Protein L: a novel reagent for the detection of chimeric antigen receptor (CAR) expression by flow cytometry.
Protein L: a novel reagent for the detection of chimeric antigen receptor (CAR) expression by flow cytometry.
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蛋白L：一种通过流式细胞仪检测嵌合抗原受体（CAR）表达的新试剂。
DOI：
10.1186/1479-5876-10-29
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发表时间：
2012-02-13
影响因子：
7.4
通讯作者：
Morgan RA
中科院分区：
文献类型：
--
作者：
Zheng Z;Chinnasamy N;Morgan RA
在过去的二十年中，针对肿瘤相关抗原的过继免疫治疗的嵌合抗原受体（CAR）的设计已经取得了显著进展。在结构上，汽车由针对肿瘤相关抗原的单链抗体片段组成，该单链抗体片段融合至细胞外间隔区和跨膜结构域，随后是T细胞胞质信号传导部分。目前，正在进行几项临床试验，使用基因修饰的外周血淋巴细胞（PBL）与针对各种肿瘤相关抗原的汽车。尽管汽车的设计有所改进，靶抗原的数量也有所增加，但目前还没有通用的流式细胞术方法可用于检测汽车在转导淋巴细胞表面的表达。目前，抗片段抗原结合（Fab）缀合物最广泛地用于通过流式细胞术测定基因修饰的淋巴细胞上汽车的表达。这些试剂的局限性在于它们中的许多不是市售的，通常它们是多克隆抗体，并且通常结果不一致。为了开发一种简单通用的流式细胞术方法来检测汽车的表达，我们采用蛋白L来确定转导淋巴细胞上汽车的表达。蛋白L是一种免疫球蛋白（IG）结合蛋白，其与IG的可变轻链（κ链）结合而不干扰抗原结合位点。蛋白L与大多数类型的IG结合，并且还与单链抗体片段（scFv）和Fab片段结合。我们使用来源于人和鼠抗体的汽车来验证这种新的基于蛋白L的流式细胞术方法，结果与其他已建立的方法相关性良好。用表达两种基于人抗体的汽车（抗EGFRvIII和抗VEGFR2）、两种鼠抗体衍生的汽车（抗CSPG4和抗CD19）和两种基于人源化小鼠抗体的汽车（抗ERBB 2和抗PSCA）的逆转录病毒载体转导活化的人PBL。转导的细胞首先用生物素标记的蛋白L染色，然后用藻红蛋白（PE）缀合的链霉亲和素（SA）染色，并通过流式细胞术分析。为了比较，用生物素缀合的山羊抗小鼠Fab或CAR特异性融合蛋白平行染色细胞。使用蛋白L，所有CAR转导的淋巴细胞表现出范围为40至80%的阳性细胞的特异性染色模式（与未转导的细胞相比），并且染色与用抗Fab抗体观察到的模式相当。我们的数据证明了采用蛋白L作为通过流式细胞术检测转导的淋巴细胞上的CAR表达的通用试剂的可行性。
There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL) with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes. Currently anti-fragment antigen binding (Fab) conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig)-binding protein that binds to the variable light chains (kappa chain) of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv) and Fab fragments. We used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2), two murine antibody derived CARs (anti-CSPG4, and anti-CD19), and two humanized mouse antibody based CARs (anti-ERBB2, and anti-PSCA). Transduced cells were stained first with biotin labeled protein L followed by phycoerythrin (PE)-conjugated streptavidin (SA) and analyzed by flow cytometry. For comparison, cells were stained in parallel with biotin conjugated goat-anti-mouse Fab or CAR specific fusion proteins. Using protein L, all CAR transduced lymphocytes exhibited specific staining pattern ranging from 40 to 80% of positive cells (compared to untransduced cells) and staining was comparable to the pattern observed with anti-Fab antibodies. Our data demonstrate the feasibility of employing Protein L as a general reagent for the detection of CAR expression on transduced lymphocytes by flow cytometry.
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DOI：
10.1097/cji.0b013e3181ac6138
发表时间：
2009-09
期刊：
Journal of immunotherapy (Hagerstown, Md. : 1997)
影响因子：
--
作者：
Kochenderfer JN;Feldman SA;Zhao Y;Xu H;Black MA;Morgan RA;Wilson WH;Rosenberg SA
通讯作者：
Rosenberg SA
影响因子：
2.2
作者：
NILSON, BHK;LOGDBERG, L;AKERSTROM, B
通讯作者：
AKERSTROM, B
影响因子：
11.2
作者：
Burns WR;Zhao Y;Frankel TL;Hinrichs CS;Zheng Z;Xu H;Feldman SA;Ferrone S;Rosenberg SA;Morgan RA
通讯作者：
Morgan RA
DOI：
10.4049/jimmunol.0900447
发表时间：
2009-11-01
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Zhao Y;Wang QJ;Yang S;Kochenderfer JN;Zheng Z;Zhong X;Sadelain M;Eshhar Z;Rosenberg SA;Morgan RA
通讯作者：
Morgan RA
DOI：
10.1073/pnas.86.24.10024
发表时间：
1989-12-01
影响因子：
11.1
作者：
GROSS, G;WAKS, T;ESHHAR, Z
通讯作者：
ESHHAR, Z