Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of Medicine, 866 Yuhangtang Road, Hangzhou 310058, China; Liangzhu Laboratory, Zhejiang University Medical Center, 1369 West Wenyi Road, Hangzhou 311121, China; Institute of Hematology, Zhejiang University and Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou 310058, Zhejiang, China.
Department of Bioengineering, Institute of Engineering in Medicine, University of California, San Diego, San Diego, CA 92093, USA.
Mol Ther. 2023 Jan 4;31(1):35-47. doi: 10.1016/j.ymthe.2022.08.018. Epub 2022 Aug 31.
CD19-targeting chimeric antigen receptors (CARs) with CD28 and CD3ζ signaling domains have been approved by the US FDA for treating B cell malignancies. Mutation of immunoreceptor tyrosine-based activation motifs (ITAMs) in CD3ζ generated a single-ITAM containing 1XX CAR, which displayed superior antitumor activity in a leukemia mouse model. Here, we investigated whether the 1XX design could enhance therapeutic potency against solid tumors. We constructed both CD19- and AXL-specific 1XX CARs and compared their in vitro and in vivo functions with their wild-type (WT) counterparts. 1XX CARs showed better antitumor efficacy in both pancreatic and melanoma mouse models. Detailed analysis revealed that 1XX CAR-T cells persisted longer in vivo and had a higher percentage of central memory cells. With fluorescence resonance energy transfer (FRET)-based biosensors, we found that decreased ITAM numbers in 1XX resulted in similar 70-kDa zeta chain-associated protein (ZAP70) activation, while 1XX induced higher Ca elevation and faster extracellular signal-regulated kinase (Erk) activation than WT CAR. Thus, our results confirmed the superiority of 1XX against two targets in different solid tumor models and shed light on the underlying molecular mechanism of CAR signaling, paving the way for the clinical applications of 1XX CARs against solid tumors.
CD19 靶向嵌合抗原受体 (CAR) 与 CD28 和 CD3ζ 信号域已被美国 FDA 批准用于治疗 B 细胞恶性肿瘤。CD3ζ 中的免疫受体酪氨酸激活基序 (ITAM) 的突变产生了一个含有 1XX 的 CAR，在白血病小鼠模型中显示出优异的抗肿瘤活性。在这里，我们研究了 1XX 设计是否可以增强对实体瘤的治疗效力。我们构建了 CD19 和 AXL 特异性的 1XX CAR，并将其与野生型 (WT) 进行了比较。1XX CAR 在胰腺和黑色素瘤小鼠模型中均显示出更好的抗肿瘤疗效。详细分析表明，1XX CAR-T 细胞在体内的持久性更长，且中央记忆细胞的比例更高。通过荧光共振能量转移 (FRET) 基的生物传感器，我们发现 1XX 中 ITAM 数量的减少导致类似的 70-kDa zeta 链相关蛋白 (ZAP70) 激活，而 1XX 诱导的 Ca 升高和细胞外信号调节激酶 (Erk) 激活速度高于 WT CAR。因此，我们的结果证实了 1XX 在两种不同的实体瘤模型中的优越性，并阐明了 CAR 信号的潜在分子机制，为 1XX CAR 针对实体瘤的临床应用铺平了道路。
