T-cell Therapy for B-lineage Acute Lymphoblastic Leukemia

描述（由申请人提供）：这项修订后的R01拨款解决了异基因造血干细胞移植（HSCT）后b系急性淋巴细胞白血病（B-ALL）复发的问题。我们假设异体造血干细胞移植后癌症复发的发生率可以通过移植后B-ALL最小残留病（MRD）来降低，通过对供体来源的T细胞进行基因修饰，使其特异性针对CD19。为了巩固HSCT，我们设计了下一代嵌合抗原受体（CAR），命名为CD19RCD28，将T细胞的特异性转移到b细胞谱系限制性细胞表面分子CD19上，而不依赖于主要组织相容性复合体（MHC）。基因修饰的CD19RCD28+ T细胞通过嵌合CD28和CD3-6裂解B-ALL，以car调控的方式上调IL-2和抗凋亡基因的产生。睡美人（SB）系统与电穿孔相结合，引入CAR以及共表达HSV-1胸苷激酶（TK），用于正电子发射断层扫描（PET）成像。Aim #1的研究现在将评估是否可以开发出一种全人类cd19特异性CAR (hCD19RCD28)，该CAR通过SB转位基因修饰的T细胞中的cd19依赖性杀伤、细胞因子产生和持续增殖来提供完全有效的激活信号。采用一种弥散性b系肿瘤的异种小鼠模型，利用无创生物发光成像（BLI）和<PET纵向评估注入CAR+TK+细胞的持久性和抗肿瘤效果，以确定过继治疗的可行性和安全性。目的2将评估高风险CD19+ B-ALL的同种异体造血干细胞移植后，输注逐渐增加剂量的供体来源的hCD19RCD28+ T细胞（有/没有TK表达）的安全性、可行性和持久性。表达TK的T细胞将通过PET成像。如果有必要，更昔洛韦（GCV）将给予TK+细胞在严重毒性事件的条件消融。Aim #3中的相关研究将通过对连续获得的标本进行载体特异性Q-PCR和TCR光谱分型分析，描述在规定的T细胞剂量水平下转移的T细胞的大小和持久性。其他相关研究将评估过继移植T细胞到骨髓样本（BM）的运输以及移植T细胞在MRD解剖部位的功能状态。使用2'-脱氧-20-[18F]fluoro-5-乙基-1-2- d -arabinofuranosyluracil （[18F]- feau）在输注的CAR+ T细胞中共表达TK代谢/捕获的人PET成像，将用于评估过继转移T细胞的分布。总的来说，这些研究的结果将促进用供体来源的cd19特异性T细胞靶向hsct后MRD的发展，以提高B-ALL患者的无病生存期。摘要：我们将在移植后注入cd19特异性T细胞以提高急性淋巴细胞白血病患者的生存率。

DESCRIPTION (provided by applicant): This revised R01 grant addresses the problem of relapse of B-lineage acute lymphoblastic leukemia (B-ALL) after allogeneic hematopoietic stem-cell transplantation (HSCT). We hypothesize that the incidence of cancer relapse following allogeneic HSCT can be reduced by targeting post-transplant B-ALL minimal residual disease (MRD) with adoptively transferred donor-derived T cells genetically modified to be specific for CD19. To consolidate HSCT, we have designed a next-generation chimeric antigen receptor (CAR), designated CD19RCD28, to redirect specificity of T cells to the B-cell lineage-restricted cell-surface molecule CD19 independent of major histocompatibility complex (MHC). Genetically modified CD19RCD28+ T cells activated through chimeric CD28 and CD3-6 lyse B-ALL, upregulate production of IL-2 and anti-apoptotic genes, in a CAR-regulated manner. The Sleeping Beauty (SB) system has been combined with electroporation to introduce the CAR as well as co-express HSV-1 thymidine kinase (TK) for imaging by positron emission tomography (PET). The studies in Aim #1 will now evaluate whether an all-human CD19-specific CAR can be developed (hCD19RCD28) that provides a fully-competent activation signal as determined by CD19-dependent killing, cytokine production, and sustained proliferation in T cells that have been genetically modified by SB transposition. A xenogeneic mouse model of disseminated B-lineage tumor will be used to ascertain the feasibility and safety of adoptive therapy using non-invasive bioluminescent imaging (BLI) and <PET to longitudinally asses the persistence of the infused CAR+TK+ cells and the anti-tumor effect. Aim #2 will evaluate the safety, feasibility and persistence, of infusing escalating doses of donor-derived hCD19RCD28+ T cells with/without TK expression, after allogeneic HSCT for high-risk CD19+ B-ALL. T cells expressing TK will be imaged by PET. If necessary, ganciclovir (GCV) will be given for conditional ablation of TK+ cells in the event of serious toxicity. Correlative studies in Aim #3 will delineate the magnitude and persistence of transferred T cells at the prescribed T-cell Dose Levels using vector-specific Q-PCR and TCR spectratyping analyses on serially acquired specimens. Other correlative studies will evaluate the trafficking to sampled bone marrow (BM) of adoptively transferred T cells and the functional status of transferred T cells in this anatomic site of MRD. Human PET imaging using 2'-Deoxy-20-[18F]fluoro-5-ethyl-1-2-D-arabinofuranosyluracil ([18F]-FEAU) metabolized/trapped by TK co-expressed in infused CAR+ T cells, will be used to evaluate the distribution of adoptively transferred T cells. In aggregate, the results of the studies will facilitate the evolution of targeting post-HSCT MRD with donor-derived CD19-specific T cells for enhanced disease-free survival of patients with B-ALL. LAY SUMMARY: We will infuse CD19-specific T cells after transplantation to improve survival for patients with acute lymphoblastic leukemia.