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

该项目结合了输注自体抗cd19嵌合抗原受体转导T细胞的临床试验，以及对从接受了嵌合抗原受体转导T细胞输注的患者身上获得的细胞进行的实验室实验。2010年，该项目在《Blood》杂志上发表了一篇题为“用基因工程改造的识别CD19的自体T细胞治疗患者的b系细胞根除和淋巴瘤消退”的论文。该项目还产生了两份在主要国际会议上提出的摘要。本节的其余部分总结了该项目。化疗耐药的b细胞恶性肿瘤需要新的治疗方法。表达特异性识别b细胞抗原CD19的嵌合抗原受体（CARs）的基因工程T细胞过继转移是治疗b细胞恶性肿瘤的一种有前途的新方法。我们正在进行一项临床试验，患者接受输注自体T细胞，这些细胞被编码抗cd19 CAR的γ -逆转录病毒转导。CAR由抗cd19抗体的可变区域、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，随后输注抗cd19 - car转导的T细胞和一个疗程的高剂量IL-2。我们在这项临床试验中治疗了8名患者。慢性淋巴细胞白血病（CLL） 4例，b细胞淋巴瘤4例。所有患者都产生了特异性识别表达cd19的靶细胞的抗cd19 car转导T细胞。给每个患者的细胞总数为每公斤体重0.5x10e7至5.5x10e7个细胞。平均54%的给药细胞表达抗cd19 CAR。一名CLL患者在治疗15个月后获得完全缓解。5名患者获得部分缓解，3名患者正在进行中。1例CLL患者病情稳定。一名淋巴瘤患者死于经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例出现了CD19+ b系细胞的显著缺失。这种b细胞耗竭持续了15个月，并且在4例患者中有3例持续。由于b细胞耗竭的持续时间较长，这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接受我们的治疗方案之前，其多克隆血B细胞水平正常。治疗6个月后，患者血B细胞计数为1 /微升（正常范围为61-321 /微升）。一名CLL患者在化疗和car -转导T细胞治疗后32天内腺病消退。通过定量PCR检测8例患者血液中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，但在2例患者中，在输注后超过90天，外周血单个核细胞中检测到CAR基因的比例超过0.1%。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%。在car -转导T细胞输注后的前10天，患者出现了明显的毒性。最突出的毒性是低血压。通过定量PCR检测8例患者血液中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，但在2例患者中，在输注后超过90天，外周血单个核细胞中检测到CAR基因的比例超过0.1%。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%。在car -转导T细胞输注后的前10天，患者出现了明显的毒性。最突出的毒性是低血压。这些结果表明，表达car的T细胞可以特异性地消除靶细胞，并在人体中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.