背景:嵌合抗原受体(CAR)T细胞治疗靶受体酪氨酸激酶样孤儿受体1(ROR1)在血液肿瘤和实体瘤中广泛表达,然而,具有单链可变片段(scFv)-R12靶向域的临床特征的ROR1-CART细胞未能诱导持久的缓解,部分是由于免疫抑制肿瘤微环境(TME)。在这里,我们描述了一种改进的ROR1-CAR的开发,全人scFv9靶向域,并增强了TGFβRIIDN装甲,可抵抗主要的TME因素,转化生长因子β(TGFβ)。
方法:通过慢病毒转导富集的CD4+和CD8+T细胞产生CART细胞,并且在体外和体内将新型的基于scFv9的ROR1-CAR-1与临床表征的基于ROR1-R12-scFv的CAR-2进行了比较。
结果:CAR-1T细胞表现出比CAR-2更高的CAR表面密度,并产生更多的干扰素(IFN)-γ肿瘤坏死因子(TNF)-α和白细胞介素(IL)-2响应于血液学(Jeko-1,RPMI-8226)和实体(OVCAR-3,Capan-2,NCI-H226)体外肿瘤细胞系。在体内,CAR-1和CAR-2均清除了血液系统Jeko-1淋巴瘤异种移植物,然而,只有CAR-1完全拒绝卵巢实体OVCAR-3肿瘤,与CD8+和CD4+CART细胞的更大扩增相一致,并富集中枢和效应记忆表型。当配备TGFβ防护装甲TGFβRIIDN时,CAR-1T细胞抵抗TGFβ介导的pSmad2/3磷酸化,与单独的CAR-1相比。当与ROR-1+AsPC-1胰腺癌细胞系在TGFβ1的存在下共培养时,装甲CAR-1表现出改善的杀伤功能恢复,IFN-γ,TNF-α和IL-2分泌。与单独的CAR-1相比,在过表达TGFβ1的小鼠AsPC-1胰腺肿瘤异种移植物中,装甲的CAR-1,实现了完整的肿瘤缓解,加速了CAR+T细胞的扩增,循环活性TGFβ1减少,无明显毒性或体重减轻。出乎意料的是,在无TGFβ过表达的AsPC-1异种移植物中,ROR-1-CART细胞与ROR-1阳性肿瘤细胞相互作用特异性诱导TGFβ1的产生,TGFβRIIDN装甲加速了肿瘤清除。
结论:新型的完全人TGFβRIIDN装甲的ROR1-CAR-1T细胞对ROR1阳性肿瘤具有高度的效力,并耐受TGFβ在固体TME中的抑制作用。此外,TGFβ1诱导代表了一种新的,固体TME中的CAR诱导检查点,这可以通过在T细胞上共表达TGβRIIDN装甲来规避。
BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy target receptor tyrosine kinase-like orphan receptor 1 (ROR1) is broadly expressed in hematologic and solid tumors, however clinically-characterized ROR1-CAR T cells with single chain variable fragment (scFv)-R12 targeting domain failed to induce durable remissions, in part due to the immunosuppressive tumor microenvironment (TME). Herein, we describe the development of an improved ROR1-CAR with a novel, fully human scFv9 targeting domain, and augmented with TGFβRIIDN armor protective against a major TME factor, transforming growth factor beta (TGFβ).
METHODS: CAR T cells were generated by lentiviral transduction of enriched CD4+ and CD8+ T cells, and the novel scFv9-based ROR1-CAR-1 was compared with the clinically-characterized ROR1-R12-scFv-based CAR-2 in vitro and in vivo.
RESULTS: CAR-1 T cells exhibited greater CAR surface density than CAR-2 when normalized for %CAR+, and produced more interferon (IFN)-γ tumor necrosis factor (TNF)-α and interleukin (IL)-2 in response to hematologic (Jeko-1, RPMI-8226) and solid (OVCAR-3, Capan-2, NCI-H226) tumor cell lines in vitro. In vivo, CAR-1 and CAR-2 both cleared hematologic Jeko-1 lymphoma xenografts, however only CAR-1 fully rejected ovarian solid OVCAR-3 tumors, concordantly with greater expansion of CD8+ and CD4+CAR T cells, and enrichment for central and effector memory phenotype. When equipped with TGFβ-protective armor TGFβRIIDN, CAR-1 T cells resisted TGFβ-mediated pSmad2/3 phosphorylation, as compared with CAR-1 alone. When co-cultured with ROR-1+ AsPC-1 pancreatic cancer line in the presence of TGFβ1, armored CAR-1 demonstrated improved recovery of killing function, IFN-γ, TNF-α and IL-2 secretion. In mouse AsPC-1 pancreatic tumor xenografts overexpressing TGFβ1, armored CAR-1, in contrast to CAR-1 alone, achieved complete tumor remissions, and yielded accelerated expansion of CAR+ T cells, diminished circulating active TGFβ1, and no apparent toxicity or weight loss. Unexpectedly, in AsPC-1 xenografts without TGFβ overexpression, TGFβ1 production was specifically induced by ROR-1-CAR T cells interaction with ROR-1 positive tumor cells, and the TGFβRIIDN armor conferred accelerated tumor clearance.
CONCLUSIONS: The novel fully human TGFßRIIDN-armored ROR1-CAR-1 T cells are highly potent against ROR1-positive tumors, and withstand the inhibitory effects of TGFß in solid TME. Moreover, TGFβ1 induction represents a novel, CAR-induced checkpoint in the solid TME, which can be circumvented by co-expressing the TGβRIIDN armor on T cells.