50 mL离心管中轻摇混匀后 400g ，5-10 min低温离心，弃上清并重新添加HBSS，重复次3次；
A3.转移到含有高糖DMEM培养基的10cm 无菌培养皿中并进一步剪切到直径3-5mm；
A4.将得到的细胞悬液以及组织块用100um 细胞滤网过滤到50mL 离心管中 162g离心4min；
A5.弃上清，并添加5-20 mL DMEM培养基重悬细胞；
A6.用排枪将重悬好的细胞转移到96孔细胞培养板中150uL/孔培养7天，然后转移到低附着性培养瓶中继续培养，每4天换一次液，直到细胞群体积开始变大，之后每7天换一次液，21天之后原代培养基中可以不加入ROCK 抑制剂；
A7.当细胞密度长到100个细胞群/mL 时,冻存到含有10% DMSO液氮中。
进一步的，步骤A3中，所述DMEM培养基含有10%的血清。
进一步的，所述DC细胞的获得步骤为：患者注射肿瘤新抗原疫苗后采血50mL，用传统密度梯度离心法获外周血单个核细胞，利用磁珠分离并诱导成抗原负载的成熟DC细胞，收集DC细胞并冻存在液氮。
进一步的，所述肿瘤新抗原特异性T细胞获得步骤包括：
C1.患者注射肿瘤新抗原疫苗后采血50mL，密度梯度离心法获外周血单个核细胞3-5×108，用抗人CD14抗体磁珠获得纯度大于90% 的CD14 阳性单个核细胞，CD14阴性淋巴细胞冻存；
C2.单个核细胞2×106培养在内含1000U/mL 粒细胞巨噬细胞集落生子因子、1000U/mL重组人白介素4的无血清GMP CellGro培养基；
C3.第3天加入10 mmol/mL肿瘤新抗原多肽，过夜；
C4.第4天加入2.5ug/mL人工合成的单磷酰脂质A和1000 U/mL医药级别的伽玛干扰素继续培养24 h；
C5.第5天收集抗原负载的DC细胞，一半冻存；
C6.将C1步骤冻存的CD14阴性淋巴细胞复苏与DC细胞以10:1的比例在含有10%自体血清的X-VIVO20培养液培养14天，在第3天和第10天加入20 ng/mL人重组白介素2；
C7.DC与T细胞共培养的第7天，加入复苏冻存的抗原负载的DC共培养；
C8.第14天，加入50 ng/mL 佛波醇12-十四酸酯13-乙酸酯、1 mg/mL 离子霉素和10 mg/mL 布雷菲德菌素A培育5 h后，收获抗原特异性CD8细胞。
进一步的，所述新抗原特异性T细胞功能检测步骤包括：
D1.向检测板中加入培养基并测定背景阻抗值；
D2.收集对数期细胞并计数，调整细胞悬液浓度，向检测板中加入一定体积的细胞，室温超净台内放置30min；
D3.将加入细胞的检测板放入检测台上，进行实时动态的细胞增殖检测；
D4.过夜检测后，向各培养孔中加入配制好的细胞因子并继续进行实时动态检测，即可获得细胞因子介导的细胞效应曲线及不同时间段IC50值。
本发明的有益技术效果是：本方法与传统相比，具有创新性，能够在短时间内分析鉴定数百个突变多肽序列，筛选出能产生特异性抗肿瘤免疫应答的突变多肽制备疫苗，提高疗效。
说明书附图
图1是实施例2利用本申请方法预测一位获得的20个突变肽序列自体肺腺癌细胞的实时杀伤实验结果；图中数字表示多肽序列号，箭头表示多肽特异性细胞毒淋巴细胞加入患者原代肿瘤细胞共培养。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
一种个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法，步骤包括：
A、肿瘤原代细胞培养；
A1.在1-2 h内取下肿瘤块并用生理盐水或者PBS清洗，然后物理剪切成小块并置于高糖DMEM细胞培养基中；
A2.将组织块转移到加有HBSS的50 mL离心管中轻摇混匀后 400g ，5-10 min低温离心，弃上清并重新添加HBSS，重复次3次；
A3.转移到含有高糖DMEM培养基的10cm 无菌培养皿中并进一步剪切到直径3-5mm；所述DMEM培养基含有10%的血清；
A4.将得到的细胞悬液以及组织块用100um 细胞滤网过滤到50mL 离心管中 162g离心4min；
A5.弃上清，并添加5-20 mL DMEM培养基重悬细胞；
A6.用排枪将重悬好的细胞转移到96孔细胞培养板中150uL/孔培养7天，然后转移到低附着性培养瓶中继续培养，每4天换一次液，直到细胞群体积开始变大，之后每7天换一次液，21天之后原代培养基中可以不加入ROCK 抑制剂；
A7.当细胞密度长到100个细胞群/mL 时,冻存到含有10% DMSO液氮中。
B、可冻存的肿瘤抗原负载的DC细胞的获得；患者注射肿瘤新抗原疫苗后采血50mL，用传统密度梯度离心法获外周血单个核细胞，利用磁珠分离并诱导成抗原负载的成熟DC细胞，收集DC细胞并冻存在液氮。
C、肿瘤新抗原特异性T细胞获得；
C1.患者注射肿瘤新抗原疫苗后采血50mL，密度梯度离心法获外周血单个核细胞3-5×108，用抗人CD14抗体磁珠获得纯度大于90% 的CD14 阳性单个核细胞，CD14阴性淋巴细胞冻存；
C2.单个核细胞2×106培养在内含1000U/mL 粒细胞巨噬细胞集落生子因子、1000U/mL重组人白介素4的无血清GMP CellGro培养基；
C3.第3天加入10 mmol/mL肿瘤新抗原多肽，过夜；
C4.第4天加入2.5ug/mL人工合成的单磷酰脂质A和1000 U/mL医药级别的伽玛干扰素继续培养24 h；
C5.第5天收集抗原负载的DC细胞，一半冻存；
C6.将C1步骤冻存的CD14阴性淋巴细胞复苏与DC细胞以10:1的比例在含有10%自体血清的X-VIVO20培养液培养14天，在第3天和第10天加入20 ng/mL人重组白介素2；
C7.DC与T细胞共培养的第7天，加入复苏冻存的抗原负载的DC共培养；
C8.第14天，加入50 ng/mL 佛波醇12-十四酸酯13-乙酸酯、1 mg/mL 离子霉素和10 mg/mL 布雷菲德菌素A培育5 h后，收获抗原特异性CD8细胞。
D、新抗原特异性T细胞功能检测
D1.向检测板中加入培养基并测定背景阻抗值；
D2.收集对数期细胞并计数，调整细胞悬液浓度，向检测板中加入一定体积的细胞，室温超净台内放置30min；
D3.将加入细胞的检测板放入检测台上，进行实时动态的细胞增殖检测；
D4.过夜检测后，向各培养孔中加入配制好的细胞因子并继续进行实时动态检测，即可获得细胞因子介导的细胞效应曲线及不同时间段IC50值。
实施例2
针对一名肺癌患者，利用本申请预测方法获得的其中20个突变肽序列，为确保患者的抗肿瘤免疫应答，经本法产生的每个多肽特异性细胞毒淋巴细胞针对自体肺腺癌细胞的实时杀伤实验，选择12和15多肽序列制备多肽疫苗（如图1所示）。
本技术领域技术人员可以理解，除非另外定义，这里使用的所有术语（包括技术术语和科学术语）具有与本发明所属领域中的普通技术人员的一般理解相同的意义。还应该理解的是，诸如通用字典中定义的那些术语应该被理解为具有与现有技术的上下文中的意义一致的意义，并且除非像这里一样定义，不会用理想化或过于正式的含义来解释。
最后所应说明的是：以上实施例仅用以说明而非限制本发明的技术方案，尽管参照上述实施例对本发明进行了详细说明，本领域的普通技术人员应该理解：依然可以对本发明进行修改或者等同替换，而不脱离本发明的精神和范围的任何修改或局部替换，其均应涵盖在本发明的权利要求范围当中。
Claims (7)
1.一种个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法，其特征在于，步骤包括：
A、肿瘤原代细胞培养；
B、可冻存的肿瘤抗原负载的DC细胞的获得；
C、肿瘤新抗原特异性T细胞获得；
D、新抗原特异性T细胞功能检测。
2.根据权利要求1所述个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法，其特征在于，所述肿瘤原代细胞培养步骤包括：
A1.在1-2 h内取下肿瘤块并用生理盐水或者PBS清洗，然后物理剪切成小块并置于高糖DMEM细胞培养基中；
A2.将组织块转移到加有HBSS的50 mL离心管中轻摇混匀后 400g ，5-10 min低温离心，弃上清并重新添加HBSS，重复次3次；
A3.转移到含有高糖DMEM培养基的10cm 无菌培养皿中并进一步剪切到直径3-5mm；
A4.将得到的细胞悬液以及组织块用100um 细胞滤网过滤到50mL 离心管中 162g离心4min；
A5.弃上清，并添加5-20 mL DMEM培养基重悬细胞；
A6.用排枪将重悬好的细胞转移到96孔细胞培养板中150uL/孔培养7天，然后转移到低附着性培养瓶中继续培养，每4天换一次液，直到细胞群体积开始变大，之后每7天换一次液，21天之后原代培养基中不加入ROCK 抑制剂；
A7.当细胞密度长到100个细胞群/mL 时,冻存到含有10% DMSO液氮中。
3.根据权利要求1所述个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法，其特征在于，步骤A3中，所述DMEM培养基含有10%的血清。
4.根据权利要求1所述个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法，其特征在于，所述DC细胞的获得步骤为：患者注射肿瘤新抗原疫苗后采血50mL，用传统密度梯度离心法获外周血单个核细胞，利用磁珠分离并诱导成抗原负载的成熟DC细胞，收集DC细胞并冻存在液氮。
5.根据权利要求1所述个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法，其特征在于，所述肿瘤新抗原特异性T细胞获得步骤包括：
C1.患者注射肿瘤新抗原疫苗后采血50mL，密度梯度离心法获外周血单个核细胞3-5×108，用抗人CD14抗体磁珠获得纯度大于90% 的CD14 阳性单个核细胞，CD14阴性淋巴细胞冻存；
C2.单个核细胞2×106培养在内含1000U/mL 粒细胞巨噬细胞集落生子因子、1000U/mL重组人白介素4的无血清GMP CellGro培养基；
C3.第3天加入10 mmol/mL肿瘤新抗原多肽，过夜；
C4.第4天加入2.5ug/mL人工合成的单磷酰脂质A和1000 U/mL医药级别的伽玛干扰素继续培养24 h；
C5.第5天收集抗原负载的DC细胞，一半冻存；
C6.将C1步骤冻存的CD14阴性淋巴细胞复苏与DC细胞以10:1的比例在含有10%自体血清的X-VIVO20培养液培养14天，在第3天和第10天加入20 ng/mL人重组白介素2；
C7.DC与T细胞共培养的第7天，加入复苏冻存的抗原负载的DC共培养；
C8.第14天，加入50 ng/mL 佛波醇12-十四酸酯13-乙酸酯、1 mg/mL 离子霉素和10mg/mL 布雷菲德菌素A培育5 h后，收获抗原特异性CD8细胞。
6.根据权利要求1所述个体化肿瘤新抗原介导抗肿瘤细胞免疫应答的体外预测方法，其特征在于，所述新抗原特异性T细胞功能检测步骤包括：
D1.向检测板中加入培养基并测定背景阻抗值；
D2.收集对数期细胞并计数，调整细胞悬液浓度，向检测板中加入一定体积的细胞，室温超净台内放置30min；
D3.将加入细胞的检测板放入检测台上，进行实时动态的细胞增殖检测；
D4.过夜检测后，向各培养孔中加入配制好的细胞因子并继续进行实时动态检测，即可获得细胞因子介导的细胞效应曲线及不同时间段IC50值。
7.根据权利要求1-6任一项所述预测方法在生产特异性抗肿瘤疫苗中的应用。
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