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CAR阳性表达率检测

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2018-02-27 13:52

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。

四种CAR-T阳性率检测试剂优劣势对比

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
案例汇总

案例名称 数据来源
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
(点击对应案例即可跳转案例详情)
案例详情

案例一、利用PE标记CD19蛋白检测anti-CD19 CAR 阳性表达率

返回案例汇总

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:

  • PE标记的CD19蛋白(PE-Labeled Human CD19 (20-291) Protein, His Tag (Site-specific conjugation) ACROBiosystems, Cat. No. CD9-HP2H3);
  • 简要流程:

    • 1.用含10% FBS的DMEM培养基在37℃,5% CO2培养箱中培养Anti-CD19 CAR-293细胞。
    • 2.收集细胞,进行细胞计数及存活率测定,等分细胞悬液,每管 2×105 活细胞(Anti-CD19-CAR阳性表达率为98%,注意细胞存活率必须大于等于95%)。
    • 3.加入100 µL稀释后的PE标记CD19蛋白(ACROBiosystems, Cat. No. CD9-HP2H3)(1:50稀释),4℃避光孵育1小时。
    • 4.用含2% BSA的PBS缓冲液洗涤细胞三次,并用200 µL PBS重悬细胞。
    • 5.转移细胞至流式管进行流式细胞分析。


    检测结果:结果显示Anti-CD19 CAR阳性表达率为100%

    FACS Analysis of anti-BCMA CAR expression

    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

    案例二、利用PE标记BCMA蛋白检测anti-BCMA CAR 阳性表达率

    返回案例汇总

    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:

  • PE标记的BCMA蛋白(PE-Labeled Human BCMA / TNFRSF17 Protein, His Tag (Site-specific conjugation) ACROBiosystems, Cat. No. BCA-HP2H2);
  • 简要流程:

    • 1.用含10% FBS的DMEM培养基在37℃,5% CO2培养箱中培养Anti-BCMA CAR-293细胞。
    • 2.收集细胞,进行细胞计数及存活率测定,等分细胞悬液,每管 2×105 活细胞(Anti-BCMA-CAR阳性表达率为98%,注意细胞存活率必须大于等于95%)。
    • 3.加入100 µL稀释后的PE标记BCMA蛋白(ACROBiosystems, Cat. No. BCA-HP2H2)(1:50稀释),4℃避光孵育1小时。
    • 4.用含2% BSA的PBS缓冲液洗涤细胞三次,并用200 µL PBS重悬细胞。
    • 5.转移细胞至流式管进行流式细胞分析。


    检测结果:结果显示Anti-BCMA CAR阳性表达率为100%

    FACS Analysis of anti-BCMA CAR expression

    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

    案例三、利用FITC标记anti-FMC63 antibody检测anti-CD19(FMC63) CAR 阳性表达率

    返回案例汇总

    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:

  • FITC标记的抗FMC63抗体(FITC-Labeled Monoclonal Anti-FMC63 scFv Antibody, Mouse IgG1 (Y45) ACROBiosystems, Cat. No. FM3-FY45);
  • 简要流程:

    • 1.用含10% FBS的DMEM培养基在37℃,5% CO2培养箱中培养Anti-CD19 CAR-293细胞。
    • 2.收集细胞,进行细胞计数及存活率测定,等分细胞悬液,每管 2×105 活细胞(Anti-CD19-CAR阳性表达率为98%,注意细胞存活率必须大于等于95%)。
    • 3.加入100 µL稀释后的FITC标记抗FMC63抗体(ACROBiosystems, Cat. No. FM3-FY45)(1:50稀释),4℃避光孵育1小时。
    • 4.用含2% BSA的PBS缓冲液洗涤细胞三次,并用200 µL PBS重悬细胞。
    • 5.转移细胞至流式管进行流式细胞分析。


    检测结果:结果显示Anti-CD19 CAR阳性表达率为100%

    FACS Analysis of anti-BCMA CAR expression

    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

    案例四、利用生物素标记hBCMA检测anti-BCMA CAR阳性表达率

    返回案例汇总

    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:

  • 生物素标记的hBCMA蛋白 (Biotinylated Human BCMA / TNFRSF17 Protein, Fc Tag, Avi Tag (Avitag™), ACROBiosystems, Cat.No. BC7-H82F0);
  • PE标记的链霉亲和素(PE Streptavidin , Biolegend, Cat.No. 405204);

  • 简要流程:

    • 1. 将anti-BCMA CAR基因通过慢病毒质粒转染,整合到来自患者的自体T细胞基因中;
    • 2. 利用anti-BCMA CAR特异性结合BCMA蛋白的特性,将生物素标记的hBCMA蛋白(ACROBiosystems, Cat.No. BC7-H82F0)标记到anti-BCMA CAR-T细胞表面;
    • 3. 利用生物素特异性结合亲和素的特性,将PE标记的链霉亲和素(Biolegend, Cat.No. 405204) 作为荧光二抗,标记到anti-BCMA CAR-T细胞表面;
    • 4. 用BD FACSCalibur流式细胞分析仪进行检测分析。


    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%.

    FACS Analysis of anti-BCMA CAR expression

    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)

    案例五、利用生物素标记hCD19检测anti-CD19 CAR阳性表达率

    返回案例汇总

    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:

  • 生物素标记的hCD19蛋白 (Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling), ACROBiosystems, Cat.No. CD9-H8259);
  • PE标记的链霉亲和素(PE Streptavidin, Biolegend);
  • Anti-CD3-BV711抗体(Brilliant Violet 711™ anti-human CD3 Antibody, BioLegend).

  • 简要流程:

    • 1. 利用CD19 CAR能特异性结合CD19蛋白的特性,如果待检T细胞正常表达了CD19 CAR,生物素标记的CD19-Fc蛋白就能被标记到待检T细胞表面;
    • 2. 利用生物素能特异性结合亲和素(PE Streptavidin)和Anti-CD3-BV711抗体能特异性结合CD3分子的特性,如果待检T细胞表面有生物素和CD3分子,链霉亲和素上的PE和Anti-CD3抗体上的BV711荧光标记就能被标记到待检T细胞表面;
    • 3. 将制备好的单细胞悬液上机进行流式细胞分析。


    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。

    FACS Analysis of anti-CD19 CAR expression

    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.

    案例六、不同供应商hCD19, Fc tag蛋白的结合特异性验证

    返回案例汇总

    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:

  • 蛋白样本1:Human CD19 Protein, Fc Tag (ACROBiosystems, Cat.No. CD9-H5259);
  • 蛋白样本2:Human CD19 Protein, Fc Tag (Company N);
  • 阴性对照蛋白:Human PD-L1 Protein, Fc Tag (ACROBiosystems, Cat.No. PD1-H5258);
  • 荧光二抗:FITC anti-human IgG Fc antibody (Biolegend, Cat.No. 409310).

  • 简要流程:

    • 1. 用Human CD19 Fc tag蛋白分别标记R1013-C6 细胞、Expi 293细胞和Jurkat E6.1细胞;
    • 2. 将FITC anti-human IgG Fc antibody作为荧光二抗,对以上细胞进行二次标记;
    • 3. 用NovoCyteTM Flow Cytometer流式细胞分析仪进行检测分析。


    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


    Binding specificity analysis of ACROBiosystems hCD19(C-Fc tag) protein

    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

    Binding specificity analysis of Company N hCD19(C-Fc tag) protein

    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.
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