CAR陽性表達率檢測是CAR-T療法質(zhì)量控制的重要一環(huán)，常用的檢測方案有利用Protein L、Anti-Fab抗體或靶點蛋白結(jié)合流式細胞術(shù)進行檢測。其中，靶點蛋白結(jié)合流式細胞術(shù)的檢測方案因其靶點特異性強的優(yōu)勢而備受青睞，許多業(yè)內(nèi)人士預(yù)期靶點特異性的檢測將會被監(jiān)管機構(gòu)納入CAR-T質(zhì)量控制監(jiān)管考量的范疇。本文將對CAR陽性表達率檢測相關(guān)案例做一匯總以供大家參考。

三種CAR-T陽性率檢測試劑優(yōu)劣勢對比

檢測試劑	檢測原理	優(yōu)點	缺點
Protein L	結(jié)合抗體κ輕鏈	成本低，且市面有能簡化操作的直標Protein L	背景值高，無靶點特異性，不能檢測輕鏈λ型CAR
Anti-Fab抗體	結(jié)合抗體Fab段	成本低，且市面有能簡化操作的直標抗體	背景值高，無靶點特異性
靶點蛋白	特異性結(jié)合scFv抗原識別區(qū)	具靶點特異性，且可以評估親和力	成本相對高，不同靶點需建立不同方法，市面少有直標靶點蛋白

案例匯總

	案例名稱	數(shù)據(jù)來源
案例一	利用生物素標記hBCMA檢測anti-BCMA CAR陽性表達率	深圳普瑞金	點擊查看
案例二	利用生物素標記hCD19檢測anti-CD19 CAR陽性表達率	ACROBiosystems	點擊查看
案例三	利用生物素標記hCD19檢測anti-CD19 CAR陽性表達率	MacLeod DT	點擊查看
案例四	利用hCD19, His Tag蛋白檢測anti-CD19 CAR陽性表達率	ACROBiosystems	點擊查看
案例五	利用hCD19, Fc tag蛋白檢測anti-CD19 CAR陽性表達率	ACROBiosystems	點擊查看
案例六	不同供應(yīng)商hCD19, Fc tag蛋白的結(jié)合特異性驗證	ACROBiosystems	點擊查看

(點擊對應(yīng)案例即可跳轉(zhuǎn)案例詳情)

ACROBiosystems積極與CAR-T相關(guān)的醫(yī)藥研發(fā)人員緊密合作， 構(gòu)建CAR-T相關(guān)實驗體系建設(shè)，如果您有CAR-T靶點的需求可以申請免費試用，或?qū)σ韵路桨阜窒碛惺裁匆蓡枺梢约皶r，我們將持續(xù)分享該領(lǐng)域的成功案例以加速CAR-T藥物的研發(fā)進程。

 

案例詳情

案例一、利用生物素標記hBCMA檢測anti-BCMA CAR陽性表達率 

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應(yīng)用案例由深圳普瑞金生物藥業(yè)公司友情提供！

檢測方法：流式細胞術(shù)

檢測儀器：BD FACSCalibur流式細胞分析儀

樣本信息：轉(zhuǎn)染anti-BCMA CAR的人原代T淋巴細胞

主要試劑：

• 生物素標記的hBCMA蛋白 (Biotinylated Human BCMA / TNFRSF17 Protein, Fc Tag, Avi Tag (Avitag™), ACROBiosystems, Cat.No. BC7-H82F0)；
• PE標記的鏈霉親和素（PE Streptavidin , Biolegend, Cat.No. 405204)；

 

簡要流程：

• 1. 將anti-BCMA CAR基因通過慢病毒質(zhì)粒轉(zhuǎn)染，整合到來自患者的自體T細胞基因中；
• 2. 利用anti-BCMA CAR特異性結(jié)合BCMA蛋白的特性，將生物素標記的hBCMA蛋白（ACROBiosystems, Cat.No. BC7-H82F0）標記到anti-BCMA CAR-T細胞表面；
• 3. 利用生物素特異性結(jié)合親和素的特性，將PE標記的鏈霉親和素（Biolegend, Cat.No. 405204) 作為熒光二抗，標記到anti-BCMA CAR-T細胞表面；
• 4. 用BD FACSCalibur流式細胞分析儀進行檢測分析。

 

檢測結(jié)果：結(jié)果顯示1號患者anti-BCMA-CAR T細胞陽性率在52.72%，2號患者anti-BCMA-CAR T細胞陽性率在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)

相關(guān)產(chǎn)品：

BC7-H82F0 (Biotinylated Human BCMA, Fc & Avi Tag)

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案例二、利用生物素標記hCD19檢測anti-CD19 CAR陽性表達率 

 

應(yīng)用案例由ACRO應(yīng)用開發(fā)團隊自主研發(fā)提供！

檢測方法：流式細胞術(shù)

檢測儀器：NovoCyte^TM Flow Cytometer, ACEA Biosciences

樣本信息：R1013-C6細胞(過表達Anti-CD19[FMC63] scFv & RFP的Expi 293細胞)

主要試劑：

• 生物素標記的hCD19蛋白 (Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling), ACROBiosystems, Cat.No.；
• FITC標記的鏈霉親和素（FITC Streptavidin, Biolegend, Cat.No. 405201)；

 

簡要流程：

• 1. 將Anti-CD19[FMC63] scFv & RFP基因通過質(zhì)粒轉(zhuǎn)染，整合到Expi 293細胞基因中，命名為R1013-C6細胞；
• 2. 利用anti-CD19[FMC63] scFv特異性結(jié)合CD19蛋白的特性，將生物素標記的hCD19蛋白（ACROBiosystems, Cat.No. CD9-H8259）標記到R1013-C6細胞表面；
• 3. 利用生物素特異性結(jié)合親和素的特性，將FITC標記的鏈霉親和素（FITC Streptavidin, Biolegend, Cat.No. 405201)作為熒光二抗，標記到R1013-C6細胞表面；
• 4. 用NovoCyte^TM Flow Cytometer流式細胞分析儀進行檢測分析。

 

檢測結(jié)果：結(jié)果顯示生物素標記的CD19蛋白能夠與 R1013-C6細胞膜表面的anti-CD19 [FMC63] scFv特異性結(jié)合，這種結(jié)合能夠被游離的抗CD19的抗體FMC63拮抗。

FACS Analysis of anti-CD19 CAR expression

293 cells were transfected with FMC63-scFv and RFP tag . 2x10⁵ of the cells were first incubated with A. Biotinylated protein control. B. Recombinant biotinylated human CD19 (Cat.No. CD9-H8259, 10ug/ml ) . C. Recombinant biotinylated human CD19 (Cat.No. CH8259, 10ug/ml) and FMC63(Mouse anti-CD19 antibody). FITC Streptavidin was used to analyse with FACS. RFP was used to evaluate CAR(FMC63-scFv) expression and FITC was used to evaluate the binding activity of recombinant biotinylated human CD19 (Cat.No. CD9-H8259) .

相關(guān)產(chǎn)品：

CD9-H8259 Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling)

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案例三、利用生物素標記hCD19檢測anti-CD19 CAR陽性表達率 

 

應(yīng)用案例來自 MacLeod DT, et al., 2017, Mol Ther. 25(4):949-961.doi: 10.1016/j.ymthe.2017.02.005.

檢測方法：流式細胞術(shù)

檢測儀器：BD Fortessa flow cytometer (BD Biosciences)

樣本信息：TRC1-2-treated, AAV:TRAC:CAR-transduced T cells

主要試劑：

• 生物素標記的hCD19蛋白 (Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling), ACROBiosystems, Cat.No. CD9-H8259)；
• PE標記的鏈霉親和素（PE Streptavidin, Biolegend)；
• Anti-CD3-BV711抗體（Brilliant Violet 711™ anti-human CD3 Antibody, BioLegend).

 

簡要流程：

• 1. 利用CD19 CAR能特異性結(jié)合CD19蛋白的特性，如果待檢T細胞正常表達了CD19 CAR，生物素標記的CD19-Fc蛋白就能被標記到待檢T細胞表面；
• 2. 利用生物素能特異性結(jié)合親和素（PE Streptavidin）和Anti-CD3-BV711抗體能特異性結(jié)合CD3分子的特性，如果待檢T細胞表面有生物素和CD3分子，鏈霉親和素上的PE和Anti-CD3抗體上的BV711熒光標記就能被標記到待檢T細胞表面；
• 3. 將制備好的單細胞懸液上機進行流式細胞分析。

 

檢測結(jié)果：結(jié)果顯示CD3陰性T細胞群中CD19 CAR表達的比例要高于CD3陽性T細胞群。

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.

相關(guān)產(chǎn)品：

CD9-H8259 Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling)

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案例四、利用hCD19, His Tag蛋白檢測anti-CD19 CAR陽性表達率 

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驗證數(shù)據(jù)由ACRO應(yīng)用開發(fā)團隊自主研發(fā)提供！

檢測方法：流式細胞術(shù)

細胞樣本：R1013-C6 cells (過表達Anti-CD19[FMC63] scFv & RFP的Expi 293細胞)

主要試劑：

• Human CD19, His Tag蛋白（ACROBiosystems, Cat.No. CD9-H52H2）；
• FITC anti-6xHis tag antibody（Abcam, Cat.No. ab1206）.

 

簡要流程：

• 1. 將anti-CD19[FMC63] scFv & RFP基因通過質(zhì)粒轉(zhuǎn)染，整合到Expi 293細胞基因中，命名為R1013-C6細胞；
• 2. 利用anti-CD19[FMC63] scFv特異性結(jié)合CD19蛋白的特性，將hCD19, His Tag蛋白（ACROBiosystems, Cat.No. ）標記到R1013-C6細胞表面；
• 3. 將熒光二抗FITC anti-6xHis tag antibody （Abcam, Cat.No. ab1206）標記到R1013-C6細胞表面；
• 4. 用Accuri C6 Flow Cytometer流式細胞分析儀進行檢測分析。

 

檢測結(jié)果：結(jié)果顯示hCD19, His Tag蛋白能夠與 R1013-C6細胞膜表面的anti-CD19 [FMC63] scFv特異性結(jié)合，這種結(jié)合能夠被游離的抗CD19的抗體FMC63拮抗。

FACS Analysis of anti-CD19 CAR expression

293 cells were transfected with FMC63-scFv and RFP tag . 2x105 of the cells were first incubated with A. His Tag-protein control. B. Recombinant His Tag-human CD19  C. The mixture of recombinant His Tag-human CD19 ( and FMC63(Mouse anti-CD19 antibody), followed by FITC anti-6xHis tag antibody, and then analyzed using Accuri C6 Flow Cytometer. RFP was used to evaluate CAR(FMC63-scFv) expression and FITC was used to evaluate the binding activity of recombinant His Tag-human CD19 (Cat.No. ).

相關(guān)產(chǎn)品：

(Human CD19 Protein, His Tag)

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案例五、利用hCD19, Fc tag蛋白檢測anti-CD19 CAR陽性表達率 

 

驗證數(shù)據(jù)由ACRO應(yīng)用開發(fā)團隊自主研發(fā)提供！

檢測方法：流式細胞術(shù)

細胞樣本：R1013-C6 cells (過表達Anti-CD19[FMC63] scFv & RFP的Expi 293細胞)

主要試劑：

• Human CD19, Fc tag蛋白 （Acrobiosystems, Cat.No. ）；
• FITC anti-human IgG Fc antibody ( Biolegend, Cat.No. 409310）.

 

簡要流程：

• 1. 將anti-CD19[FMC63] scFv & RFP基因通過質(zhì)粒轉(zhuǎn)染，整合到Expi 293細胞基因中，命名為R1013-C6細胞；
• 2. 利用anti-CD19[FMC63] scFv特異性結(jié)合CD19蛋白的特性，將hCD19, Fc tag蛋白（Acrobiosystems, Cat.No.）標記到R1013-C6細胞表面；
• 3. 將熒光二抗FITC anti-human IgG Fc antibody ( Biolegend, Cat.No. 409310）標記到R1013-C6細胞表面；
• 4. 用NovoCyte^TM Flow Cytometer流式細胞分析儀進行檢測分析。

 

檢測結(jié)果：結(jié)果顯示hCD19, Fc tag蛋白能夠與 R1013-C6細胞膜表面的anti-CD19 [FMC63] scFv特異性結(jié)合，這種結(jié)合能夠被游離的抗CD19的抗體FMC63拮抗。

FACS Analysis of anti-CD19 CAR expression

293 cells were transfected with FMC63-scFv and RFP tag . 2x10⁵ of the cells were first incubated with A. Human Fc tag control. B. Recombinant human CD19,Fc Tag (Cat.No., 10ug/ml ). C. Recombinant human CD19,Fc Tag(Cat.No. , 10ug/ml ) and FMC63(Mouse anti-CD19 antibody) . The FITC anti-human IgG Fc was used to analyse with FACS. RFP was used to evaluate CAR(FMC63-scFv) expression and FITC was used to evaluate the binding activity of recombinant human CD19,Fc Tag .

相關(guān)產(chǎn)品：

(Human CD19 Protein, Fc Tag)

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案例六、不同供應(yīng)商hCD19, Fc tag蛋白的結(jié)合特異性驗證 

 

驗證數(shù)據(jù)由ACRO應(yīng)用開發(fā)團隊自主研發(fā)提供！

檢測方法：流式細胞術(shù)

細胞樣本： R1013-C6 cells (過表達Anti-CD19[FMC63] scFv & RFP的Expi 293細胞)；Expi 293 cells；Jurkat E6.1 cells.

主要試劑：

• 蛋白樣本1：Human CD19 Protein, Fc Tag (ACROBiosystems, Cat.No.);
• 蛋白樣本2：Human CD19 Protein, Fc Tag (Company N)；
• 陰性對照蛋白：Human PD-L1 Protein, Fc Tag (ACROBiosystems, Cat.No. 
• 熒光二抗：FITC anti-human IgG Fc antibody (Biolegend, Cat.No. 409310).

 

簡要流程：

• 1. 用Human CD19 Fc tag蛋白分別標記R1013-C6 細胞、Expi 293細胞和Jurkat E6.1細胞；
• 2. 將FITC anti-human IgG Fc antibody作為熒光二抗，對以上細胞進行二次標記；
• 3. 用NovoCyte^TM Flow Cytometer流式細胞分析儀進行檢測分析。

 

檢測結(jié)果：結(jié)果顯示ACROBiosystems的Human CD19 Protein, Fc Tag蛋白僅能特異性識別R1013-C6細胞表面的Anti-CD19[FMC63] scFv，不能與Expi 293細胞和Jurkat E6.1細胞發(fā)生非特異性結(jié)合反應(yīng)；而同等實驗條件下，Company N的Human CD19 Protein, Fc Tag蛋白與Expi 293細胞和Jurkat E6.1細胞發(fā)生了較強的非特異性結(jié)合。

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

FACS analysis of human CD19 protein, Fc Tag (ACROBiosystems, Cat.No.) 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.  followed by FITC anti-human IgG Fc antibody, and then analyzed using NovoCyte^TM Flow Cytometer. The data were analyzed with FCS Express 6Plus and GraphPad Prism 5 software.

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 NovoCyte^TM Flow Cytometer. The data were analyzed with FCS Express 6Plus and GraphPad Prism 5 software.

相關(guān)產(chǎn)品：

CD9-H5259 (Human CD19 Protein, Fc Tag)

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參考文獻

• (1) MacLeod DT, et al., 2017, Mol Ther. 25(4):949-961.doi: 10.1016/j.ymthe.2017.02.005..