肿瘤细胞治疗研究
英文原题：GMP development and preclinical validation of CAR-T cells targeting a lytic EBV antigen for therapy of EBV-associated malignancies.
这些结果支持将用 ZT002 生成的 gp350CAR-T 细胞作为创新新药，用于治疗实体和血液 EBV 相关恶性肿瘤患者。
引言：爱泼斯坦–巴尔病毒（EBV）广泛传播，与淋巴增殖性疾病、B/T/NK细胞淋巴瘤、鼻咽癌（NPC）和胃癌（GC）相关。EBV裂解再激活可导致NPC基因组不稳定、炎症和肿瘤发生，并促进癌症进展。标准治疗无效的NPC患者生存结局很差。EBV gp350是一种包膜蛋白，可在NPC样本中于细胞内及恶性细胞膜上检测到，是T细胞靶向免疫疗法的潜在病毒抗原。既往研究已证明，靶向gp350的嵌合抗原受体（CAR）工程化T细胞对EBV阳性淋巴增殖性疾病具有活性。方法：本研究进一步推进这种针对NPC的病毒特异性CAR-T免疫疗法的临床前和非临床开发，将不同gp350 CAR设计插入慢病毒载体（LV）骨架。结果：研究选择了表达scFv 7A1-抗gp350、并包含CD8跨膜结构域及CD28/CD3信号结构域的构建体ZT002。在无血清培养基中使用HEK 293T/17悬浮细胞，按照药品生产质量管理规范（GMP）开展大规模生产，获得高滴度ZT002（约1×10⁸ TU/ml）。LV感染复数（MOI）为1时，可获得较高比例的功能性gp350 CAR阳性T细胞（>70%），且基因组载体拷贝数较低（<2）。因此，研究采用ZT002建立gp350 CAR-T批次生产方法。多批次GMP规模化生产及T细胞转导、扩增验证显示，每批平均获得3×10⁹个gp350 CAR-T细胞。超过80%的CD3⁺ gp350 CAR-T细胞可结合纯化gp350蛋白。体外细胞毒性和细胞因子分泌实验（IFN和TNF）证实，gp350 CAR-T细胞可特异性识别表达gp350的NPC、GC和淋巴瘤靶细胞。研究将表达gp350的EBV阳性NPC细胞系C666.1皮下注射至免疫缺陷B-NDG小鼠（NOD.CB17-PrkdcscidIl2rgtm1/Bcgen），随后给予递增剂量gp350 CAR-T细胞或未转导T细胞治疗。gp350 CAR-T细胞诱导抗肿瘤应答，可分布至多个组织、浸润肿瘤并排斥gp350阳性肿瘤细胞。讨论：这些结果支持将采用ZT002制备的gp350 CAR-T细胞作为创新药物，用于治疗实体及液体EBV相关恶性肿瘤患者。
INTRODUCTION: Epstein-Barr virus (EBV) is a widely spread pathogen associated with lymphoproliferative diseases, B/ T/ NK cell lymphomas, nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). EBV lytic reactivations contribute to the genomic instability, inflammation and tumorigenesis of NPC, promoting cancer progression. Patients with NPC refractory to standard therapies show dismal survival. EBV gp350 is an envelope protein detectable in NPC specimens intracellularly and on the cell membrane of malignant cells, and is a potential viral antigen for T cell-directed immunotherapies. The potency of T cells engineered with a chimeric antigen receptor (CAR) targeting gp350 against EBV + lymphoproliferative disease was previously shown. METHODS: Here, we advanced towards preclinical and non-clinical developments of this virus-specific CAR-T cell immunotherapy against NPC. Different gp350CAR designs were inserted into a lentiviral vector (LV) backbone. RESULTS: A construct expressing the scFv 7A1-anti-gp350 incorporating the CD8 transmembrane and CD28.CD3 signaling domain (ZT002) was selected. High titer ZT002 (~1x10 8 TU/ml) was manufactured in HEK 293T/17 suspension cells in serum free media as large-scale production under good manufacturing practices (GMP). A LV multiplicity of infection (MOI) of 1 resulted in high frequencies of functional gp350CAR + T cells (>70%) at a low (<2) vector copy numbers in the genome. ZT002 was therefore used to establish gp350CAR-T batch run production methods. GMP upscaling and validation of T cell transduction and expansion in several runs resulted in average 3x10 9 gp350CAR-T cells per batch. >80% CD3 + gp350CAR-T cells bound to purified gp350 protein. In vitro cytotoxicity and cytokine secretion assays (IFN- and TNF- ) confirmed the specificity of gp350CAR-T cells against gp350 + NPC, GC and lymphoma cell targets. Immunocompromised B-NDG mice (NOD.CB17- PrkdcscidIl2rgtm1 /Bcgen) were challenged s.c. with a EBV + NPC C666.1 cell line expressing gp350 and then treated with escalating doses of gp350CAR-T cells or with non-transduced T cells. gp350CAR-T cells promoted antitumor responses, bio-distributed in several tissues, infiltrated in tumors and rejected gp350 + tumor cells. DISCUSSION: These results support the use of gp350CAR-T cells generated with ZT002 as an Innovative New Drug to treat patients with solid and liquid EBV-associated malignancies.
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