The efficacy of LCMV-based cancer vaccines is unleashed by intratumoral injections of polyI:C (II). The efficacy of LCMV-based cancer vaccines is unleashed by intratumoral injections of polyI:C (II)

Background Lymphocytic choriomeningitis virus (LCMV) belongs to the Arenavirus family known for inducing strong cytotoxic T-cell responses in both mice and humans. LCMV has been engineered for the development of anti-tumor vaccines, currently undergoing evaluation in phase I/II clinical trials. Initial findings have demonstrated safety and an exceptional ability to induce and expand tumor-specific T lymphocytes. Combination strategies to maximize the anti-tumor effectiveness of LCMV-based vaccines are required. Methods We assessed the anti-tumor therapeutic effects of intratumoral administration of polyinosinic:polycytidylic acid (poly(I:C)) and systemic vaccination using an LCMV-vector expressing non-oncogenic versions of the E6 and E7 antigens of human papillomavirus 16 (artLCMV-E7E6) in a bilateral model engrafting TC-1/A9 cells. This cell line, derived from the parental TC-1, exhibits low MHC class I expression and is highly immune-resistant. The mechanisms underlying the combination's efficacy were investigated through bulk RNAseq, flow cytometry analyses of the tumor microenvironment, selective depletions using antibodies and clodronate liposomes, Batf3 deficient mice, and in vivo bioluminescence experiments. Finally, we assessed the anti-tumor effectiveness of the combining of LCMV-E6E7 with BO-112, a GMP-grade poly(I:C) formulated in polyethyleneimine, currently under evaluation in clinical trials. Results Intratumoral injection of poly(I:C) enhanced the anti-tumor efficacy of artLCMV-E7E6 in both injected and non-injected tumor lesions. The combined treatment resulted in a significant delay in tumor growth and often complete eradication of several tumor lesions, leading to significantly improved survival compared to monotherapies. While intratumoral administration of poly(I:C) did not impact LCMV vector biodistribution or transgene expression, it significantly modified leukocyte infiltrates within the tumor microenvironment and amplified systemic efficacy through proinflammatory cytokines/chemokines such as CCL3, CCL5, CXCL10, TNF, IFNα, and IL12p70. Upregulation of MHC on tumor cells and a reconfiguration of the gene expression programs related to tumor vasculature, leukocyte migration, and the activation profile of tumor-infiltrating CD8+ T lymphocytes were observed. Indeed, the anti-tumor effect relied on the functions of CD8+ T lymphocytes and macrophages. The synergistic efficacy of the combination was further confirmed when BO-112 was included. Conclusion Intratumoral injection of poly(I:C) sensitizes MHClow tumors to the anti-tumor effects of artLCMV-E7E6, resulting in a potent therapeutic synergy Overall design: TC1/A9 tumor-bearing mice received intravenous treatment with 1×105 RCV FFU artLCMV-E7E6 on day 7 and intratumoral injections of 50 μg of poly(I:C) or BO-112

背景 淋巴细胞脉络丛脑膜炎病毒（Lymphocytic choriomeningitis virus, LCMV）属于沙粒病毒科，其特征为可在小鼠与人类体内诱导强烈的细胞毒性T细胞（cytotoxic T-cell）应答。LCMV已被工程化用于抗肿瘤疫苗的研发，目前正处于I/II期临床试验评估阶段。初步研究结果证实了其安全性，以及诱导并扩增肿瘤特异性T淋巴细胞（tumor-specific T lymphocytes）的卓越能力。亟需开发联合策略以最大化基于LCMV的疫苗的抗肿瘤效果。

方法 我们在植入TC-1/A9细胞的双侧肿瘤模型中，评估了瘤内注射聚肌苷酸:聚胞苷酸（polyinosinic:polycytidylic acid, poly(I:C)）联合系统性接种表达人乳头瘤病毒16型（human papillomavirus 16）非致癌型E6、E7抗原的LCMV载体（artLCMV-E7E6）的抗肿瘤治疗效果。该细胞系源自亲本TC-1细胞，MHC I类分子表达水平较低，且具有高度免疫抵抗性。我们通过批量RNA测序（bulk RNAseq）、肿瘤微环境流式细胞术（flow cytometry）分析、抗体及氯膦酸盐脂质体（clodronate liposomes）选择性耗竭实验、Batf3缺陷型小鼠（Batf3 deficient mice）模型以及体内生物发光实验（in vivo bioluminescence experiments），对该联合疗法的起效机制进行了探究。最后，我们评估了LCMV-E6E7与BO-112联合的抗肿瘤效果——BO-112是一种以聚乙烯亚胺（polyethyleneimine）为载体配制的GMP级poly(I:C)，目前正处于临床试验评估阶段。

结果 瘤内注射poly(I:C)可增强artLCMV-E7E6对注射侧与非注射侧肿瘤病灶的抗肿瘤活性。联合治疗可显著延缓肿瘤生长，且常可完全清除多个肿瘤病灶，相较单一疗法可显著提升小鼠生存率。尽管瘤内注射poly(I:C)不会影响LCMV载体的生物分布或转基因表达，但可显著重塑肿瘤微环境内的白细胞浸润情况，并通过CCL3、CCL5、CXCL10、TNF、IFNα、IL12p70等促炎细胞因子/趋化因子放大系统抗肿瘤效应。研究还观察到肿瘤细胞表面MHC分子的上调，以及与肿瘤血管系统、白细胞迁移及肿瘤浸润CD8+ T细胞活化谱相关的基因表达程序重编程。进一步验证表明，该抗肿瘤效果依赖于CD8+ T细胞与巨噬细胞的功能。当联合使用BO-112时，该联合疗法的协同抗肿瘤效果得到了进一步证实。

结论 瘤内注射poly(I:C)可使MHC低表达肿瘤对artLCMV-E7E6的抗肿瘤效应产生敏感性，从而形成强效的治疗协同作用。

整体实验设计：在第7天，携带TC1/A9肿瘤的小鼠经静脉接种1×10^5 RCV灶形成单位（FFU）的artLCMV-E7E6，并瘤内注射50 μg poly(I:C)或BO-112。