肿瘤细胞治疗研究
英文原题：Inducible localized delivery of an anti-PD-1 scFv enhances anti-tumor activity of ROR1 CAR-T cells in TNBC.
我们下一代靶向 ROR1 的诱导型装甲 CAR 平台，仅在靶肿瘤细胞存在时释放免疫刺激载荷，从而增强 CAR-T 细胞的治疗活性。
背景：嵌合抗原受体（CAR）T 细胞可在血液系统恶性肿瘤患者中诱导强烈免疫反应，但治疗实体瘤的成功有限，部分原因是免疫抑制性肿瘤微环境（TME）会限制包括 CAR-T 在内的TIL（肿瘤浸润淋巴细胞）活性。我们开发了新一代装甲 CAR（F i-CAR），靶向受体酪氨酸激酶样孤儿受体 1（ROR1）；ROR1 在包括预后较差的三阴性乳腺癌（TNBC）在内的多种侵袭性肿瘤中高表达。该 F i-CAR-T 设计为在 TME 内 CAR-T 活化时释放抗 PD-1 检查点抑制剂，从而促进 CAR-T 和 TIL 活化并限制毒性。 方法：为增强效力，我们开发了一种 F i-CAR 构型：经 ROR1 介导活化后，在肿瘤微环境内通过 IL-2 介导、NFAT 诱导分泌抗 PD-1 单链可变片段（scFv）。采用 ELISA 和流式细胞术，在体外分析其对 TNBC 细胞系的细胞毒反应以及释放载荷的水平和结合功能，并在 TNBC NSG 小鼠模型中体内评估 F i-CAR-T 的效力。 结果：F i-CAR-T 细胞与肿瘤细胞表面 ROR1 结合 5 小时后可释放可测量的抗 PD-1 载荷，并在具有挑战性的 1:10 效靶比下增强细胞毒作用。对已形成的 PD-L1 阳性 TNBC 异种移植模型进行治疗后，与单独给予靶向 ROR1 的非装甲 CAR 细胞（F CAR-T；小鼠生存 49 天）或其联合全身给药抗 PD-1 抗体（57 天）相比，给予 F i-CAR-T 显著抑制肿瘤生长并延长小鼠生存期（71 天）。关键的是，F i-CAR-T 使肿瘤浸润 T 细胞增加 3 倍，并伴随细胞毒性、迁移和增殖相关基因表达升高。 结论：新一代靶向 ROR1 的诱导型装甲 CAR 平台仅在存在靶肿瘤细胞时释放免疫刺激载荷，从而增强 CAR-T 的治疗活性。该技术在 TNBC 异种移植模型中显著改善生存；结合其潜在安全性，值得在 TNBC 患者中进一步开展临床评估。
BACKGROUND: Chimeric antigen receptor (CAR)-T cells can induce powerful immune responses in patients with hematological malignancies but have had limited success against solid tumors. This is in part due to the immunosuppressive tumor microenvironment (TME) which limits the activity of tumor-infiltrating lymphocytes (TILs) including CAR-T cells. We have developed a next-generation armored CAR (F i-CAR) targeting receptor tyrosine kinase-like orphan receptor 1 (ROR1), which is expressed at high levels in a range of aggressive tumors including poorly prognostic triple-negative breast cancer (TNBC). The F i-CAR-T is designed to release an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation within the TME, facilitating activation of CAR-T cells and TILs while limiting toxicity. METHODS: To bolster potency, we developed a F i-CAR construct capable of IL-2-mediated, NFAT-induced secretion of anti-PD-1 single-chain variable fragments (scFv) within the tumor microenvironment, following ROR1-mediated activation. Cytotoxic responses against TNBC cell lines as well as levels and binding functionality of released payload were analyzed in vitro by ELISA and flow cytometry. In vivo assessment of potency of F i-CAR-T cells was performed in a TNBC NSG mouse model. RESULTS: F i-CAR-T cells released measurable levels of anti-PD-1 payload with 5 h of binding to ROR1 on tumor and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Treatment of established PDL1 + TNBC xenograft model with F i-CAR-T cells resulted in significant abrogation in tumor growth and improved survival of mice (71 days), compared to non-armored CAR cells targeting ROR1 (F CAR-T) alone (49 days) or in combination with systemically administered anti-PD-1 antibody (57 days). Crucially, a threefold increase in tumor-infiltrating T cells was observed with F i-CAR-T cells and was associated with increased expression of genes related to cytotoxicity, migration and proliferation. CONCLUSIONS: Our next-generation of ROR1-targeting inducible armored CAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells. This technology provided a significant survival advantage in TNBC xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach in TNBC patients.
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