非病毒介导的OX40配体基因转导用于肿瘤免疫治疗

免疫检查点阻断（ICB）正迅速成为多种癌症治疗的标准方案。然而，对这种治疗反应的患者子集有限。促进抗肿瘤免疫的另一途径是使用免疫刺激分子，如细胞因子或T细胞共刺激因子。全身性免疫治疗药物的系统性给药会引发显著的免疫相关不良事件（irAEs），因此需要局部抗肿瘤作用。一种实现途径是肿瘤内非病毒基因免疫治疗，这能实现持续且局部的基因表达及多次药物给药。在本研究中，我们结合先前描述的非病毒基因递送系统PEG-PEI-TAT共聚物（PPT）与编码小鼠OX40L的质粒DNA。所得OX40L/PPT纳米颗粒通过凝胶迁移率检测、动态光散射分析和体外转染效率评估进行表征。利用皮下（s.c.）植入的CT26（结肠癌）、B16F0（黑色素瘤）和4T1（乳腺癌）肿瘤模型评估纳米颗粒的抗肿瘤效果。通过流式细胞术分析肿瘤间质免疫细胞群的动态变化。体重减轻和恶病质作为irAE指标。将OX40L/PPT与腹腔注射的PD-1免疫检查点阻断（ICB）联合应用于CT26肿瘤模型，观察其效果。所得OX40L/PPT纳米颗粒具有良好的细胞转染性质，在三种癌症模型中均能体外表达OX40L蛋白。结果显示，OX40L/PPT治疗成功抑制了B16F0和CT26肿瘤的生长，且对4T1肿瘤也呈现抑制趋势。在B16F0模型中，OX40L/PPT增强了抗肿瘤的效应NK和杀伤T细胞的活性，减少了促肿瘤的髓系细胞群。在所有三种模型中未观察到irAE的迹象，表明治疗具有良好的耐受性。OX40L/PPT与PD-1 ICB的联合应用显著提高了CT26结肠癌模型的治疗效果，为CT26结肠癌细胞提供了保护性免疫。总体而言，OX40L非病毒基因治疗单独或联合ICB表现出强大的抗肿瘤潜力，有望引领癌症基因治疗的变革，推动癌症治疗领域的突破性进展。

Immune checkpoint blockade (ICB) is rapidly becoming a standard of care in the treatment of many cancer types. However, the subset of patients who respond to this type of therapy is limited. Another way to promote antitumoral immunity is the use of immunostimulatory molecules, such as cytokines or T cell co-stimulators. The systemic administration of immunotherapeutics leads to significant immune-related adverse events (irAEs), therefore, the localized antitumoral action is needed. One way to achieve this is intratumoral non-viral gene-immune therapy, which allows for prolonged and localized gene expression, and multiple drug administration. In this study, we combined the previously described non-viral gene delivery system, PEG-PEI-TAT copolymer, PPT, with murine OX40L-encoding plasmid DNA.The resulting OX40L/PPT nanoparticles were characterized via gel mobility assay, dynamic light scattering analysis and in vitro transfection efficiency evaluation. The antitumoral efficacy of intratumorally (i.t.) administered nanoparticles was estimated using subcutaneously (s.c.) implanted CT26 (colon cancer), B16F0 (melanoma) and 4T1 (breast cancer) tumor models. The dynamics of stromal immune cell populations was analyzed using flow cytometry. Weight loss and cachexia were used as irAE indicators. The effect of combination of i.t. OX40L/PPT with intraperitoneal PD-1 ICB was estimated in s.c. CT26 tumor model.The obtained OX40L/PPT nanoparticles had properties applicable for cell transfection and provided OX40L protein expression in vitro in all three investigated cancer models. We observed that OX40L/PPT treatment successfully inhibited tumor growth in B16F0 and CT26 tumor models and showed a tendency to inhibit 4T1 tumor growth. In B16F0 tumor model, OX40L/PPT treatment led to the increase in antitumoral effector NK and T killer cells and to the decrease in pro-tumoral myeloid cells populations within tumor stroma. No irAE signs were observed in all 3 tumor models, which indicates good treatment tolerability in mice. Combining OX40L/PPT with PD-1 ICB significantly improved treatment efficacy in the CT26 subcutaneous colon cancer model, providing protective immunity against CT26 colon cancer cells.Overall, the anti-tumor efficacy observed with OX40L non-viral gene therapy, whether administered alone or in combination with ICB, highlights its potential to revolutionize cancer gene therapy, thus paving the way for unprecedented advancements in the cancer therapy field.