Department of Pediatrics, University of California, Los Angeles (UCLA), 10833 Le Conte Avenue, A2-410 MDCC, MC 175217, Los Angeles, CA 90095-1752, USA.
J Transl Med. 2013 Jan 29;11:23. doi: 10.1186/1479-5876-11-23.
Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain capable of activating T-cells. CARs displayed on the surface of transduced cells perform non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of mature T-lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B-lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models.
A specific reagent for the detection of anti-CD19 CAR expression was developed, a fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (CD19sIg). Genes encoding CD19sIg fusion proteins were constructed by fusing either exons 1 to 3 (CD19sIg1-3) or exons 1 to 4 (CD19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T-cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The CD19sIg proteins were produced from 293 T cells by stable lentiviral vector transduction and purification from culture medium.
ELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule CD19sIg1-4, but no binding by CD19sIg1-3. Conjugation of the CD19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays, detecting as low as 0.5% of CAR-modified primary cells with minimal background staining.
This fusion molecule is a sensitive reagent for detection of anti-CD19 CAR derived from any monoclonal antibody present in CAR-modified T-cells.
嵌合抗原受体（CARs）由单克隆抗体的抗原识别部分与能够激活 T 细胞的细胞内信号结构域融合而成。转导细胞表面展示的 CAR 进行非 MHC 限制的抗原识别，并激活细胞内信号通路，诱导靶细胞溶解、细胞因子分泌和增殖。目前正在进行临床试验，评估使用靶向 CD19 抗原的 CAR 转导的成熟 T 淋巴细胞治疗 B 细胞谱系恶性肿瘤。CD19 是免疫治疗的一个有吸引力的靶点，因为它在大多数 B 淋巴细胞起源的成熟和恶性肿瘤阶段持续且特异性表达，但不在造血干细胞上表达。针对 CAR 分子胞外结构域的抗体（抗-Fab、Fc 或独特型）已用于通过流式细胞术检测研究和临床样本中的 CAR 表达，但可能需要针对每个构建体进行开发，并在来自异种移植模型的样本中存在显著的背景。
开发了一种用于检测抗 CD19 CAR 表达的特异性试剂，该试剂是由人 CD19 胞外结构域和人 IgG1 Fc 区组成的融合蛋白（CD19sIg）。通过将人 CD19 cDNA 的外显子 1 至 3（CD19sIg1-3）或外显子 1 至 4（CD19sIg1-4）融合到人 IgG1Fc 片段中，构建编码 CD19sIg 融合蛋白的基因。这些融合蛋白的作用方式与用于鉴定抗原特异性 T 细胞的 MHC 四聚体类似，并且在研究携带抗 CD19 CAR 的细胞的激活方面也可能具有其他应用。通过稳定的慢病毒载体转导 293T 细胞生产 CD19sIg 蛋白，并从培养基中纯化。
使用几种不同的抗 CD19 单克隆抗体进行 ELISA 检测，证明融合分子 CD19sIg1-4 具有剂量相关的特异性结合，但 CD19sIg1-3 没有结合。将 CD19sIg1-4 融合蛋白与 Alexa Fluor 488 缀合，允许对携带抗 CD19 CAR 的细胞进行流式细胞术检测的特异性和灵敏性染色，可检测到低至 0.5%的 CAR 修饰的原代细胞，背景染色最小。
该融合分子是一种灵敏的试剂，可用于检测任何存在于 CAR 修饰的 T 细胞中的抗 CD19 CAR 衍生的单克隆抗体。
