CAR-T靶点蛋白
嵌合抗原受体T（CAR-T）细胞疗法是一种基于体外改造T细胞，使其表面表达特异性识别肿瘤表面抗原的受体片段的癌症免疫疗法，改造后的T细胞输入患者体内，不需要抗原递呈细胞（APC）的帮助，直接靶向体内癌细胞并发挥免疫杀伤作用。
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，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。
ACROBiosystems作为专注于医药研发领域的蛋白供应商，利用专业的蛋白研发平台、蛋白标记平台、稳定株开发平台和流式细胞分析平台，开发了一系列包括非标记、生物素标记、荧光标记等多种形式的CAR-T靶点蛋白以及配套的流式细胞法检测CAR阳性率的protocol，以助力CAR-T的研发，加速CAR-T研发的进程。目前产品已覆盖CD19、BCMA、CD22、MSLN和EGFR在内的近40个CAR-T热门靶点。
CAR-T阳性率检测策略及蛋白产品设计
直接检测法 —— 荧光标记蛋白
产品特点
靶抗原预先标记了荧光素基团
可简化检测流程，节省检测时间
可避免因二抗交叉反应而引起的非特异性背景
可简化检测流程，节省检测时间
可避免因二抗交叉反应而引起的非特异性背景
> PE-labeled （点击分子即可查看产品详情）
BCMA C11D5.3 scFv Carbonic Anhydrase IX CD19 CD27 Ligand CD30 CD4 CD7 CD79B CLEC12A DLL3 EGF R EGFRvIII FAP FMC63 FOLR1 Glypican 3 GUCY2C H-2Kb & B2M H-2Kb & B2M & OVA (SIINFEKL) H-2Kd & B2M Her2 HLA-A*0201 & B2M HLA-A*0201 & B2M & AFP (FMNKFIYEI) HLA-A*0201 & B2M & CMV pp65 (NLVPMVATV) HLA-A*0201 & B2M & EBV EBNA3C (LLDFVRFMGV) HLA-A*0201 & B2M & EBV LMP1 (YLLEMLWRL) HLA-A*0201 & B2M & EBV LMP2 (FLYALALLL) HLA-A*0201 & B2M & EBV LMP2A (CLGGLLTMV) HLA-A*0201 & B2M & HIV Gag (SLYNTVATL) HLA-A*0201 & B2M & hTERT (ILAKFLHWL) HLA-A*0201 & B2M & KRASG12V (KLVVVGAVGV) HLA-A*0201 & B2M & MAGE-A10 (GLYDGMEHL) HLA-A*0201 & B2M & p53 (HMTEVVRHC) HLA-A*0201 & B2M & PAP (ALDVYNGLL) HLA-A*0201 & B2M & Survivin (TLPPAWQPFL) HLA-A*0201 | B2M | HPV16-E6 HLA-A*0201 | B2M | HPV16-E7 HLA-A*0201 | B2M | MAGE-A4 (KVLEHVVRV) HLA-A*0201 | B2M | NY-ESO-1 HLA-A*0301 & B2M HLA-A*0301 & B2M & KRASG12V (VVGAVGVGK) HLA-A*1101 & B2M HLA-A*1101 & B2M & EBV (AVFDRKSDAK) HLA-A*1101 & B2M & EBV LMP2 (SSCSSCPLSK) HLA-A*1101 & B2M & HPV16-E6 (TTLEQQYNK) HLA-A*1101 & B2M & HPV16-E7 (IVCPICSQK) HLA-A*1101 & B2M & KRAS (VVVGAGGVGK) HLA-A*1101 & B2M & KRASG12D (VVVGADGVGK) HLA-A*1101 & B2M & KRASG12V (VVVGAVGVGK) HLA-A*2402 & B2M HLA-A*2402 & B2M & EBV EBNA3A (RYSIFFDYM) HLA-A*2402 & B2M & EBV EBNA3B (TYSAGIVQI) HLA-A*2402 & B2M & p53 (TYSPALNKMF) HLA-E*0103 & B2M HLA-E*0103 & B2M & CMV UL40 (VMAPRTLLL) HLA-E*0103 & B2M & CMV UL40 (VMAPRTVIL) HLA-E*0103 & B2M & CMV UL40 (VMAPRTVLL) HLA-E*0103 & B2M & CMV UL40 (VMPPRTVIL) IL-3 R alpha Mamu-A*01 & B2M Mesothelin Protein L PSMA ROR1 Siglec-2 Siglec-3 SIRP alpha SLAMF7 uPAR VEGF R2
> FITC-labeled （点击分子即可查看产品详情）
B7-H3 (4Ig) BCMA Carbonic Anhydrase IX CD19 CD27 Ligand CD30 CD37 CD38 CD4 CD47 CD5 CD7 CD72 CD8 alpha & beta CD98 CD99 CEACAM-5 CLEC12A DLL3 EGF R EGFRvIII EMMPRIN EpCAM ErbB3 FAP Flt-3 FMC63 FOLR1 G4S linker Glypican 3 GUCY2C Her2 HGF R IL-13 R alpha 2 IL-3 R alpha LILRB4 Mesothelin NCAM-1 Nectin-4 NKG2D PSMA Rituximab ROBO1 ROR1 Siglec-2 Siglec-3 SLAMF7 Syndecan-1 uPAR VEGF R2
> 应用案例
- 使用FITC标记CD19蛋白检测Anti-CD19-CAR表达阳性率
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- 293 cells were transfected with anti-CD19-scFv and RFP tag. 2e5 of the cells were stained with B. FITC-labeled Human CD19 (20-291) (Cat. No. CD9-HF2H2, 10 µg/ml) and C. FITC-labeled protein control. A. Non-transfected 293 cells and C. FITC-labeled protein control were used as negative control. RFP was used to evaluate CAR (anti-CD19-scFv) expression and FITC was used to evaluate the binding activity of FITC-labeled Human CD19 (20-291) (Cat. No. CD9-HF2H2).
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Protocol
基于生物素-亲和素的检测法 —— 生物素标记蛋白
产品特点
靶抗原预先标记了生物素，随后可使用荧光标记的链霉亲和素检测
链霉亲和素对生物素具有极高的亲和力，两者的结合具有高度特异性
可高灵敏度和高特异性地检测CAR阳性表达率
链霉亲和素对生物素具有极高的亲和力，两者的结合具有高度特异性
可高灵敏度和高特异性地检测CAR阳性表达率
> Biotinylated proteins -> Specially designed
CA125 Carbonic Anhydrase IX CD19 CD30 CD38 CD4 CD5 CD7 CD72 CD8 alpha CD8 alpha & beta CEACAM-5 Chimeric HLA-A*0201 (H-2Kb α3) & B2M & GP100(YLEPGPVTA) CLEC12A DLL3 EGF R EGFRvIII EMMPRIN EpCAM EphA2 ErbB3 FAP FMC63 FOLR1 GUCY2C GUCY2D H-2Db & B2M H-2Kb & B2M & OVA (SIINFEKL) TRBC1 TRBC2 TRDC TRGC1 TRGC2 uPAR VEGF R2
> 应用案例
- 使用Human CD19, Fc Tag蛋白检测Anti-CD19-CAR表达阳性率
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- 293 cells were transfected with FMC63-scFv and RFP tag. 2e5 of the cells were first stained with B. Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity) (Cat. No. CD9-H5251, 3 µg/ml) and C. Human Fc Tag Protein Control, followed by FITC-conjugated Anti-human IgG Fc Antibody. A. Non-transfected 293 cells and C. Human Fc Tag Protein Control were used as negative control. RFP was used to evaluate CAR (anti-CD19-scFv) expression and FITC was used to evaluate the binding activity of Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity) (Cat. No. CD9-H5251).
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Protocol
- CAR-T靶点蛋白
- CAR-T阳性率检测策略及蛋白产品设计