PO.CL07.04 · 临床研究
甲基硒酸增强抗FN14 CAR-T细胞的效应功能，并将细胞毒性重新导向富含TGF-beta的肾脏、前列腺和脑肿瘤
Methylseleninic acid enhances anti-FN14 CAR-T cell effector function and redirects cytotoxicity against TGF-beta-rich kidney, prostate, and brain tumors
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：透明细胞肾细胞癌(ccRCC)、转移性去势抵抗性前列腺癌(mCRPC)和胶质母细胞瘤(GBM)是治疗难治性肿瘤，其特征为高水平的TGF-beta1，后者是抗肿瘤免疫和抗原呈递的主要抑制因子。药理浓度的甲基硒酸(MSA)可强效抑制TGF-beta1、PD-L1和VEGF，且无显著的脱靶毒性。FN14即TWEAK受体，是一种转移驱动因子，在这些恶性肿瘤中选择性过表达，使其成为高特异性的CAR-T靶点。
方法：我们开发了具有CD28或4-1BB共刺激及CD3ζ信号传导的第二代抗FN14 CAR，并通过慢病毒转导入人CD4+/CD8+ T细胞。使用LDH释放和xCELLigence实验测定对FN14+ ccRCC、mCRPC、GBM细胞系及患者来源肿瘤的细胞毒性。通过胞内细胞因子、表型分析和多重分析评估效应功能。在生理浓度下检测MSA，以评估其对CAR-T活力、代谢、功能和肿瘤易感性的影响。
结果：抗FN14 CAR-T细胞在所有肿瘤类型中介导了快速的抗原特异性裂解，在1:1的效靶比下实现了>50%的特异性裂解。MSA(2.5–5 μM)使肿瘤PD-L1和TGF-beta降低了>40%，并改善了CAR-T的代谢活性，增强了IL-2和IFN-gamma的分泌。用MSA(2.5 μM)制造CAR-T细胞相比对照增加了细胞毒性和持久性(p < 0.01)。MSA的效应在临床相容浓度下呈剂量和时程依赖性，且无可检测的脱靶毒性。
结论：MSA通过抑制肿瘤来源的TGF-beta1和PD-L1并直接增强CAR-T活性，增强了抗FN14 CAR-T的细胞毒性、代谢适应性和持续的效应功能。作为一种临床相关的TGF-beta1抑制剂和活性硒代-L-甲硫氨酸代谢产物，MSA代表了一种易于转化的策略，可减少CAR-T耗竭并改善其在富含TGF-beta的实体瘤中的疗效。
查看英文原文 English abstract
Background: Clear cell renal cell carcinoma (ccRCC), metastatic castration-resistant prostate cancer (mCRPC), and glioblastoma (GBM) are treatment-refractory tumors marked by high TGF-beta1, a master suppressor of antitumor immunity and antigen presentation. Pharmacologic methylseleninic acid (MSA) potently inhibits TGF-beta1, PD-L1, and VEGF without significant off-target toxicity. FN14, the TWEAK receptor and a metastasis driver, is selectively overexpressed in these malignancies, making it a high-specificity CAR-T target.
Methods: We developed second-generation anti-FN14 CARs with CD28 or 4-1BB costimulation and CD3ζ signaling and lentivirally transduced them into human CD4⁺/CD8⁺ T cells. Cytotoxicity against FN14⁺ ccRCC, mCRPC, GBM lines and patient-derived tumors was measured using LDH release and xCELLigence assays. Effector function was assessed by intracellular cytokines, phenotyping, and multiplex analysis. MSA was tested at physiologic concentrations to evaluate effects on CAR-T viability, metabolism, function, and tumor susceptibility.
Results: Anti-FN14 CAR-T cells mediated rapid, antigen-specific lysis across all tumor types, achieving >50% specific lysis at an E:T ratio of 1:1. MSA (2.5-5 µM) reduced tumor PD-L1 and TGF-beta by >40% and improved CAR-T metabolic activity, enhancing IL-2 and IFN-gamma secretion. Manufacturing CAR-T cells with MSA (2.5 µM) increased cytotoxicity and persistence versus controls (p < 0.01). MSA's effects were dose- and schedule-dependent at clinically compatible concentrations without detectable off-target toxicity.
Conclusion: MSA augments anti-FN14 CAR-T cytotoxicity, metabolic fitness, and sustained effector function by suppressing tumor-derived TGF-beta1 and PD-L1 and directly enhancing CAR-T activity. As a clinically relevant TGF-beta1 inhibitor and active seleno-L-methionine metabolite, MSA represents a readily translatable strategy to reduce CAR-T exhaustion and improve efficacy in TGF-beta-rich solid tumors.
利益披露 Disclosure
G. B. Kim, None..
O. B. Amissah, None..
C. R. Ager, None..
Y. Zakharia, None.