Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor.
Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor.
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DOI：
10.1097/cji.0b013e3181ac6138
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发表时间：
2009-09
期刊：
影响因子：
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通讯作者：
Rosenberg SA
中科院分区：
文献类型：
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作者：
Kochenderfer JN;Feldman SA;Zhao Y;Xu H;Black MA;Morgan RA;Wilson WH;Rosenberg SA
T细胞可以被工程化以表达识别肿瘤相关抗原的嵌合抗原受体（汽车）的基因。我们构建并比较了两种含有识别CD 19的单链可变区部分（scFv）的汽车。一种CAR含有4-1BB分子的信号传导部分，而另一种则没有。我们选择了不含4-1BB部分的CAR用于进一步的临床前开发。我们证明了编码这种受体的γ逆转录病毒可以感染人类T细胞。抗CD 19-CAR转导的CD 8+和CD 4 + T细胞特异性响应于CD 19+靶细胞产生干扰素-γ和白细胞介素-2。转导的T细胞特异性地杀死原代慢性淋巴细胞白血病（CLL）细胞。我们从以前接受过化疗的CLL患者中转导T细胞。我们诱导这些T细胞充分增殖，为临床过继性T细胞转移提供足够的细胞，其方案包括在转导前用抗CD 3单克隆抗体（OKT 3）进行初始刺激，然后在转导后7天进行第二次OKT 3刺激。该方案通过在初始OKT 3刺激之前耗尽CD 19+细胞而成功地适用于具有高外周血白血病细胞计数的CLL患者。在准备将招募晚期B细胞恶性肿瘤患者的临床试验时，我们产生了产生编码抗CD 19 CAR的逆转录病毒的生产细胞克隆，并且我们在良好生产规范（GMP）条件下产生了用于所提议的临床试验的足够的逆转录病毒上清液。
T cells can be engineered to express the genes of chimeric antigen receptors (CARs) that recognize tumor-associated antigens. We constructed and compared two CARs that contained a single chain variable region moiety (scFv) that recognized CD19. One CAR contained the signaling moiety of the 4-1BB molecule and the other did not. We selected the CAR that did not contain the 4-1BB moiety for further pre-clinical development. We demonstrated that gammaretroviruses encoding this receptor could transduce human T cells. Anti-CD19-CAR-transduced CD8+ and CD4+ T cells produced interferon-γ and interleukin-2 specifically in response to CD19+ target cells. The transduced T cells specifically killed primary chronic lymphocytic leukemia (CLL) cells. We transduced T cells from CLL patients that had been previously treated with chemotherapy. We induced these T cells to proliferate sufficiently to provide enough cells for clinical adoptive T cell transfer with a protocol consisting of an initial stimulation with an anti-CD3 monoclonal antibody (OKT3) prior to transduction followed by a second OKT3 stimulation seven days after transduction. This protocol was successfully adapted for use in CLL patients with high peripheral blood leukemia cell counts by depleting CD19+ cells prior to the initial OKT3 stimulation. In preparation for a clinical trial that will enroll patients with advanced B cell malignancies, we generated a producer cell clone that produces retroviruses encoding the anti-CD19 CAR, and we produced sufficient retroviral supernatant for the proposed clinical trial under good manufacturing practice (GMP) conditions.