36109 | 对于CAR-T细胞来说，发挥肿瘤杀伤作用的有效成分是CAR阳性的T细胞，CAR-T细胞产品的包装规格及临床使用剂量是以CAR-T阳性细胞数表示的，因此，CAR转染阳性率是CAR-T细胞产品质量控制的必检项，监管部门建议应采用流式细胞法检测CAR转染阳性率。目前有针对CAR不同结构区域的检测方法，包括针对CAR抗原结合位点的，比如CD19抗原和抗独特型抗体，或针对轻链或铰链区的抗Fab抗体或Protein L蛋白，其中，针对抗原结合部位的CAR阳性率检测方法因其具有更好专属性而被广泛使用。本文将对CAR阳性表达率检测相关案例做一汇总以供大家参考。
| 试剂 | 检测机制 | 优势 | 劣势 |
|---|---|---|---|
| Protein L | 结合抗体κ轻链 | 具有一定的通用性，可检测不同靶点的CAR | 仅能识别人VκI VκIII和VκIV亚型和鼠VκI亚型，但不能识别人VκII亚型和鼠Ig的其他亚型轻链 |
| Anti-Fab抗体 | 结合抗体Fab段 | 具有一定的通用性，可检测不同靶点的CAR | 背景值高，商品化产品质量参差不齐且多数为多抗，批间一致性较差 |
| Anti-idiotype抗体 | 特异性结合scFv抗原识别区 | 高特异性、高灵敏度，低背景 | 少有商品化产品，需定制开发，定制周期较长（6个月左右） |
| 靶点蛋白 | 特异性结合scFv抗原识别区 | 具靶点专属性，可以评估CAR与靶标的结合能力 | 检测灵敏度受scFv与靶点蛋白间的亲和力限制 |
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案例名称 | 数据来源 |
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|---|---|---|---|
| 案例一 | 利用PE标记CD19蛋白检测anti-CD19 CAR 阳性表达率 | ACROBiosystems | 点击查看 |
| 案例二 | 利用PE标记BCMA蛋白检测anti-BCMA CAR 阳性表达率 | ACROBiosystems | 点击查看 |
| 案例三 | 利用FITC标记anti-FMC63 antibody检测anti-CD19(FMC63) CAR 阳性表达率 | ACROBiosystems | 点击查看 |
| 案例四 | 利用生物素标记hBCMA检测anti-BCMA CAR阳性表达率 | 深圳普瑞金 | 点击查看 |
| 案例五 | 利用生物素标记hCD19检测anti-CD19 CAR阳性表达率 | MacLeod DT | 点击查看 |
| 案例六 | 不同供应商hCD19, Fc tag蛋白的结合特异性验证 | ACROBiosystems | 点击查看 |
检测方法：流式细胞术
检测仪器：BD FACS Celesta流式细胞分析仪 (BD Biosciences)
样本信息：Anti-CD19 CAR-293细胞
主要试剂：
简要流程：
检测结果：结果显示Anti-CD19 CAR阳性表达率为100%
1e6 of the anti-CD19 CAR-293 cells were stained with 100 μL of 1:50 dilution (2 μL stock solution in 100 μL FACS buffer) of PE-Labeled Human CD19 (20-291), His Tag (Cat. No. CD9-HP2H3). PE Streptavidin was used as negative control (QC tested).
Protocol
检测方法：流式细胞术
检测仪器：BD FACS Celesta流式细胞分析仪 (BD Biosciences)
样本信息：Anti-BCMA CAR-293细胞
主要试剂：
简要流程：
检测结果：结果显示Anti-BCMA CAR阳性表达率为100%
1e6 of the Anti-BCMA CAR-293 cells were stained with 100 μL of 1:50 dilution (2 μL stock solution in 100 μL FACS buffer) of PE-Labeled Human BCMA Protein, His Tag (Cat. No. BCA-HP2H2) and negative control protein respectively, PE signal was used to evaluate the binding activity (QC tested).
Protocol
检测方法：流式细胞术
检测仪器：BD FACS Celesta流式细胞分析仪 (BD Biosciences)
样本信息：Anti-CD19 CAR-293细胞
主要试剂：
简要流程：
检测结果：结果显示Anti-CD19 CAR阳性表达率为100%
2e5 of Anti-CD19 CAR-293 cells were stained with 100 μL of 1:50 dilution (2 μL stock solution in 100 μL FACS buffer) FITC-Labeled Monoclonal Anti-FMC63 scFv Antibody, Mouse IgG1 (Cat. No. FM3-FY45) and isotype control respectively. FITC signal was used to evaluate the binding activity (QC tested).
Protocol
应用案例由深圳普瑞金生物药业公司友情提供！
检测方法：流式细胞术
检测仪器：BD FACSCalibur流式细胞分析仪
样本信息：转染anti-BCMA CAR的人原代T淋巴细胞
主要试剂：
简要流程：
检测结果：结果显示1号患者anti-BCMA-CAR T细胞阳性率在52.72%，2号患者anti-BCMA-CAR T细胞阳性率在73.49%.
Human T cells were transfected with anti-BCMA CAR and cultured for 3 days. Three days post-transfection, 1x10 6 cells were first incubated with 50ul biotinylated human BCMA protein (Cat.No. BC7-H82F0, 8ug/ml ), washed and
then stained with PE Streptavidin and analyzed by flow cytometry. (Data are kindly provided by PREGENE Biopharma)
应用案例来自 MacLeod DT, et al., 2017, Mol Ther. 25(4):949-961.doi: 10.1016/j.ymthe.2017.02.005.
检测方法：流式细胞术
检测仪器：BD Fortessa flow cytometer (BD Biosciences)
样本信息：TRC1-2-treated, AAV:TRAC:CAR-transduced T cells
主要试剂：
简要流程：
检测结果：结果显示CD3阴性T细胞群中CD19 CAR表达的比例要高于CD3阳性T细胞群。
Activated T cells were electroporated with TRC1-2 mRNA and transduced with AAV:TRAC:CAR at an MOI of 400,000 vg/cell and cultured for 5 days in the presence of IL-2. Five days post-transduction, cells were stained for expression of the CAR using abiotinylated CD19-Fc reagent and CD3, with TRC1-2-treated, mock-transduced cells used as a control for gating of CAR expression. CD3+ cells were then depleted. Enriched CD3 cells were cultured for 3 additional days in the presence of IL-15 and IL-21 and then analyzed again by flow cytometry for CD3 and CAR expression.
验证数据由ACRO应用开发团队自主研发提供！
检测方法：流式细胞术
细胞样本： R1013-C6 cells (过表达Anti-CD19[FMC63] scFv & RFP的Expi 293细胞)；Expi 293 cells；Jurkat E6.1 cells.
主要试剂：
简要流程：
检测结果：结果显示ACROBiosystems的Human CD19 Protein, Fc Tag蛋白仅能特异性识别R1013-C6细胞表面的Anti-CD19[FMC63] scFv，不能与Expi 293细胞和Jurkat E6.1细胞发生非特异性结合反应；而同等实验条件下，Company N的Human CD19 Protein, Fc Tag蛋白与Expi 293细胞和Jurkat E6.1细胞发生了较强的非特异性结合。
Protocol
FACS analysis of human CD19 protein, Fc Tag (ACROBiosystems, Cat.No. CD9-H5259) binding to A. R1013-C6 cells, B. Expi 293 cells, C. Jurkat E6.1 cells. Cells were first stained with human CD19 protein, Fc Tag (ACROBiosystems, Cat.No. CD9-H5259) followed by FITC anti-human IgG Fc antibody, and then analyzed using NovoCyteTM Flow Cytometer. The data were analyzed with FCS Express 6Plus and GraphPad Prism 5 software.
Protocol
FACS analysis of human CD19 protein, Fc Tag (Company N) binding to A. R1013-C6 cells, B. Expi 293 cells, C. Jurkat E6.1 cells. Cells were first stained with human CD19 protein, Fc Tag (Company N) followed by FITC anti-human IgG Fc antibody, and then analyzed using NovoCyteTM Flow Cytometer. The data were analyzed with FCS Express 6Plus and GraphPad Prism 5 software.
