Cellular Immunotherapy Program, Cancer Center, Massachusetts General Hospital, Boston, Massachusetts, USA.
Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.
J Immunother Cancer. 2020 Sep;8(2). doi: 10.1136/jitc-2020-000990.
Adoptive cell therapy with chimeric antigen receptor T cells (CAR-T) has become a standard treatment for patients with certain aggressive B cell malignancies and holds promise to improve the care of patients suffering from numerous other cancers in the future. However, the high manufacturing cost of CAR-T cell therapies poses a major barrier to their broader clinical application. Among the key cost drivers of CAR-T production are single-use reagents for T cell activation and clinical-grade viral vector. The presence of variable amounts of contaminating monocytes in the starting material poses an additional challenge to CAR-T manufacturing, since they can impede T cell stimulation and transduction, resulting in manufacturing failure.
We created K562-based artificial antigen-presenting cells (aAPC) with genetically encoded T cell stimulation and costimulation that represent an inexhaustible source for T cell activation. We additionally disrupted endogenous expression of the low-density lipoprotein receptor (LDLR) on these aAPC (aAPC-ΔLDLR) using CRISPR-Cas9 gene editing nucleases to prevent inadvertent lentiviral transduction and avoid the sink effect on viral vector during transduction. Using various T cell sources, we produced CD19-directed CAR-T cells via aAPC-ΔLDLR-based activation and tested their in vitro and in vivo antitumor potency against B cell malignancies.
We found that lack of LDLR expression on our aAPC-ΔLDLR conferred resistance to lentiviral transduction during CAR-T production. Using aAPC-ΔLDLR, we achieved efficient expansion of CAR-T cells even from unpurified starting material like peripheral blood mononuclear cells or unmanipulated leukapheresis product, containing substantial proportions of monocytes. CD19-directed CAR-T cells that we produced via aAPC-ΔLDLR-based expansion demonstrated potent antitumor responses in preclinical models of acute lymphoblastic leukemia and B-cell lymphoma.
Our aAPC-ΔLDLR represent an attractive approach for manufacturing of lentivirally transduced T cells that may be simpler and more cost efficient than currently available methods.
嵌合抗原受体 T 细胞（CAR-T）的过继细胞疗法已成为某些侵袭性 B 细胞恶性肿瘤的标准治疗方法，有望在未来改善众多其他癌症患者的治疗效果。然而，CAR-T 细胞疗法的高制造成本是其广泛临床应用的主要障碍。CAR-T 生产的关键成本驱动因素包括 T 细胞激活的即用型试剂和临床级别的病毒载体。起始材料中存在数量不定的污染单核细胞，给 CAR-T 制造带来了额外的挑战，因为它们会阻碍 T 细胞的刺激和转导，导致制造失败。
我们使用基因编码的 T 细胞刺激和共刺激创建了基于 K562 的人工抗原呈递细胞（aAPC），这是 T 细胞激活的无尽来源。我们还使用 CRISPR-Cas9 基因编辑核酸酶破坏这些 aAPC 上内源性低密度脂蛋白受体（LDLR）的表达（aAPC-ΔLDLR），以防止无意中的慢病毒转导，并避免转导过程中对病毒载体的吸收效应。我们使用各种 T 细胞来源，通过 aAPC-ΔLDLR 激活生产 CD19 定向 CAR-T 细胞，并测试了它们针对 B 细胞恶性肿瘤的体外和体内抗肿瘤效力。
我们发现，我们的 aAPC-ΔLDLR 上 LDLR 表达的缺失赋予了 CAR-T 生产过程中对慢病毒转导的抗性。使用 aAPC-ΔLDLR，即使从外周血单核细胞或未经处理的白细胞分离物等未纯化的起始材料中，我们也能实现 CAR-T 细胞的高效扩增，其中含有相当比例的单核细胞。我们通过 aAPC-ΔLDLR 扩增生产的 CD19 定向 CAR-T 细胞在急性淋巴细胞白血病和 B 细胞淋巴瘤的临床前模型中表现出强大的抗肿瘤反应。
我们的 aAPC-ΔLDLR 为制造慢病毒转导的 T 细胞提供了一种有吸引力的方法，与目前可用的方法相比，这种方法可能更简单、更具成本效益。
