Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA
Department of Bioengineering, Rice University, Houston, TX, USA
Haematologica. 2021 Mar 1;106(3):847-858. doi: 10.3324/haematol.2019.233882.
Allogeneic hematopoietic stem cell transplantation is an effective therapy for high-risk leukemias. In children, graft manipulation based on the selective removal of aβ T cells and B cells has been shown to reduce the risk of acute and chronic graft-versus-host disease, thus allowing the use of haploidentical donors which expands the population of recipients in whom allogeneic hematopoietic stem cell transplantation can be used. Leukemic relapse, however, remains a challenge. T cells expressing chimeric antigen receptors can potently eliminate leukemia, including those in the central nervous system. We hypothesized that by engineering the donor aβ T cells that are removed from the graft by genome editing to express a CD19-specific chimeric antigen receptor, while simultaneously inactivating the T-cell receptor, we could create a therapy that enhances the anti-leukemic efficacy of the stem cell transplant without increasing the risk of graft-versus-host disease. Using genome editing with Cas9 ribonucleoprotein and adeno-associated virus serotype 6, we integrated a CD19-specific chimeric antigen receptor inframe into the TRAC locus. More than 90% of cells lost T-cell receptor expression, while >75% expressed the chimeric antigen receptor. The initial product was further purified with less than 0.05% T-cell receptorpositive cells remaining. In vitro, the chimeric antigen receptor T cells efficiently eliminated target cells and produced high cytokine levels when challenged with CD19+ leukemia cells. In vivo, the gene-modified T cells eliminated leukemia without causing graft-versus-host disease in a xenograft model. Gene editing was highly specific with no evidence of off-target effects. These data support the concept that the addition of aβ T-cell-derived, genome-edited T cells expressing CD19-specific chimeric antigen receptors could enhance the anti-leukemic efficacy of aβ T-celldepleted haploidentical hematopoietic stem cell transplantation without increasing the risk of graft-versus-host disease.
异基因造血干细胞移植是治疗高危白血病的有效方法。在儿童中，基于选择性去除αβ T 细胞和 B 细胞的移植物处理已被证明可降低急性和慢性移植物抗宿主病的风险，从而允许使用半相合供体，从而扩大可接受异基因造血干细胞移植的受者人群。然而，白血病复发仍然是一个挑战。表达嵌合抗原受体的 T 细胞可以有效地消除白血病，包括中枢神经系统中的白血病。我们假设通过对从移植物中去除的供体αβ T 细胞进行基因组编辑，使其表达 CD19 特异性嵌合抗原受体，同时使 T 细胞受体失活，我们可以创建一种治疗方法，在不增加移植物抗宿主病风险的情况下增强干细胞移植的抗白血病疗效。我们使用 Cas9 核糖核蛋白和腺相关病毒血清型 6 进行基因组编辑，将 CD19 特异性嵌合抗原受体整入 TRAC 基因座。超过 90%的细胞失去 T 细胞受体表达，而超过 75%的细胞表达嵌合抗原受体。初始产物进一步用少于 0.05%的 TCR 阳性细胞进行纯化。在体外，嵌合抗原受体 T 细胞有效地消除了靶细胞，并在受到 CD19+白血病细胞挑战时产生了高水平的细胞因子。在体内，基因修饰的 T 细胞在异种移植模型中消除了白血病，而没有引起移植物抗宿主病。基因编辑具有高度特异性，没有证据表明存在脱靶效应。这些数据支持这样的概念，即添加源自αβ T 细胞的、经基因组编辑表达 CD19 特异性嵌合抗原受体的 T 细胞可以增强αβ T 细胞耗尽的半相合造血干细胞移植的抗白血病疗效，而不增加移植物抗宿主病的风险。
