利用“睡美人”转座子将嵌合抗原受体酪氨酸激酶样孤儿...

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Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations

Abstract

T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.

利用“睡美人”转座子将嵌合抗原受体酪氨酸激酶样孤儿受体1（ROR1）整合到各种记忆T细胞群

摘要:

靶向CD19的嵌合抗原受体修饰的T细胞证明了其对B细胞淋巴肿瘤的杀伤活性。然而，这经常伴随着正常 CD19 阳性B细胞和体液免疫的丢失。受体酪氨酸激酶样孤儿受体（ROR1）在B细胞恶性肿瘤和实体瘤亚群表面表达，但不在正常的B细胞或正常产后组织上表达。因此，特异性靶向ROR1的过继T细胞治疗有消除肿瘤细胞和不损伤正常组织的能力。为了检验这一假说，我们开发了靶向ROR1的CAR-T细胞来对抗恶性细胞。由2代ROR1-特异性CARs信号区域连接CD3ζ和CD28（设计成ROR1RCD28）或者CD137（设计成ROR1RCD137）组成两个“睡美人”转座子，并将其转导到T细胞中。我们通过使用γ射线激活以及和繁殖细胞(AaPC)共培养来筛选共表达ROR1和共刺激分子CAR的T细胞。在可溶性白细胞介素IL-2和IL-21存在的条件下，超过1个月和AaPC共培养，对细胞进行扩增，然后通过非酶数字阵列测量（NanoString）和多面板流式细胞仪检测不同记忆表型的CAR-T细胞。这种T细胞产生干扰素γ并且对ROR1阳性肿瘤具有特殊的活性。此外，这些细胞可以消除ROR1 +肿瘤异种移植，特别是表达ROR1RCD137的T细胞。临床试验将探讨ROR1特异性CAR- T细胞特异性杀伤肿瘤细胞的同时，保持正常的B细胞群的能力。

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