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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。 2017年是CAR-T细胞疗法的元年。这一年两款突破性CAR-T疗法——诺华公司用于治疗25岁以下青少年复发或难治性急性淋巴细胞白血病的Kymriah和Kite Pharma公司用于治疗特殊类型成人大B细胞淋巴瘤的Yescarta,相继获美国FDA批准上市;这一年截止到年底全球正在开展的CAR-T临床试验已超过400项(根据ClinicalTrials数据)。

然而,CAR-T细胞作为一种“活的药物”与传统化药和生物药有着巨大的差别,如何监管是目前亟待解决的一大难题。在CAR-T细胞规范化生产和质量控制尚无标准可依的现阶段,制药企业纷纷制定了各自企业内部的质控标准。据了解,CAR阳性表达率检测作为质控的重要一环,常用的检测方案有利用Protein L、Anti-Fab抗体或靶点蛋白结合流式细胞术进行检测。其中,靶点蛋白结合流式细胞术的检测方案因其靶点特异性强的优势而备受青睐,许多业内人士预期靶点特异性的检测将会被监管机构纳入CAR-T质量控制监管考量的范畴。

三种CAR-T阳性率检测试剂优劣势对比

检测试剂 检测原理 优点 缺点
Protein L 结合抗体κ轻链 成本低,且市面有能简化操作的直标Protein L 背景值高,无靶点特异性,不能检测轻链λ型CAR
Anti-Fab抗体 结合抗体Fab段 成本低,且市面有能简化操作的直标抗体 背景值高,无靶点特异性
靶点蛋白 特异性结合scFv抗原区识别 具靶点特异性,且可以评估亲和力 成本相对高,不同靶点需建立不同方法,市面少有直标靶点蛋白

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和生物素标记平台,开发了一系列适用于特异性检测CAR阳性表达率的CAR-T靶点蛋白,其中生物素预先标记的蛋白在Biopanning筛选scFv,以及流式细胞技术检测CAR表达和筛选CAR表达的细胞中独具优势。目前产品已覆盖BCMACD19ROR1EGFRVIII等20余个CAR-T热门靶点。

应用实例:

Anti-BCMA CAR阳性表达率检测

检测数据由深圳普瑞金生物药业公司友情提供。检测结果显示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)

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Anti-CD19 CAR表达阳性率检测

检测数据由ACRO应用开发团队自主研发提供。检测结果显示生物素标记的CD19蛋白能够与 R1013-C6细胞膜表面的anti-CD19 CAR 特异性结合,这种结合能够被游离的抗CD19的抗体FMC63拮抗。

FACS Analysis of anti-CD19 CAR expression

293 cells were transfected with FCM63-scFv and RFP tag . 2x10 5 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. CD9-H8259, 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 biotinylated human CD19 (Cat. No. CD9-H8259) .

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Anti-CD19 CAR阳性表达率检测

检测数据由MacLeod DT, et al.发表于Journal of Molecular therapy。检测结果显示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。(更多详情请参见 MacLeod DT, et al., 2017, Mol Ther. 25(4):949-961.doi: 10.1016/j.ymthe.2017.02.005.

CAR-expression.png

Figure A. 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 a biotinylated 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.

推荐产品

Targets Diseases Recommended Products
BCMA 多发性骨髓瘤、白血病、B细胞淋巴瘤 BC7-H82F0 ( Biotinylated Human BCMA)
BCA-H522Y (Human BCMA)
ROR1 白血病,乳腺癌r RO1-H821y ( Biotinylated Human ROR1)
RO1-H522y (Human ROR1)
CD33 急性髓系白血病 CD3-H82E7 ( Biotinylated Human CD33)
CD3-H5226 (Human CD33)
EGFRVIII 胶质母细胞瘤 EGR-H82E0 ( Biotinylated Human EGFRVIII)
EGI-H52H4 (Human EGFRvIII)
CD30 白血病,B细胞淋巴瘤 CD0-H82E6 ( Biotinylated Human CD30)
CD0-H5229 (Human CD30)
EGFR 非小细胞肺癌,上皮癌、胶质瘤 EGR-H82E3 ( Biotinylated Human EGFR)
EGR-H5222 (Human EGFR)
FOLR1 卵巢癌 FO1-M82E9 ( Biotinylated Mouse FOLR1)
FO1-H5229 (Human FOLR1)
HER2 卵巢癌,乳腺癌,胶质母细胞瘤,骨肉瘤 HE2-H822R ( Biotinylated Human Her2)
HE2-H5225 (Human Her2)
c-MET/HGFR 恶性黑色素瘤,乳腺癌 MET-H82E1 ( Biotinylated Human HGF R)
MET-H5227 (Human HGF R)
CAIX 肾细胞癌 CA9-H5226(Human CA9 (38-414))
CD19 急性白血病、B细胞淋巴瘤 CD9-H8259 ( Biotinylated Human CD19)
CD20 白血病,B细胞淋巴瘤 CD0-H5268 (Human MS4A1 / CD20, Fc Tag)
CD22 白血病,B细胞淋巴瘤 SI2-H5228 (Human Siglec-2 / CD22)
EpCAM 肝肿瘤,胃肿瘤 EPM-H82E8 ( Biotinylated Human EpCAM)
EPM-H5223 (Human EpCAM)
GPC3 原发性肝细胞癌 GP3-H5223 (Human Glypican 3)
IL13Rα2 胶质瘤 IL2-H5257 (Human IL13Rα2, Fc Tag)
MSLN 间皮瘤,卵巢癌 MSN-H82E9 ( Biotinylated Human MSLN)
MSN-H5223 (Human MSLN)
MUC1 精囊癌 MU1-H5252 (Human MUC-1, Fc Tag)
CD138 多发性骨髓瘤 SD1-H5228 (Human Syndecan-1 / CD138)
CD38 B细胞恶性肿瘤 CD8-H82E7 ( Biotinylated Human CD38)
CD8-H5224 (Human CD38)

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