尽管免疫检查点阻断治疗在治疗癌症方面取得了成功，但许多患者的应答不够充分。我们旨在通过优化抗体和它们的递送途径，利用基于森林蚊病毒（SFV）的自我扩增RNA（saRNA）载体定向递送小单结构域抗体（纳米抗体），从而改善该疗法。我们创造了针对PD-1和PD-L1的纳米抗体，能够抑制人类和小鼠之间的相互作用。融合二聚化结构域可使PD-1/PD-L1 IC50值分别降低8倍和40倍，针对抗PD-L1和抗PD-1纳米抗体的作用都得以减弱。表达二聚体纳米抗体的SFV病毒颗粒在MC38模型中表现出强大的抗肿瘤反应，导致>50% 的完全回归，并且与表达传统抗体的载体相比，治疗疗效更好。这些效果在B16黑色素瘤模型中也得到了观察。虽然纳米抗体的短暂表达是由于saRNA载体的细胞病理性导致的，但足以在肿瘤中产生强烈的促炎反应，增加NK和CD8+T细胞浸润。通过局部质粒电穿孔传递表达二聚体纳米抗体的SFV载体，该方法更容易应用于临床，并且也显示出强大的抗肿瘤效果。© 2023 Elsevier B.V. 保留所有权利。

Despite the success of immune checkpoint blockade for cancer therapy, many patients do not respond adequately. We aimed to improve this therapy by optimizing both the antibodies and their delivery route, using small monodomain antibodies (nanobodies) delivered locally with a self-amplifying RNA (saRNA) vector based on Semliki Forest virus (SFV). We generated nanobodies against PD-1 and PD-L1 able to inhibit both human and mouse interactions. Incorporation of a dimerization domain reduced PD-1/PD-L1 IC50 by 8- and 40-fold for anti-PD-L1 and anti-PD-1 nanobodies, respectively. SFV viral particles expressing dimeric nanobodies showed a potent antitumor response in the MC38 model, resulting in >50% complete regressions, and showed better therapeutic efficacy compared to vectors expressing conventional antibodies. These effects were also observed in the B16 melanoma model. Although a short-term expression of nanobodies was observed due to the cytopathic nature of the saRNA vector, it was enough to generate a strong proinflammatory response in tumors, increasing infiltration of NK and CD8+ T cells. Delivery of the SFV vector expressing dimeric nanobodies by local plasmid electroporation, which could be more easily translated to the clinic, also showed a potent antitumor effect.Copyright © 2023. Published by Elsevier B.V.