免疫检查点阻断 (ICB) 正在迅速成为治疗多种癌症类型的护理标准。然而，对这种治疗有反应的患者子集是有限的。促进抗肿瘤免疫的另一种方法是使用免疫刺激分子，例如细胞因子或 T 细胞共刺激剂。免疫治疗药物的全身给药会导致严重的免疫相关不良事件（irAE），因此需要局部抗肿瘤作用。实现这一目标的一种方法是瘤内非病毒基因免疫治疗，它允许延长和局部基因表达，以及多种药物给药。在本研究中，我们将先前描述的非病毒基因传递系统、PEG-PEI-TAT 共聚物、PPT 与鼠 OX40L 编码质粒 DNA 结合起来。通过凝胶迁移率测定、动态光散射分析对所得 OX40L/PPT 纳米颗粒进行了表征。以及体外转染效率评价。使用皮下（s.c.）植入的 CT26（结肠癌）、B16F0（黑色素瘤）和 4T1（乳腺癌）肿瘤模型评估瘤内（i.t.）施用纳米颗粒的抗肿瘤功效。使用流式细胞术分析基质免疫细胞群的动态。体重减轻和恶病质被用作irAE指标。它的组合效果。 OX40L/PPT 与腹膜内 PD-1 ICB 的估计是皮下注射。 CT26 肿瘤模型。获得的 OX40L/PPT 纳米颗粒具有适用于细胞转染的特性，并在所有三种研究的癌症模型中提供了 OX40L 蛋白的体外表达。我们观察到 OX40L/PPT 治疗成功抑制了 B16F0 和 CT26 肿瘤模型中的肿瘤生长，并显示出抑制 4T1 肿瘤生长的趋势。在 B16F0 肿瘤模型中，OX40L/PPT 治疗导致肿瘤基质内抗肿瘤效应 NK 和 T 杀伤细胞增加，促肿瘤骨髓细胞群减少。在所有 3 个肿瘤模型中均未观察到 irAE 迹象，这表明小鼠具有良好的治疗耐受性。 OX40L/PPT与PD-1 ICB联合显着提高了CT26皮下结肠癌模型的治疗效果，提供了针对CT26结肠癌细胞的保护性免疫。总体而言，OX40L非病毒基因治疗观察到的抗肿瘤功效，无论是单独给药还是单独给药与 ICB 相结合，凸显了其彻底改变癌症基因治疗的潜力，从而为癌症治疗领域前所未有的进步铺平了道路。版权所有 © 2024 Rakitina, Kuzmich, Bezborodova, Kondratieva, Pleshkan, Zinovyeva, Didych, Sass, Snezhkov, Kostina,科克沙罗夫和阿列克谢恩科。

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.Copyright © 2024 Rakitina, Kuzmich, Bezborodova, Kondratieva, Pleshkan, Zinovyeva, Didych, Sass, Snezhkov, Kostina, Koksharov and Alekseenko.