Improved Chimeric Antigen Receptor Therapies B-cell Malignancies

在过去的一年中，该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组成部分组成，抗原识别片段通常来自单克隆抗体，连接抗原识别片段与跨膜部分的细胞外区域，共刺激结构域如4-1BB和CD28，以及T细胞活化结构域如CD3-zeta。我们是首批测试抗cd19 CAR - 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，报告了抗cd19 CAR - T细胞治疗晚期淋巴瘤患者的总有效率为73%。这项工作还报道了血清白细胞介素-15水平与抗淋巴瘤反应之间的重要联系。我们还报道了用抗cd19 CAR - T细胞治疗弥漫性大b细胞淋巴瘤的最长持续完全缓解。在Molecular Therapy杂志上发表的一篇论文中报道了4例完全缓解38至56个月的患者。在过去的两年中，我们已经构建了多个新的car来测试car的各个组件。T细胞通过使用γ逆转录病毒载体与各种car进行转导，并进行体外检测。其目的是找到赋予T细胞杀死癌细胞和增殖能力而不产生大量潜在毒性炎症细胞因子的car。我们发现，改变铰链和跨膜区域、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，我们发现具有CD8铰链和跨膜区域的car细胞比具有CD28铰链和跨膜区域的car细胞产生更低水平的细胞因子和激活诱导的细胞死亡。具有CD8或CD28铰链和跨膜区域的car都可以消除小鼠体内的肿瘤。这项工作已经在《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类抗cd19 CAR，目前正在临床试验中进行测试。我们已经招募了16名患者进行这项工作产生的CAR的临床试验。这种CAR被称为Hu19-CD828Z。它有铰链和CD8分子的跨膜区域。在16例接受治疗的患者中，到目前为止，15例患者的抗淋巴瘤反应可评估。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，严重神经毒性发生率降低。这项工作在2016年12月的美国血液学学会会议上进行了口头报告。我们已经完成了新的Hu19-CD828Z CAR与我们以前使用的FMC63-28Z CAR的正式比较。我们发现新的Hu19-CD828Z CAR的3级或4级临床神经毒性发生率为5%，而旧的FMC63-28Z CAR的3级或4级临床神经毒性发生率为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car。我们已经开发了3种靶向b细胞抗原CD20的功能性car。我们计划利用这些抗CD20 CAR的知识来进行抗CD20 CAR的临床试验或设计一种双特异性CAR，包括CD20结合和靶向另一种b细胞抗原的结构域。同时靶向1种以上抗原是很重要的，因为许多淋巴瘤病例不表达CD19。我们选择了一种针对CD19和CD20的最佳双频CAR结构。双频结构编码2个CAR，其中一个CAR是前面描述的Hu19-CD828Z CAR。第二种CAR是一种抗cd20 CAR，命名为Hu20-CD8BBZ。整个双链结构体被命名为Hu19-CD828-Hu20BB，该结构体由一个γ逆转录病毒载体编码。我们已经向FDA提交了一份新药研究申请（IND），以便在人体中测试用这种双频结构转导的T细胞。我们计划在2019年秋季启动用这些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.