肿瘤细胞治疗研究
英文原题：Targeted delivery of IFN-α-anti-GPC3 fusion protein via mRNA-LNP platform elicits potent anti-tumor immunity in hepatocellular carcinoma.
重复给药（6 或 10 mpk）确定了最大耐受剂量（MTD）为 6 mpk，至少比最小有效剂量（MED，0.15 mpk）高 40 倍。
本研究旨在利用mRNA-脂质纳米颗粒（mRNA-LNP）平台，实现干扰素-γ（IFN-γ）/抗磷脂酰肌醇蛋白聚糖3（抗GPC3）融合蛋白（GPA01）在肝脏原位表达，增强IFN-γ的靶向性和抗肿瘤活性，为GPC3阳性肝细胞癌（HCC）提供精准治疗策略。设计并合成编码GPC3/IFN-γ双特异性融合蛋白的mRNA，将其包封于脂质纳米颗粒中并转染HepG2 HCC细胞系，在体外表征蛋白表达、结合活性和基因诱导作用。建立HepG2-luc原位HCC模型及Hepa 1-6/hGPC3-hi小鼠皮下肿瘤模型，评估mRNA-LNP或对照药物治疗后的肿瘤生长、生存和免疫细胞浸润。在人IFNAR转基因小鼠中评估安全性。体外实验显示，mRNA-LNP成功转染并表达具有生物活性的融合蛋白；转染细胞上清可特异性结合GPC3并诱导干扰素刺激基因（ISG）。体内研究显示，与对照相比，GPC3/IFN-γ mRNA-LNP显著抑制肿瘤生长、延长中位生存期并增加肿瘤内CD8 T细胞和NK细胞浸润，且安全性良好。联合PD-1抗体（PD-1 Ab）产生协同抗肿瘤作用，该作用主要依赖CD8 T细胞浸润。人IFNAR转基因小鼠安全性评估显示，单次1–10 mg/kg剂量耐受性良好，仅引起部分生物标志物短暂变化。重复给药（6或10 mg/kg）确定最大耐受剂量（MTD）为6 mg/kg，至少为最低有效剂量（MED，0.15 mg/kg）的40倍。mRNA-LNP介导IFN-γ-抗GPC3融合蛋白递送可实现靶向肝脏原位表达，并显著增强抗肿瘤活性，具有较宽治疗窗。该策略为HCC精准免疫治疗提供了新途径，具有显著临床转化潜力。
This study aimed to utilize the mRNA-lipid nanoparticle (mRNA-LNP) platform to achieve in situ hepatic expression of an interferon- (IFN- )/anti-glypican-3 (anti-GPC3) fusion protein (GPA01), enhancing IFN- targeting and antitumor activity to provide a precision therapy strategy for GPC3-positive hepatocellular carcinoma (HCC). mRNA encoding a GPC-3/IFN- bispecific fusion protein was designed and synthesized, encapsulated in lipid nanoparticles, and transfected into HCC cell lines (HepG2) for in vitro characterization of protein expression, binding activity, and gene induction. Orthotopic HCC models (HepG2-luc) and subcutaneous tumor model (Hepa 1-6/hGPC3-hi) were established in mice to evaluate tumor growth, survival, and immune cell infiltration following treatment with mRNA-LNP or control agents. Safety was assessed in human IFNAR transgenic mice. In vitro experiments demonstrated successful transfection and bioactive fusion protein expression by mRNA-LNP, with transfected supernatants showing specific GPC3 binding and interferon-stimulated gene (ISG) induction. In vivo studies revealed that GPC-3/IFN- mRNA-LNP significantly inhibited tumor growth, prolonged median survival, and increased intratumoral CD8 T cell and NK cell infiltration compared to controls, with favorable safety profiles. Combination therapy with PD-1 antibody (PD-1 Ab) exerted synergistic antitumor effects, primarily dependent on CD8 T cell infiltration. Safety evaluations in human IFNAR transgenic mice showed good tolerability at single doses of 1-10 mpk, with transient changes in select biomarkers. Repeated dosing (6 or 10 mpk) identified a maximum tolerated dose (MTD) of 6 mpk, at least 40-fold higher than the minimal effective dose (MED, 0.15 mpk). mRNA-LNP-mediated delivery of IFN- -anti-GPC3 fusion protein achieves targeted in situ hepatic expression, significantly enhancing antitumor activity with a broad therapeutic window. This strategy offers a novel approach for precision immunotherapy in HCC, holding substantial potential for clinical translation.
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