Improved Chimeric Antigen Receptor Therapies B-cell Malignancies

在过去的一年里，这个项目包括测试可能影响嵌合抗原受体(CARS)功能的多种因素。嵌合抗原受体由几个部分组成，通常来源于单抗的抗原识别部分，连接抗原识别部分和跨膜部分的胞外区，共刺激结构域，如4-1BB和CD28，以及T细胞激活结构域，如CD3-Zeta。我们是第一批测试抗CD19汽车T细胞的研究人员之一。在过去的一年里，我们在临床肿瘤学杂志上发表了重要的研究结果，报告了晚期淋巴瘤患者接受抗CD19 CAR T细胞治疗的总有效率为73%。这项研究还报道了血清白介素15水平和抗淋巴瘤反应之间的重要关联。我们还报道了用抗CD19 CAR T细胞治疗的弥漫性大B细胞淋巴瘤持续缓解时间最长的病例。在分子治疗杂志上发表的一篇论文中报道了4名完全缓解38至56个月的患者。在过去的两年里，我们制造了多辆新车来测试汽车的各种部件。用不同的CARS通过伽玛逆转录病毒载体转导T细胞，并进行体外检测。其目的是找到能够赋予T细胞杀死癌细胞并在不产生大量潜在有毒炎症细胞因子的情况下进行增殖的CAR。我们发现，改变铰链区和跨膜区、共刺激结构域或T细胞激活结构域都会导致CAR功能的深刻差异。经过广泛的体外测试，我们发现带有CD8铰链和跨膜区的CARS比带有CD28铰链和跨膜区的CARS产生的细胞因子和激活诱导的细胞死亡水平更低。带有CD8或CD28铰链和跨膜区的CARS都可以消除小鼠的肿瘤。这项工作在《分子疗法》杂志上发表了一篇论文。这项工作还导致了一种新的全人抗CD19汽车，目前正在进行临床试验。我们已经招募了16名患者参加这项工作产生的CAR的临床试验。这款车的名字是Hu19-CD828Z。它有CD8分子的铰链区和跨膜区。在16名接受治疗的患者中，到目前为止有15名患者的抗淋巴瘤反应是可评估的。总有效率为73%，完全缓解率为47%。一个值得注意的初步发现是，与之前的抗CD19 CAR临床试验相比，严重神经毒性的比率降低了。这项工作导致在2016年12月的美国血液病学会会议上进行了口头陈述。我们已经完成了新款Hu19-CD828Z轿车与我们之前使用的FMC63-28Z轿车的正式比较。我们发现，新的Hu19-CD828Z CAR的3级或4级临床神经毒性的发生率为5%，而旧的FMC63-28Z CAR的3级或4级临床神经毒性的发生率为50%。我们发现，表达Hu19-CD828Z的T细胞比表达FMC63-28Z的T细胞产生的细胞因子水平更低。该项目的另一个方面是开发针对CD19以外的B细胞抗原的CARS。我们已经开发了3种针对B细胞抗原CD20的功能汽车。我们计划利用这一抗CD20 CAR的知识来进行抗CD20 CAR的临床试验，或者设计一种包括CD20结合和针对另一种B细胞抗原的区域的双特异性CAR。同时靶向一种以上的抗原很重要，因为许多淋巴瘤病例不表达CD19。我们已经选择了一种针对CD19和CD20的最佳双顺反子汽车结构。双顺反子结构编码2辆车，其中一辆车是前面描述的Hu19-CD828Z车。第二辆车是一辆名为Hu20-CD8BBZ的反CD20车。整个双顺反子构建体命名为Hu19-CD828-Hu20BB，该构建体由伽玛逆转录病毒载体编码。我们已经向FDA提交了一份研究新药申请(IND)，以便在人类中测试用这种双顺反子结构转导的T细胞。我们计划在2019年秋季启动一项用这些T细胞治疗淋巴瘤患者的临床试验。

In the past year, this project has consisted of testing multiple factors that could affect the function of chimeric antigen receptors (CARs). Chimeric antigen receptors consist of several components, the antigen-recognition moiety that is usually derived from a monoclonal antibody, a extracellular region that connects the antigen-recognition moiety to the transmembrane portion, costimulatory domains such as 4-1BB and CD28, and T cell activation domains such as CD3-zeta. We were among the first investigators to test anti-CD19 CAR T cells. In the past year, we published important results in the Journal of Clinical Oncology reporting a 73% overall response rate when patients with advanced lymphoma were treated with anti-CD19 CAR T cells. This same work also reported the important association between serum interleukin-15 levels and anti-lymphoma responses. We also reported the longest continuous complete remissions of diffuse large B-cell lymphoma that was treated with anti-CD19 CAR T cells. Four patients with complete remissions of 38 to 56 months were reported in a paper that is in press at Molecular Therapy. We have constructed multiple new CARs over the past 2 years to test various components of CARs. T cells are transduced with the various CARs by using a gammaretroviral vector, and in vitro assays are carried out. The aim is to find CARs that impart T cells with the ability to kill cancer cells and proliferate without producing large amounts of potentially toxic inflamatory cytokines. We have found that changing the hinge and transmembrane regions, costimulatory domains, or T cell activation domains all cause profound differences in CAR function. Following extensive in vitro testing, we found that CARs with hinge and transmembrane regions from CD8 led to lower levels of cytokine production and activation-induced cell death than CARs with hinge and transmembrane regions from CD28. CARs with either CD8 or CD28 hinge and transmembrane regions could both eliminate tumors from mice. This work has resulted a paper in press at Molecular Therapy. This work has also lead to a new fully-human anti-CD19 CAR that is currently being tested in a clinical trial. We have enrolled 16 patients on a clinical trial of the CAR resulting from this work. The CAR is called Hu19-CD828Z. It has hinge and transmembrane regions from the CD8 molecule. Of 16 treated patients, 15 patients are evaluable for anti-lymphoma response so far. The overall response rate is 73% and the complete remission rate is 47%. One notable preliminary finding is a decreased rate of severe neurologic toxicity compared to previous anti-CD19 CAR clinical trials. This work resulted in an oral presentation at the American Society of Hematology meeting in December 2016. We have completed a formal comparison of the new Hu19-CD828Z CAR to our previously-used FMC63-28Z CAR. We found a 5% rate of Grade 3 or 4 clinical neurologic toxicity with teh new Hu19-CD828Z CAR versus a 50% rate of Grade 3 or 4 clinical neurologic toxicity with the older FMC63-28Z CAR. We have found lower levels of cytokine production with T cells expressing Hu19-CD828Z versus T cells expressing FMC63-28Z. Another aspect of this project is developing CARs against B-cell antigens other than CD19. We have developed 3 functional CARs that target the B-cell antigen CD20. We plan to utilize this knowledge of anti-CD20 CARs to either conduct a clinica trial of an anti-CD20 CAR or to design a bispecific CAR including CD20 binding and a domain targeting another B-cell antigen. Targeting more than 1 antigen simultaneously is important because many cases of lymphoma do not express CD19. We have selected an optimal bicistronic CAR construct that targets both CD19 and CD20. The bicistronic construct encodes 2 CARs, one of the CARs is the previously -described Hu19-CD828Z CAR. The second CAR is an anti-CD20 CAR designated Hu20-CD8BBZ. The entire bicistronic construct is designated Hu19-CD828-Hu20BB, and the construct is encoded by a gammaretroviral vector. We have submitted an Investigational New Drug Application (IND) to the FDA in order to test T cells transduced with this bicistronic construct in humans. We plan to initiate a clinical trial treating lymphoma patients with these T cells in the fall of 2019.