由于其直接感染和杀死肿瘤并增强抗肿瘤免疫反应的能力，溶瘤病毒在临床上引起了重要的关注。我们之前开发了KLS-3010，这是一种新型溶瘤病毒，源于国际卫生部-白色（IHD-W）株疫苗天花病毒，具有强大的抗肿瘤效果。在本研究中，我们通过将三个转基因物（透明质酸酶[PH-20]、白细胞介素-12[IL-12]和可溶性programmed cell death 1与Fc结构域融合[sPD1-Fc]）插入KLS-3010中，生成了重组溶瘤病毒KLS-3020，并研究了其对CT26.WT和B16F10小鼠肿瘤模型的抗肿瘤效力和诱导抗肿瘤免疫反应的能力。KLS-3020的单次注射显著减少了肿瘤生长。使用单独表达每个转基因物的病毒以及KLS-3020，探究了转基因物的作用。PH-20促进了病毒的扩散和肿瘤免疫细胞浸润，IL-12激活并重新编程T细胞为炎症表型，sPD1-Fc增加了肿瘤内活化T细胞的种群。在CT26.WT肿瘤模型中，KLS-3020引起了肿瘤特异性的全身免疫反应和邻位肿瘤控制。将转基因物插入KLS-3020增加了其抗肿瘤效力，支持进一步将KLS-3020作为一种新型溶瘤免疫疗法进行临床研究。© 2023 The Authors.

Oncolytic viruses are of significant clinical interest due to their ability to directly infect and kill tumors and enhance the anti-tumor immune response. Previously, we developed KLS-3010, a novel oncolytic virus derived from the International Health Department-White (IHD-W) strain vaccinia virus, which has robust tumoricidal effects. In the present study, we generated a recombinant oncolytic virus, KLS-3020, by inserting three transgenes (hyaluronidase [PH-20], interleukin-12 [IL-12], and soluble programmed cell death 1 fused to the Fc domain [sPD1-Fc]) into KLS-3010 and investigated its anti-tumor efficacy and ability to induce anti-tumor immune responses in CT26.WT and B16F10 mouse tumor models. A single injection of KLS-3020 significantly decreased tumor growth. The roles of the transgenes were investigated using viruses expressing each single transgene alone and KLS-3020. PH-20 promoted virus spread and tumor immune cell infiltration, IL-12 activated and reprogrammed T cells to inflammatory phenotypes, and sPD1-Fc increased intra-tumoral populations of activated T cells. The tumor-specific systemic immune response and the abscopal tumor control elicited by KLS-3020 were demonstrated in the CT26.WT tumor model. The insertion of transgenes into KLS-3020 increased its anti-tumor efficacy, supporting further clinical investigation of KLS-3020 as a novel oncolytic immunotherapy.© 2023 The Authors.