In situ vaccination is a promising strategy to convert the immunosuppressive tumor microenvironment into an immunostimulatory one with limited systemic exposure and side effect. However, sustained clinical benefits require long-term and multidimensional immune activation including innate and adaptive immunity. Here, we develop a probiotic food-grade Lactococcus lactis-based in situ vaccination (FOLactis) expressing a fusion protein of Fms-like tyrosine kinase 3 ligand and co-stimulator OX40 ligand. Intratumoural delivery of FOLactis contributes to local retention and sustained release of therapeutics to thoroughly modulate key components of the antitumour immune response, such as activation of natural killer cells, cytotoxic T lymphocytes, and conventional-type-1-dendritic cells in the tumors and tumor-draining lymph nodes. In addition, intratumoural administration of FOLactis induces a more robust tumor antigen-specific immune response and superior systemic antitumour efficacy in multiple poorly immune cell-infiltrated and anti-PD1-resistant tumors. Specific depletion of different immune cells reveals that CD8⁺ T and natural killer cells are crucial to the in situ vaccine-elicited tumor regression. Our results confirm that FOLactis displays an enhanced antitumour immunity and successfully converts the ‘cold’ tumors to ‘hot’ tumors.

原位疫苗接种是一种很有前途的策略，可以将免疫抑制性肿瘤微环境转变为具有有限全身暴露和副作用的免疫刺激性微环境。然而，持续的临床益处需要长期和多维的免疫激活，包括先天免疫和适应性免疫。在这里，我们开发了一种益生菌食品级乳酸乳球菌基于原位疫苗接种 (FOLactis) 表达 Fms 样酪氨酸激酶 3 配体和共刺激物 OX40 配体的融合蛋白。FOLactis 的肿瘤内递送有助于治疗药物的局部保留和持续释放，以彻底调节抗肿瘤免疫反应的关键成分，例如激活肿瘤和肿瘤中的自然杀伤细胞、细胞毒性 T 淋巴细胞和常规 1 型树突状细胞-引流淋巴结。此外，FOLactis 的肿瘤内给药可在多种免疫细胞浸润和抗 PD1 耐药的肿瘤中诱导更强大的肿瘤抗原特异性免疫反应和卓越的全身抗肿瘤功效。不同免疫细胞的特异性耗竭表明 CD8 ⁺T 细胞和自然杀伤细胞对于原位疫苗引起的肿瘤消退至关重要。我们的结果证实，FOLactis 显示出增强的抗肿瘤免疫力，并成功地将“冷”肿瘤转化为“热”肿瘤。