Autologous T cells Transduced with an Anti-CD19 Chimeric Antigen Receptor

该项目结合了对自体抗CD19嵌合抗原受体转导T细胞输注的临床试验和对从接受嵌合抗原受体转导T细胞输注的患者获得的细胞进行的实验室实验。2010年期间，该项目在《血液》杂志上发表了一篇题为《B系细胞的根除和淋巴瘤的消退》的文章，该患者接受了识别CD19的自体T细胞的基因工程治疗。该项目还产生了两份摘要，在主要国际会议上提交。本节的其余部分总结了该项目。对化疗耐药的B细胞恶性肿瘤需要新的治疗方法。基因工程T细胞过继转移表达嵌合抗原受体(CARS)，特异性识别B细胞抗原CD19，是治疗B细胞恶性肿瘤的一种新方法。我们正在进行一项临床试验，患者接受自体T细胞的输注，这些T细胞被编码抗CD19 CAR的伽马逆转录病毒转导。CAR由抗CD19抗体的可变区、CD28分子的一部分和CD3-Zeta分子的一部分组成。我们的临床方案包括环磷酰胺加氟达拉滨化疗，然后输注抗CD19转导的T细胞和一个疗程的大剂量IL-2。在这次临床试验中，我们已经治疗了8名患者。其中4例为慢性淋巴细胞白血病(CLL)，4例为B细胞淋巴瘤。为所有患者生产了抗CD19-CAR转导的T细胞，该T细胞特异性地识别CD19表达的靶细胞。每名患者接受的细胞总数从每公斤体重0.5x10e7到5.5x10e7不等。平均54%的受试细胞表达抗CD19 CAR。1例慢性淋巴细胞性白血病患者在治疗15个月后完全缓解。5名患者获得部分缓解，3名患者正在进行中。1例慢性淋巴细胞性白血病患者病情稳定。一名淋巴瘤患者死于培养证实的甲型流感肺炎，无法评估淋巴瘤反应。8例患者中有4例CD19+B细胞明显耗尽。这种B细胞耗竭持续了长达15个月，并且在4名患者中有3名正在进行。由于B细胞耗竭的持续时间较长，不能归因于患者接受的化疗。例如，一名滤泡性淋巴瘤患者在按照我们的方案进行治疗之前，多克隆血B细胞水平正常。治疗6个月后，他的血液B细胞计数为每微升1个(正常范围为每微升61-321个B细胞)。1例慢性淋巴细胞性白血病患者在化疗和接受CAR转导的T细胞治疗后的头32天内腺病消退。定量聚合酶链式反应检测8例患者血液中均可检测到CAR转导细胞。外周血单个核细胞(PBMC)中含有CAR基因的百分比差异很大，但有2例患者在输注90天后超过0.1%的PBMC中检测到CAR基因。在输注后的早期时间点，表达CAR的T细胞占所有血液T细胞的20%。患者在CAR转导的T细胞输注后的前10天内有显著的毒性。最突出的毒性是低血压。定量聚合酶链式反应检测8例患者血液中均可检测到CAR转导细胞。外周血单个核细胞(PBMC)中含有CAR基因的百分比差异很大，但有2例患者在输注90天后超过0.1%的PBMC中检测到CAR基因。在输注后的早期时间点，表达CAR的T细胞占所有血液T细胞的20%。患者在CAR转导的T细胞输注后的前10天内有显著的毒性。最突出的毒性是降压。这些结果表明，表达CAR的T细胞可以特异性地清除靶细胞，并在人体内引起显著的细胞因子介导的毒性。

This project is a combination of a clinical trial of infusions of autologous anti-CD19 chimeric-antigen-receptor-transduced T cells and laboratory experiments performed on cells obtained from patients that received infustions of the chimeric-antigen-receptor-transduced T cells. During 2010 this project resulted in one publication in Blood entitled "Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically-engineered to recognize CD19". The project also resulted in 2 abstracts that were presented at major international meetings. The rest of this section summarizes the project. New therapies are needed for chemotherapy-resistant B-cell malignancies. Adoptive transfer of T cells genetically-engineered to express chimeric antigen receptors (CARs) that specifically recognize the B-cell antigen CD19 is a promising new approach for treating B-cell malignancies. We are conducting a clinical trial in which patients receive infusions of autologous T cells that are transduced with gamma-retroviruses encoding an anti-CD19 CAR. The CAR is made up of the variable regions of an anti-CD19 antibody, a portion of the CD28 molecule, and a portion of the CD3-zeta molecule. Our clinical protocol consists of cyclophosphamide plus fludarabine chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of high-dose IL-2. We have treated 8 patients on this clinical trial. Four of the patients had chronic lymphocytic leukemia (CLL), and 4 patients had B-cell lymphoma. Anti-CD19-CAR-transduced T cells that specifically recognized CD19-expressing target cells were produced for all patients. The total number of cells administered to each patient ranged from 0.5x10e7 to 5.5x10e7 cells per kg of bodyweight. A mean of 54 percent of the administered cells expressed the anti-CD19 CAR. One patient with CLL obtained a complete remission that is ongoing 15 months after treatment. Five patients obtained partial remissions that are ongoing in 3 patients. One patient with CLL had stable disease. One patient with lymphoma died of culture-proven influenza A pneumonia and is not evaluable for lymphoma response. A striking depletion of CD19+ B-lineage cells occurred in 4 of 8 patients. This B-cell depletion lasted for up to 15 months, and it is ongoing in 3 of 4 patients. Because of the long duration of B-cell depletion, it cannot be attributed to the chemotherapy that the patients received. For example, a patient with follicular lymphoma had a normal level of polyclonal blood B cells before treatment on our protocol. Six months after treatment, he had a blood B cell count of 1 per microliter (normal range 61-321 B cells per microliter). A patient with CLL had a regression of adenopathy in the first 32 days after chemotherapy and CAR-transduced T cell administration. CAR-transduced cells were detected in the blood of all 8 patients by quantitative PCR. The percentage of peripheral blood mononuclear cells (PBMC) containing the CAR gene varied widely, but in 2 patients the CAR gene was detected in greater than 0.1 percent of PBMC more than 90 days after infusion. At early time-points after infusion, CAR-expressing T cells constituted up to 20 percent of all blood T cells. Patients had significant toxicity during the first 10 days after CAR-transduced T cell infusion. The most prominent toxicity was hypotension. CAR-transduced cells were detected in the blood of all 8 patients by quantitative PCR. The percentage of peripheral blood mononuclear cells (PBMC) containing the CAR gene varied widely, but in 2 patients the CAR gene was detected in greater than 0.1 percent of PBMC more than 90 days after infusion. At early time-points after infusion, CAR-expressing T cells constituted up to 20 percent of all blood T cells. Patients had significant toxicity during the first 10 days after CAR-transduced T cell infusion. The most prominent toxicity was hypotension.These results demonstrate that CAR-expressing T cells can specifically eliminate targeted cells and cause significant cytokine-mediated toxicity in humans.