CN112698044A - 靶向治疗后免疫状态评价装置及方法 - Google Patents
靶向治疗后免疫状态评价装置及方法 Download PDFInfo
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Abstract
本发明提供了一种靶向治疗后免疫状态评价装置及方法,具体而言,本发明提供了检测个体以下细胞因子水平的试剂在制备评估靶向治疗后免疫状态的评价装置中的应用:IFN‑γ,IL‑1β,IL‑2,IL‑6,IL‑10,IL‑12p70,TNFα,IL‑4,IL‑5,IL‑8,IL‑17F,IL‑22,IL‑2RA,MCP‑1,GM‑CSF,IL‑15,Granzyme B,REG3a,ST‑2,TNFRI,以及Elafin。本发明进一步提供靶向治疗后免疫状态检测试剂盒、评价装置及评价方法。本发明能客观地评价靶向治疗后免疫状态,尽早发现细胞因子释放综合征等并发症,对于靶向治疗及预后具有重要意义。
Description
技术领域
本发明是关于一种靶向治疗后免疫状态评价装置及方法,具体而言,本发明是关于一种基于流式细胞术检测血清高通量细胞因子评价靶向治疗后免疫状态的装置、所用到的抗体组合物包被微珠及相关应用,属于生物医药技术领域。
背景技术
近年来,造血淋巴系统肿瘤以及各种非造血系统肿瘤的发病率明显增加,由于进展快、死亡率高、治疗成本高,肿瘤是目前危害人类健康的重大疾病之一,引起全世界的关注。特别是近十几年来恶性肿瘤的发病死亡均呈持续上升态势。虽然各种手术、放疗、化疗、造血干细胞移植等取得一定效果,但是依旧有很高比例的恶性肿瘤难以使用常规治疗方法达到缓解。本世纪开展的嵌合抗原受体T细胞(Chimeric antigen receptor T cells,CAR-T)治疗以及其他抗体性药物构成的靶向治疗,在免疫学、生物学、临床医师的共同努力下,成为本世纪最成功、进展最快的治疗方法。2015年以后中国开始广泛开展,并且后来居上取得很大成绩,尤其是在急性淋巴细胞白血病的治疗中,缓解率高达90%以上,一半以上患者取得长期生存。但是CAR-T为代表的靶向治疗,是一种免疫治疗,因此取得疗效的同时,其副作用也不容忽视,其中发生率最高,最为凶险的是细胞因子释放综合征(Cytokine ReleaseSyndrome,CRS),此外细胞因子作为某些疾病状态的诊断标准和免疫治疗判断治疗效果的依据,也是靶向治疗不可缺少的重要检测项目。
如何客观地评价靶向治疗后免疫状态,尽早发现CRS并发症,对于靶向治疗及预后具有重要意义。
发明内容
本发明的一个目的在于提供一种可用于评价靶向治疗后免疫状态的评价装置(系统)。
本发明的另一目的在于提供一种评价靶向治疗后免疫状态的方法。
本发明的另一目的在于提供一种靶向治疗后免疫状态检测试剂盒。
本发明中,所述评价靶向治疗后免疫状态包括对细胞因子释放综合征(CytokineRelease Syndrome,CRS)等并发症辅助判断。
本案发明人通过大量的研究与实际检测分析试验,确定了一组与靶向治疗后免疫状态相关的细胞因子,通过检测这些细胞因子的表达水平,可以客观地评价靶向治疗后免疫状态。
具体而言,一方面,本发明提供了检测个体以下细胞因子水平的试剂在制备评估靶向治疗后免疫状态的评价装置中的应用:
IFN-γ,IL-1β,IL-2,IL-6,IL-10,IL-12p70,TNFα,IL-4,IL-5,IL-8,IL-17F,IL-22,IL-2RA,MCP-1,GM-CSF,IL-15,Granzyme B,REG3a,ST-2,TNFRI,以及Elafin。
根据本发明的具体实施方案,所述检测个体细胞因子水平的试剂包括在蛋白水平检测各细胞因子水平的试剂。
根据本发明的具体实施方案,可以利用现有技术任何可行的方法检测上述细胞因子的表达水平。
根据本发明的具体实施方案,所述检测个体细胞因子水平的试剂包括利用流式细胞术检测各细胞因子水平的试剂。
根据本发明的具体实施方案,本发明中,利用流式细胞术检测各细胞因子水平的试剂可以包括A组分、B组分、C组分和D组分,其中:
A组分包括A1组分和A2组分,其中,A1组分包括由抗 IFN-γ抗体、抗IL-1β抗体、抗IL-2抗体、抗IL-6抗体、抗IL-10抗体、抗IL-12p70抗体、抗TNFα抗体包被的第一微珠;A2组分包括由抗IL-4抗体、抗IL-5抗体、抗IL-8抗体、抗IL-17F抗体、抗IL-22抗体包被的第二微珠;第一微珠与第二微珠粒径不同(通常粒径差异1-3微米以便于流式技术区分);
B组分包括B1组分和B2组分,B1组分包括抗IL-2RA抗体、抗MCP-1抗体包被的第三微珠;B2组分包括抗GM-CSF抗体、抗IL-15抗体、抗Granzyme B抗体、抗REG3a抗体、抗ST-2抗体、抗TNFRI抗体、抗Elafin抗体包被的第四微珠;第三微珠与第四微珠粒径不同(通常粒径差异1-3微米以便于流式技术区分);
C组分包括C1组分和C2组分,C1组分包括生物素化的抗 IFN-γ抗体、抗IL-1β抗体、抗IL-2抗体、抗IL-6抗体、抗IL-10抗体、抗IL-12p70抗体、抗TNFα抗体;C2组分包括生物素化的抗IL-4抗体、抗IL-5抗体、抗IL-8抗体、抗IL-17F抗体、抗IL-22抗体;
D组分包括D1组分和D2组分,D1组分包括生物素化抗IL-2RA抗体、抗MCP-1抗体;D2组分包括生物素化抗GM-CSF抗体、抗IL-15抗体、抗Granzyme B抗体、抗REG3a抗体、抗ST-2抗体、抗TNFRI抗体、抗Elafin抗体。
根据本发明的具体实施方案,本发明中,所述各抗体均为单克隆抗体。
根据本发明的具体实施方案,本发明中,利用流式细胞术检测各细胞因子水平时,可以包括以下步骤:
96孔板中,甲孔加入A1和A2组分,乙孔加入B1和B2组分;
将待测血清样本加入板孔中,封板,避光室温震荡孵育;
甲孔加入C1、C2组分,乙孔加入D1、D2组分,封板,避光室温震荡孵育;
加入藻红蛋白标记的链霉亲和素,封板,避光孵育;
加入读数液,转移到流式管内,上机检测。
根据本发明的具体实施方案,本发明中,所述各试剂的用量参照厂商推荐剂量或按照所属领域的常规操作进行。
在本发明的一些更具体实施方案中,利用流式细胞术检测各细胞因子水平时,按照以下操作进行:
(1)96孔板中,甲孔、标准品孔丙1-丙8孔加入45微升A1和A2组分,乙孔、标准品孔丁1-丁8孔加入45微升B1和B2组分,用抽滤器去除孔中液体;
同时分别取不同浓度的标准品加入对应的16个标准品孔中,其中,丙1~丙8为A组分(A1和A2组分)、C组分(C1和C2组分)的标准品,丁1~丁8为B组分(B1和B2组分)、D组分(D1和D2组分)的标准品;
(2)将不抗凝外周血静置5-30分钟后得到的血清样本,进行1:1稀释;待不同浓度标准品移入对应的标准品板孔中后,甲、乙样本孔每孔加入稀释好的45微升血清样本;
(3)封板,避光室温震荡孵育60分钟;
(4)用吸滤器移除16个标准品孔和甲、乙样本孔中液体,加入100微升洗液,抽滤去除洗液,重复共三次;
(5)每孔加入25微升1X二抗混合液(甲孔和丙1~丙8标准品孔加入C1、C2组分,乙孔和丁1~丁8标准品孔加入D1、D2组分),封板,避光室温震荡孵育30分钟;
(6)用吸滤器移除孔中液体,加入100微升洗液,抽滤去除洗液,重复共三次;
(7)加入25微升/孔藻红蛋白标记的链霉亲和素(SA-PE),封板,避光孵育20分钟;
(8)用吸滤器移除孔中液体,加入100微升洗液,抽滤去除洗液,重复共两次;
(9)加入100-200微升1X读数液,转移到流式管内,上机检测。
本发明中,上述检测可以获得各细胞因子表达水平的定量检测结果。本发明中,基于对靶向治疗前后白血病患者血液中21种细胞因子的表达进行定量检测,进一步明确了21种细胞因子的参考范围,以协助临床判断机体所处的免疫功能状态,及时评估病情及调整治疗方案。更进一步,本发明将各细胞因子的作用进行量化,提出一种靶向治疗后免疫状态评价方法,为简单化、科学化、快速性评估高信息量数据提供一个有效工具,尤其是有利于客观判断、尽早发现CRS并发症提供依据。
根据本发明的具体实施方案,本发明中,根据各细胞因子的水平获得符合以下计算方式的靶向治疗后免疫状态评分:
靶向治疗后免疫状态评分=∑βi×Si
其中,βi是指第i个细胞因子的权重;
Si指个体第i个细胞因子是否得分的情况,根据第i个细胞因子的检测水平峰值是否超过基础值的3倍判断该细胞因子是否得分。
根据本发明的具体实施方案,本发明中,各细胞因子的权重为:
IFN-γ、IL-2、IL-6、IL-10、ST-2、IL-8、GM-CSF的权重为I类权重;
IL-2RA、IL-17F、REG3a、IL-1β、MCP-1、TNFRI的权重为II类权重;
IL-4、IL-5、IL-22、IL-15、IL-12p70的权重为III类权重;
Elafin、TNFα、Granzyme B的权重为IV类权重;
I类权重、II类权重、III类权重的数值为正数,且I类权重的数值大于II类权重,II类权重的数值大于III类权重;IV类权重的数值为负数。
根据本发明的更具体实施方案,本发明中,各细胞因子的权重为:
IFN-γ、IL-2、IL-6、IL-10、ST-2、IL-8、GM-CSF的权重分别为2;
IL-2RA、IL-17F、REG3a、IL-1β、MCP-1、TNFRI的权重分别为1;
IL-4、IL-5、IL-22、IL-15、IL-12p70的权重分别为0.5;
Elafin、TNFα、Granzyme B的权重分别为-1。
根据本发明的具体实施方案,本发明中,若细胞因子的检测峰值超过正常基础值的3倍则该细胞因子得分计为1,若细胞因子的检测峰值等于或低于正常基础值的3倍则该细胞因子不得分计为0。
根据本发明的具体实施方案,本发明中,靶向治疗后免疫状态评分≤8分输出诊断分类结果为CRS 0-1级,>8分且<18分输出诊断分类结果为CRS 2级,≥18分输出诊断分类结果为CRS 3-4级。
在本发明的一些具体实施方案中,对258例不同时间点的CD19-CAR-T治疗后患者进行了检测,结果发现:
(1)本发明可以准确反映靶向治疗后(最具典型代表的是CAR-T治疗)活性细胞在体内增殖、杀伤肿瘤细胞发挥作用的时间,d4开始升高,d7-d10为高峰,此后逐渐下降,d28基本上恢复正常,患者临床表现,外周血CAR-T细胞、CD3+T细胞、CD8+T细胞的比例及细胞数变化,均呈现一致性反应。
(2)本发明可以准确反映CRS的严重程度。CRS是以CAR-T为代表的靶向治疗后最显著和最严重的毒性临床综合症,根据以下3项指标来确诊:①持续发烧超过3天(~38 C°);②选择性细胞因子(如IFN-γ、IL-6、IL-10等)升高;③伴有临床毒性反应的证据:如低血压、缺氧。重度患者如果不及时处理,死亡率高。关于严重程度的临床分级,目前缺乏一致性标准,2018年发表的美国宾夕法尼亚大学标准:1级CRS定义为轻度反应,需要支持性治疗,包括退热药和止吐药。2级的CRS定义为患者可能有一些功能失调,比如2级的肌酐和3级的肝功能检查结果,需要住院治疗或者或者静脉治疗(比如抗生素或其他药物)才能控制CRS症状(包括控制嗜中性粒细胞减少症的发热)。3级CRS定义为更严重的反应,需要住院治疗来控制严重的器官功能失调。4级CRS是危及生命的,有多种并发症,如低血压需要大剂量血管升压药,或缺氧需要机械通气。但是实际上很多为临床医生的主观评价,关于细胞因子升高的程度更加没有一致性的判断标准。本发明进一步通过检测多种细胞因子的变化,研究其与靶向治疗时间点、以及不同临床级别CRS之间的关系,找到了相对客观的辅助指标,将患者进行了分级(CRS 0-1级,CRS 2级,CRS 3-4级)。经临床验证,本发明方法的总体灵敏度为89.61%,特异性为96%,阳性预测值为95.83%,阴性预测值为90%。尤其是在识别0-1级患者与其他级别患者中(0-1级与其他级别的CRS,恰恰是区分是否需要住院治疗的标准),灵敏度为91.55%,特异性为99%,阳性预测值为98.48%,阴性预测值为92%。证实本发明的评价方法简单实用,可以有效地将复杂的大量临床数据进行简单有效分析,将极大提高临床实用性。
从而,另一方面,本发明还提供了一种靶向治疗后免疫状态检测试剂盒,该试剂盒包括检测个体以下细胞因子水平的试剂:
IFN-γ,IL-1 β,IL-2,IL-6,IL-10,IL-12p70,TNFα,IL-4,IL-5,IL-8,IL-17F,IL-22,IL-2RA,MCP-1,GM-CSF,IL-15,Granzyme B,REG3a,ST-2,TNFRI,以及Elafin。
根据本发明的具体实施方案,本发明的试剂盒包括利用流式细胞术检测各细胞因子水平的试剂,具体地,可包括分别容置于不同容器中的所述的A组分、B组分、C组分和D组分。
另一方面,本发明还提供了一种靶向治疗后免疫状态评价装置,其包括检测单元和数据分析单元,其中:
所述检测单元用于检测个体的细胞因子水平,获得检测结果;所述细胞因子包括:IFN-γ,IL-1 β,IL-2,IL-6,IL-10,IL-12p70,TNFα,IL-4,IL-5,IL-8,IL-17F,IL-22,IL-2RA,MCP-1,GM-CSF,IL-15,Granzyme B,REG3a,ST-2,TNFRI,以及Elafin;
所述数据分析单元用于对检测单元的检测结果进行分析处理。
根据本发明的具体实施方案,本发明的靶向治疗后免疫状态评价装置,其中,所述检测单元包括利用流式细胞术检测各细胞因子水平的试剂,具体地,可包括分别容置于不同容器中的所述的A组分、B组分、C组分和D组分。其用于检测个体的细胞因子水平的过程可参照前述。
根据本发明的具体实施方案,本发明的靶向治疗后免疫状态评价装置,其中,所述数据分析单元对检测单元的检测结果进行分析处理时,包括:
将所述细胞因子的检测结果配以权重系数,以计算所述待测个体的靶向治疗后免疫状态评分。
根据本发明的具体实施方案,本发明的靶向治疗后免疫状态评价装置,其中,所述数据分析单元包括:
预处理模块,用于将所述细胞因子的检测结果标准化;
计算模块,用于将标准化的细胞因子检测结果带入到以下评价模型,得到待测个体的靶向治疗后免疫状态评分:
靶向治疗后免疫状态评分=∑βi×Si
其中,βi是指第i个细胞因子的权重;
Si指个体第i个细胞因子是否得分的情况,根据第i个细胞因子的检测峰值是否超过正常基础值的3倍判断该细胞因子是否得分。
根据本发明的具体实施方案,本发明的靶向治疗后免疫状态评价装置,其中,所述数据分析单元还包括:
矩阵输入模块,用于接收所述预处理模块输出的多个所述标准化的检测结果,将所述标准化的检测结果以矩阵形式输入到所述计算模块;
输出模块,用于接收所述计算模块输出的靶向治疗后免疫状态评分,并输出为诊断分类结果。
根据本发明的具体实施方案,本发明的靶向治疗后免疫状态评价装置中,所述数据分析单元对检测单元的检测结果进行分析处理时,将所述细胞因子的检测结果配以的权重系数分别为:
IFN-γ、IL-2、IL-6、IL-10、ST-2、IL-8、GM-CSF的权重分别为2;
IL-2RA、IL-17F、REG3a、IL-1β、MCP-1、TNFRI的权重分别为1;
IL-4、IL-5、IL-22、IL-15、IL-12p70的权重分别为0.5;
Elafin、TNFα、Granzyme B的权重分别为-1。
根据本发明的具体实施方案,本发明的靶向治疗后免疫状态评价装置中,预处理模块用于将所述细胞因子的检测结果标准化时,若细胞因子的检测峰值超过正常基础值的3倍则该细胞因子标准化得分计为1,若细胞因子的检测峰值等于或低于正常基础值的3倍则该细胞因子标准化不得分计为0。
根据本发明的具体实施方案,本发明的靶向治疗后免疫状态评价装置中,输出模块将靶向治疗后免疫状态评分输出为诊断分类结果时,可根据靶向治疗后免疫状态评分≤8分输出诊断分类结果为CRS 0-1级,>8分且<18分输出诊断分类结果为CRS 2级,≥18分输出诊断分类结果为CRS 3-4级。
另一方面,本发明还提供了一种计算机存储介质,所述计算机存储介质存储有计算机程序指令,所述计算机程序指令被执行时实现:基于待测个体细胞因子水平获得个体靶向治疗后免疫状态评价结果。其中,所述细胞因子及评价方法参照如前所述。
另一方面,本发明还提供了一种计算机设备,其包括存储器、处理器及存储在存储器上并可在处理器上运行的计算机程序,其中,所述处理器执行所述计算机程序时实现:基于待测个体细胞因子水平获得个体靶向治疗后免疫状态评价结果。其中,所述细胞因子及评价方法参照如前所述。
本发明的靶向治疗后免疫状态评价技术,具有以下特点:(1)灵敏度高,特异性好,信息量极为丰富,可以很好地反映CAR-T细胞、CD3+和CD8+T细胞等在体内的扩增以及产生杀伤效果分泌的细胞因子情况:例如在靶向治疗的随访中,随着CAR-T细胞回输时间,大多数细胞因子在7-10天形成高峰,随着CAR-T细胞与肿瘤细胞的解离和肿瘤细胞被大量杀死,CAR-T细胞以及CD3+T和CD8+T细胞在体内扩增减缓甚至停止,细胞因子也降低,直至一个月左右回至基值;(2)与发热、中枢神经系统症状等临床症状复合率高,可以为临床医师提供极其有效的临床信息支持,有助于早诊断早干预,有望降低CRS并发症的相关死亡率;(3)更多有效生物信息,为现在极为迫切的解决CAR-T复发,改进技术,提高疗效,避免耐药和并发症问题提供最为本质和有效的信息;(4)评价方案简单易行,并且相对客观,有望成为人工智能分析的基础,为临床医生提供更客观、便捷的CRS辅助信息。
附图说明
图1A和图1B:CAR-T治疗后不同时间点(d0、d4、d7、d14、d21、d28)血清IL-6与同一外周血标本中CAR-T细胞和CD3+T细胞占淋巴细胞、CD4+T细胞和CD8+T细胞占T细胞百分比(图1A)的关系,以及绝对计数在各时间点的改变(图1B)。
图2:CD19-CAR-T治疗后d7血清标本。使用FSC/SSC二维点图,设门圈出4μm微珠门和5μm微珠门。图片A为甲管4μm微珠门的微珠,检测的A1和C1组分标记所得的7个细胞因子(抗 IFN-γ抗体、抗IL-1β抗体、抗IL-2抗体、抗IL-6抗体、抗IL-10抗体、抗IL-12p70抗体、抗TNFα抗体)表达情况,图片B为甲管5μm微珠检测的A2和C2组分标记所得的5个细胞因子(抗IL-4抗体、抗IL-5抗体、抗IL-8抗体、抗IL-17F抗体、抗IL-22抗体)表达情况,图片C为乙管4μm微珠门的微珠,检测的B1和D1组分标记所得的2个细胞因子(抗IL-2RA抗体、抗MCP-1抗体)表达情况,图片D为乙管5μm微珠检测的B2和D2组分标记所得的7个细胞因子(抗GM-CSF抗体、抗IL-15抗体、抗Granzyme B抗体、抗REG3a抗体、抗ST-2抗体、抗TNFRI抗体、抗Elafin抗体)表达情况。
图3A、图3B和图3C:试剂盒所带不同标定浓度的标准品,使用相同方法检测不同浓度的21种细胞因子曲线。每一个测定值都有对应的细胞因子浓度。将检测标本的测定值进行比对,可以得出检测标本的细胞因子浓度。
图4:CRS 3级患者,CD19-CAR-T治疗后6个时间点的血清标本21因子检测结果曲线。
图5A和图5B:选择CD19-CAR-T治疗后出现不同级别CRS的34名患者(0级11人,1级10人,2级8人,3-4级5人),使用本发明的方法检测血清标本中细胞因子水平,观察d0、d4、d7、d14、d21、d28六个时间点的21种细胞因子表达水平变化。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解,现结合具体实施例及对本发明的技术方案进行以下详细说明,应理解这些实例仅用于说明本发明而不用于限制本发明的范围。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.检测用抗体
本实施例使用的检测试剂包括:
A组分包括A1组分和A2组分,其中,A1组分包括抗 IFN-γ抗体、抗IL-1β抗体、抗IL-2抗体、抗IL-6抗体、抗IL-10抗体、抗IL-12p70抗体、抗TNFα抗体包被的4微米微珠,A2组分包括抗IL-4抗体、抗IL-5抗体、抗IL-8抗体、抗IL-17F抗体、抗IL-22抗体包被的5微米微珠。上述试剂等比装于第一容器中。
B组分包括B1组分和B2组分,B1组分包括抗IL-2RA抗体、抗MCP-1抗体包被的4微米微珠,B2组分包括抗GM-CSF抗体、抗IL-15抗体、抗Granzyme B抗体、抗REG3a抗体、抗ST-2抗体、抗TNFRI抗体、抗Elafin抗体包被的5微米微珠。上述试剂等比装于第二容器中。
C组分包括C1组分和C2组分,C1组分包括生物素化的抗 IFN-γ抗体、抗IL-1β抗体、抗IL-2抗体、抗IL-6抗体、抗IL-10抗体、抗IL-12p70抗体、抗TNFα抗体,C2组分包括生物素化的抗IL-4抗体、抗IL-5抗体、抗IL-8抗体、抗IL-17F抗体、抗IL-22抗体。上述试剂等比装于第三容器中。
D组分包括D1组分和D2组分,D1组分包括生物素化的抗IL-2RA抗体、抗MCP-1抗体,D2组分包括生物素化的抗GM-CSF抗体、抗IL-15抗体、抗Granzyme B抗体、抗REG3a抗体、抗ST-2抗体、抗TNFRI抗体、抗Elafin抗体。上述试剂等比装于第四容器中。
PE标记的链霉亲和素装于第五容器中。
第一容器和第三容器内组分对应的标准品(所有检测细胞因子的重组蛋白冻干粉)装于第六容器中。
第二容器和第四容器内组分对应的标准品装于第七容器中。
样本稀释液(主要成分为磷酸盐缓冲液以及Proclin300抑菌剂)、标准品稀释液(主要成分为磷酸盐缓冲液加BSA以及Proclin300抑菌剂)、洗液、读数液等分别装于其他不同的容器中。
上述各试剂组分均可商购获得,本实施例中均采用购自中国北京旷博生物技术有限公司的试剂盒产品。其中样本稀释液、洗液、读数液也可以自行配制,但是建议优选厂家配套试剂盒。
2.标本的处理
本实施例中,标本的处理主要参照试剂盒说明书进行,具体过程主要包括:
(1)96孔板中,甲孔、丙1~丙8孔加入45微升A1和A2组分,乙孔、丁1~丁8孔加入45微升B1和B2组分,用抽滤器去除孔中液体。
(2)将第六容器(内装第一、第三容器的标准品)和第七容器(内装第二、第四容器的标准品)的标准品,用标准品稀释液稀释至不同浓度加入对应的16个标准品孔中(丙1~丙8为第一、第三容器的标准品孔,丁1~丁8为第二、第四容器的标准品孔):丙1为最高浓度,每种细胞因子试剂的浓度为50ng/ml,丙2-丙7的浓度依次为前一孔的1/3,即丙2为16.67ng/ml, 丙3为5.56ng/ml,丙4为1.85ng/ml,丙5为0.62ng/ml, 丙6为0.21ng/ml,丙7为0.07ng/ml,丙8为标准品稀释液(提供背景)。丁1为最高浓度,每种细胞因子试剂的浓度为50ng/ml,丁2-丁7的浓度依次为前一孔的1/3,即丁2为16.67ng/ml, 丁3为5.56ng/ml,丁4为1.85ng/ml,丁5为0.62ng/ml, 丁6为0.21ng/ml,丁7为0.07ng/ml,丁8为标准品稀释液。
(3)将不抗凝外周血静置5-30分钟后得到的血清样本,进行1:1稀释:即22.5微升样本稀释液+22.5微升血清样本;待不同浓度标准品移入对应的标准品板孔中后,甲、乙样本孔每孔加入45微升稀释好的血清样本。
(4)封板,避光室温震荡孵育60分钟。
(5)用吸滤器移除16个标准品孔和甲、乙样本孔中液体,加入100微升洗液,抽滤去除洗液,重复共三次。
(6)每孔加入25微升1X二抗混合液(甲孔和丙1~丙8标准品孔加入C1、C2组分,乙孔和丁1~丁8标准品孔加入D1、D2组分,封板,避光室温震荡孵育30分钟。
(7)用吸滤器移除孔中液体,加入100微升洗液,抽滤去除洗液,重复共三次。
(8)加入25微升/孔藻红蛋白标记的链霉亲和素(SA-PE),封板,避光孵育20分钟。
(9)用吸滤器移除孔中液体,加入100微升洗液,抽滤去除洗液,重复共两次。
(10)加入100-200微升1X读数液,转移到流式管内,上机检测。
3.标本的检测
将处理好的标本,在美国Becton Dickinson公司2激光4色FACS Calibur 流式细胞仪上机检测,每个细胞因子优选获取100个粒子以上,即优选甲管获取1200粒子以上,乙管获取900粒子以上,使用cellquest或者FCAP Array v3软件分析数据。
单个标本分析过程:
如图2所示,显示前向角光散射(Forward scatter, FSC)和侧向角光散射(Sidescatter, SSC),设门圈出4μm微珠门和5μm微珠门。图片A为甲管4μm微珠检测的A1和C1组分标记所得的7个细胞因子表达情况,图片B为甲管5μm微珠检测的A2和C2组分标记所得的5个细胞因子表达情况,图片C为乙管B1和D1组分标记所得的2个细胞因子表达情况,图片D为乙管B2和D2组分标记所得的7个细胞因子表达情况。
每个检测日同时同样方法进行8个浓度的21个细胞因子的标准品检测,得到21因子的标准品曲线(图3A、图3B和图3C),将待测标本与标准品曲线进行比对,得出测定值。
每个CAR-T患者检测6个时间点,在此基础上,进行整体分析。每个患者得出一个21因子不同时间点的检测曲线。图4示例性给出了一位CRS 3级患者CD19-CAR-T治疗后6个时间点的血清标本21因子检测结果曲线,可见绝大多数细胞因子在7天左右达到高峰,然后逐渐降低,28-30天左右恢复到0点水平。不同细胞因子升高程度不同。
4.检测结果及分析
使用上述方法于陆道培医院2020年3月开始进行检测,共检测了258例不同时间点的CD19-CAR-T治疗后患者(因最初摸索时间点以及剂量,最终可以统计的108例),男:女=70:38, 中位年龄17岁(1岁-68岁),均为难治复发B-ALL,以CAR-T时间为0点,分析所检测的时间点为6个,CD19-CAR-T细胞回输前当天(d0)、回输后第四天(d4)、回输后第七天(d7)、回输后第十四天(d14)、回输后第二十一天(d21)、回输后第二十八天(d28),遇到节假日就近选择日期。
使用SPSS 25统计学软件,采用重复测量方差分析检验本发明前述的21种细胞因子的组间和组内差异,找出在各时间点以及不同程度CRS组间有差异的细胞因子(表1)。
实际研究中,除了本发明前述的21种细胞因子外,发明人还检测了现有技术提出的靶向治疗后通常需检测的其他细胞因子,例如TNFβ、IL-17A、MIP-1α等。经分析验证,这些细胞因子在本发明的靶向治疗后免疫状态评价分析中的权重为0,因此这些因子可不必纳入本发明的靶向治疗后免疫状态评价技术。为便于参考,这些细胞因子的数值一并列于表1。
表1 一个周期(28天)内各细胞因子时间点以及不同程度CRS组间差异性分析
本发明中,发现10种细胞因子随着CD19-CAR-T治疗,在不同时间点的改变有统计学差异(均p<0.05):IL-1β、IL-2、IL-6、IL-10、IL-17F、GM-CSF、ST-2、TNFRI、REG3a和IFN-γ,其中前8种(即IL-1β、IL-2、IL-6、IL-10、IL-17F、GM-CSF、ST-2、TNFRI),除了时间改变的差异,还有时间和组间的交互作用,在不同CRS级别的患者不同时间点检测的血清浓度结果存在显著性差异(表1),说明这些指标可以很好地反映CAR-T回输后细胞因子改变情况,间接反映分泌这些细胞因子的杀伤性细胞CAR-T细胞和CD3+T细胞、CD8+T细胞增殖情况,同时还能够区分不同级别CRS。
以IL-6为例,图1A和图1B显示了 CAR-T治疗后不同时间点(d0、d4、d7、d14、d21、d28)血清IL-6与同一外周血标本中CAR-T细胞和CD3+T细胞占淋巴细胞、CD4+T细胞和CD8+T细胞占T细胞百分比(图1A)的关系,以及绝对计数在各时间点的改变。IL-6水平在d0处于最低值,d4开始升高,d7达到高峰,此后逐渐降低,CAR-T细胞和CD3+T细胞占淋巴细胞百分比,CD8+T细胞占T细胞百分比也发生同步改变,CD4+T细胞占T细胞百分比发生相反改变。CAR-T细胞、CD3+T细胞、CD8+T细胞外周血中计数(图1B)改变最明显与直接,说明IL-6细胞因子水平在CAR-T治疗后呈周期性改变,可以反映主要分泌该细胞因子的淋巴细胞增殖情况,CD4+T细胞计数变化不大。
REG-3a和IFN-γ只有时间点差异,组间差别没有显著性;IL-8在不同CRS级别的患者间存在显著性差异,但是CRS级别和时间之间不存在交互作用。
各细胞因子在不同时间点、不同CRS组别的详细检测数值范围见表2。
表2 不同程度CRS患者在不同时间点血清各细胞因子检测结果(平均值±标准差)
对上述细胞因子进行进一步的详细分析,以IL-6为例,观察每个时间点,不同级别CRS之间的差别(表3)。在时间和组别(Time*group)的成对比较中,在第1 (d0)、第6(d28)个时间点,组之间IL-6水平没有显著性差异。在第2(d4)、3(d7)、4(d14)个时间点,CRS 0-1和CRS 3-4组之间IL-6水平均存在显著性差异,在第5(d21)个时间点,CRS 0-1和CRS 2组之间IL-6水平存在显著性差异,其中第三个时间点(d7)差异最大。同法对各细胞因子进行研究,发现CRS 3-4级差别最明显,除了少数细胞因子外(IL-5、TNFβ在第二个时间点差别大,REG3a,TNFα、IL-12p70在第四个时间点差别大,IL-8不同级别时间点有差别),其他细胞因子都是第三个时间点(d7)与d0天差别最大,并且随着CRS级别升高,差别明显(图5A、图5B)。
表3不同时间点检测IL-6水平差别
但是可以看出,这种数据庞大的信息,对于临床应用甚为不便。本发明中,再次进行数据处理,根据各因子的贡献,最低点(0点)和最高点d7之间的变化差距,以及对于CRS区分的显著程度,提供一个便于临床分析以及将来大数据管理和人工智能分析的数据基础。本发明通过进一步的分析得到很好的灵敏度、特异性和阳性阴性预测值,将各细胞因子根据在细胞因子释放综合征(CRS)中起到的作用不同,按照权重分层,2分、1分、0.5分、-1分四组。判断这个细胞因子在峰值日(d7)是否超过相对正常(CRS 0-1级)的基础值(d0)的3倍,“是”得分,“否”不得分。然后将各细胞因子的权重得分相加,看处于哪个CRS分级范围:≤8分定为CRS 0-1级,>8分且<18分为CRS 2级,积分≥18分为CRS 3-4级。待测的未知标本进行检测,将检测值带入计算机系统中,即可测得该患者的CRS评分。有助于将临床数据简单化、信息化、智能化,并有可能为进一步客观化CRS临床分级提供重要理论基础。
本发明中,以图5A、图5B显示的各细胞因子不同时间点、不同CRS级别变化为依据,将这些细胞因子在第三个时间点(d7)与CRS0级第一时间点(d0)差距的显著性进行权重区分,此外,本发明观察到除了主效应结果显著的细胞因子外,还有些细胞因子在图中出现明显差异,本发明参照了多参数分析的结果,发现在多参数分析中IL-4、IL-5、IL-22、TNFβ在时间和组间存在交互作用,IL2RA、IL-12P70是时间点存在显著差异。结合细胞因子水平在不同的组间及时间点改变的倍数,降低了IL-17F、IL-1β、TNFβ的权重,提高IFN-γ、IL2RA、MCP-1、IL-15的权重,TNFRI基线水平较高,因此也降低权重。故设计评分标准如下:①IFN-γ、IL-2、IL-6、IL-10、ST-2、IL-8、GM-CSF为I类权重的细胞因子,d7检测结果比CRS 0级D0天的结果高出三倍以上计为2分;②IL2RA、IL-17F、REG3a、IL-1β、TNFRI、MCP-1为II类权重的细胞因子,d7检测结果比CRS0级D0天的结果高出三倍以上计为1分;③IL-4、IL-5、IL-22、IL-12p70、IL-15为III类权重的细胞因子,d7检测结果比CRS0级D0天的结果高出三倍以上计为0.5分;④把在CRS不同级别中变动无序的Elafin、TNFα、Granzyme B定义为IV类权重的细胞因子,记为-1分,d7检测结果比CRS 0级D0天的结果高出三倍以上计为-1(表4)。前述提及的细胞因子TNFβ、IL-17A、MIP-1α在本发明的对于CRS评价的评分系统分析中的权重为0,因此这些因子可不必纳入本发明的靶向治疗后免疫状态评分系统。
表4 21种细胞因子对于CRS评价的评分系统
以此算出不同CRS组患者的分数区间,找到每组的阈值。
根据临床34例患者(0级11人,1级10人,2级8人,3-4级5人)的检测结果,本发明把≤8分定为CRS 0-1级,>8分且<17分为CRS 2级,积分≥18分为CRS 3-4级。
临床验证:使用该抽样得出的评分系统,对其他77例患者进行试验性评价,0-1级71例,2级4例,3-4级2例。发现:本发明可以准确反映CAR-T细胞在体内增殖、杀伤肿瘤细胞发挥作用的时间,d4开始升高,d7-d10为高峰,此后逐渐下降,28d基本上恢复正常,患者临床表现,外周血CAR-T细胞、CD3+T细胞、CD8+T细胞的比例及细胞数变化,均呈现一致性反应(表1,图1 A和图1B,以IL-6为例,图4)。测得总体灵敏度为89.61%,特异性为96%,阳性预测值为95.83%,阴性预测值为90%。尤其是在识别0-1级患者与其他级别患者中,灵敏度为91.55%,特异性为99%,阳性预测值为98.48%,阴性预测值为92%。证实该算法简单实用,可以有效地将复杂的大量临床数据进行分析,辅助以计算机软件和人工智能,将极大提高该试剂盒的临床实用性。并有望获得更多信息。
实施例2
患者XXX,男,56岁,CD19-CAR-T治疗后患者,为难治复发B-ALL,以CAR-T时间为0点,分析所检测的时间点为6个,CD19-carT细胞回输前当天(d0)、回输后第四天(d4)、回输后第七天(d7)、回输后第十四天(d14)、回输后第二十一天(d21)、回输后第二十八天(d28)。检测方法参照实施例1。检测结果见表5。
表5 某患者d7检测结果表
根据表4的权重,该病患的得分情况参见表6。
表6 某患者得分情况表
据此可以得到患者的评分。如该患者评分19分,初步可判断为CRS 3-4级。临床评价3级,与临床符合。
Claims (13)
1.检测个体以下细胞因子水平的试剂在制备评估靶向治疗后免疫状态的评价装置中的应用:
IFN-γ,IL-1β,IL-2,IL-6,IL-10,IL-12p70,TNFα,IL-4,IL-5,IL-8,IL-17F,IL-22,IL-2RA,MCP-1,GM-CSF,IL-15,Granzyme B,REG3a,ST-2,TNFRI,以及Elafin。
2.根据权利要求1所述的应用,其特征在于,所述检测个体细胞因子水平的试剂包括在蛋白水平检测各细胞因子水平的试剂。
3.根据权利要求1或2所述的应用,其特征在于,所述检测个体细胞因子水平的试剂包括利用流式细胞术检测各细胞因子水平的试剂。
4.根据权利要求1或2所述的应用,其特征在于,根据各细胞因子的水平获得符合以下计算方式的靶向治疗后免疫状态评分:
靶向治疗后免疫状态评分=∑βi×Si
其中,βi是指第i个细胞因子的权重;
Si指个体第i个细胞因子是否得分的情况,根据第i个细胞因子的检测水平峰值是否超过基础值的3倍判断该细胞因子是否得分。
5.一种靶向治疗后免疫状态检测试剂盒,其特征在于,该试剂盒包括检测个体以下细胞因子水平的试剂:
IFN-γ,IL-1β,IL-2,IL-6,IL-10,IL-12p70,TNFα,IL-4,IL-5,IL-8,IL-17F,IL-22,IL-2RA,MCP-1,GM-CSF,IL-15,Granzyme B,REG3a,ST-2,TNFRI,以及Elafin。
6.根据权利要求5所述的试剂盒,其特征在于,该试剂盒包括分别容置于不同容器中的A组分、B组分、C组分和D组分,其中:
A组分包括A1组分和A2组分,其中,A1组分包括由抗 IFN-γ抗体、抗IL-1β抗体、抗IL-2抗体、抗IL-6抗体、抗IL-10抗体、抗IL-12p70抗体、抗TNFα抗体包被的第一微珠;A2组分包括由抗IL-4抗体、抗IL-5抗体、抗IL-8抗体、抗IL-17F抗体、抗IL-22抗体包被的第二微珠;第一微珠与第二微珠粒径不同;
B组分包括B1组分和B2组分,B1组分包括抗IL-2RA抗体、抗MCP-1抗体包被的第三微珠;B2组分包括抗GM-CSF抗体、抗IL-15抗体、抗Granzyme B抗体、抗REG3a抗体、抗ST-2抗体、抗TNFRI抗体、抗Elafin抗体包被的第四微珠;第三微珠与第四微珠粒径不同;
C组分包括C1组分和C2组分,C1组分包括生物素化的抗 IFN-γ抗体、抗IL-1β抗体、抗IL-2抗体、抗IL-6抗体、抗IL-10抗体、抗IL-12p70抗体、抗TNFα抗体;C2组分包括生物素化的抗IL-4抗体、抗IL-5抗体、抗IL-8抗体、抗IL-17F抗体、抗IL-22抗体;
D组分包括D1组分和D2组分,D1组分包括生物素化抗IL-2RA抗体、抗MCP-1抗体;D2组分包括生物素化抗GM-CSF抗体、抗IL-15抗体、抗Granzyme B抗体、抗REG3a抗体、抗ST-2抗体、抗TNFRI抗体、抗Elafin抗体。
7.一种靶向治疗后免疫状态评价装置,其包括检测单元和数据分析单元,其特征在于:
所述检测单元用于检测个体的细胞因子水平,获得检测结果;所述细胞因子包括:IFN-γ,IL-1β,IL-2,IL-6,IL-10,IL-12p70,TNFα,IL-4,IL-5,IL-8,IL-17F,IL-22,IL-2RA,MCP-1,GM-CSF,IL-15,Granzyme B,REG3a,ST-2,TNFRI,以及Elafin;
所述数据分析单元用于对检测单元的检测结果进行分析处理。
8.根据权利要求7所述的靶向治疗后免疫状态评价装置,其特征在于,所述检测单元包括权利要求6所述的试剂盒,其用于检测个体的细胞因子水平的过程包括:
96孔板中,甲孔加入A1和A2组分,乙孔加入B1和B2组分;
将待测血清样本加入板孔中,封板,避光室温震荡孵育;
甲孔加入C1、C2组分,乙孔加入D1、D2组分,封板,避光室温震荡孵育;
加入藻红蛋白标记的链霉亲和素,封板,避光孵育;
加入读数液,转移到流式管内,上机检测。
9.根据权利要求7所述的靶向治疗后免疫状态评价装置,其特征在于,所述数据分析单元对检测单元的检测结果进行分析处理时,包括:
将所述细胞因子的检测结果配以权重系数,以计算所述待测个体的靶向治疗后免疫状态评分。
10.根据权利要求9所述的靶向治疗后免疫状态评价装置,其特征在于,所述数据分析单元包括:
预处理模块,用于将所述细胞因子的检测结果标准化;
计算模块,用于将标准化的细胞因子检测结果带入到以下评价模型,得到待测个体的靶向治疗后免疫状态评分:
靶向治疗后免疫状态评分=∑βi×Si
其中,βi是指第i个细胞因子的权重;
Si指个体第i个细胞因子是否得分的情况,根据第i个细胞因子的检测峰值是否超过正常基础值的3倍判断该细胞因子是否得分。
11.根据权利要求10所述的靶向治疗后免疫状态评价装置,其中,所述数据分析单元还包括:
矩阵输入模块,用于接收所述预处理模块输出的多个所述标准化的检测结果,将所述标准化的检测结果以矩阵形式输入到所述计算模块;
输出模块,用于接收所述计算模块输出的靶向治疗后免疫状态评分,并输出为诊断分类结果;
其中,各细胞因子的权重为:
IFN-γ、IL-2、IL-6、IL-10、ST-2、IL-8、GM-CSF的权重分别为2;
IL-2RA、IL-17F、REG3a、IL-1β、MCP-1、TNFRI的权重分别为1;
IL-4、IL-5、IL-22、IL-15、IL-12p70的权重分别为0.5;
Elafin、TNFα、Granzyme B的权重分别为-1;
若细胞因子的检测峰值超过正常基础值的3倍则该细胞因子得分计为1,若细胞因子的检测峰值等于或低于正常基础值的3倍则该细胞因子不得分计为0;
靶向治疗后免疫状态评分≤8分输出诊断分类结果为CRS 0-1级,>8分且<18分输出诊断分类结果为CRS 2级,≥18分输出诊断分类结果为CRS 3-4级。
12.一种计算机存储介质,所述计算机存储介质存储有计算机程序指令,所述计算机程序指令被执行时实现:基于待测个体细胞因子水平获得个体靶向治疗后免疫状态评价结果;
其中,所述细胞因子包括:IFN-γ,IL-1β,IL-2,IL-6,IL-10,IL-12p70,TNFα,IL-4,IL-5,IL-8,IL-17F,IL-22,IL-2RA,MCP-1,GM-CSF,IL-15,Granzyme B,REG3a,ST-2,TNFRI,以及Elafin。
13.一种计算机设备,其包括存储器、处理器及存储在存储器上并可在处理器上运行的计算机程序,其中,所述处理器执行所述计算机程序时实现:基于待测个体细胞因子水平获得个体靶向治疗后免疫状态评价结果;
其中,所述细胞因子包括:IFN-γ,IL-1β,IL-2,IL-6,IL-10,IL-12p70,TNFα,IL-4,IL-5,IL-8,IL-17F,IL-22,IL-2RA,MCP-1,GM-CSF,IL-15,Granzyme B,REG3a,ST-2,TNFRI,以及Elafin。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108276495A (zh) * | 2018-01-24 | 2018-07-13 | 博生吉医药科技(苏州)有限公司 | 靶向csf1r嵌合抗原受体修饰的nk92mi细胞和t细胞及其制法和应用 |
CN108780084A (zh) * | 2015-09-03 | 2018-11-09 | 诺华股份有限公司 | 预测细胞因子释放综合征的生物标志物 |
CN110913895A (zh) * | 2017-05-08 | 2020-03-24 | 阿迪马布有限责任公司 | 抗cd3结合结构域和包含它们的抗体以及它们的产生及使用方法 |
CN111052247A (zh) * | 2017-06-13 | 2020-04-21 | 波士顿基因公司 | 用于由经归一化生物标志物评分鉴定癌症治疗的系统和方法 |
WO2020150567A1 (en) * | 2019-01-17 | 2020-07-23 | Figene, Llc | Fibroblasts and microvesicles thereof for reduction of toxicity associated with cancer immunotherapy |
-
2021
- 2021-03-23 CN CN202110304950.3A patent/CN112698044B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108780084A (zh) * | 2015-09-03 | 2018-11-09 | 诺华股份有限公司 | 预测细胞因子释放综合征的生物标志物 |
CN110913895A (zh) * | 2017-05-08 | 2020-03-24 | 阿迪马布有限责任公司 | 抗cd3结合结构域和包含它们的抗体以及它们的产生及使用方法 |
CN111052247A (zh) * | 2017-06-13 | 2020-04-21 | 波士顿基因公司 | 用于由经归一化生物标志物评分鉴定癌症治疗的系统和方法 |
CN108276495A (zh) * | 2018-01-24 | 2018-07-13 | 博生吉医药科技(苏州)有限公司 | 靶向csf1r嵌合抗原受体修饰的nk92mi细胞和t细胞及其制法和应用 |
WO2020150567A1 (en) * | 2019-01-17 | 2020-07-23 | Figene, Llc | Fibroblasts and microvesicles thereof for reduction of toxicity associated with cancer immunotherapy |
Non-Patent Citations (3)
Title |
---|
DIJANA MILJKOVIC 等: "Chronic Rhinosinusitis with Polyps Is Characterized by Increased Mucosal and Blood Th17 Effector Cytokine Producing Cells", 《FRONTIERS IN PHYSIOLOGY》 * |
MICHELLE H. TOWNSEND 等: "The expansion of targetable biomarkers for CAR T cell therapy", 《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》 * |
管茂静: "非血缘脐血移植后植入前综合征临床危险因素分层及干预研究和发生机制的初步探讨", 《中国优秀硕士学位论文全文数据库(电子期刊) 医药卫生科技辑》 * |
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