使用 TLR2/3 激动剂 L-pampo 进行肿瘤内免疫治疗可诱导强烈的抗肿瘤免疫反应，并增强免疫检查点阻断。

BACKGROUND: Toll-like receptors (TLRs) are critical innate immune sensors that elicit antitumor immune responses in cancer immunotherapy. Although a few TLR agonists have been approved for the treatment of patients with early-stage superficial cancers, their therapeutic efficacy is limited in patient with advanced invasive cancers. Here, we identified the therapeutic role of a TLR2/3 agonist, L-pampo (LP), which promotes antitumor immunity and enhances the immune checkpoint blockade. METHODS: We generated LP by combining a TLR2 agonist, Pam3CSK4, with a TLR3 agonist, Poly (I:C). Immune responses to stimulation with various TLR agonists were compared. Tumor-bearing mice were intratumorally treated with LP, and their tumor sizes were measured. The antitumor effects of LP treatment were determined using flow cytometry, multiplexed imaging, and NanoString nCounter immune profiling. The immunotherapeutic potential of LP in combination with α-programmed cell death protein-1 (PD-1) or α-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) was evaluated in syngeneic MC38 colon cancer and B16F10 melanoma. RESULTS: The LP treatment induced a potent activation of T helper 1 (Th1) and 2 (Th2)-mediated immunity, tumor cell apoptosis, and immunogenic tumor cell death. Intratumoral LP treatment effectively inhibited tumor progression by activating tumor-specific T cell immunity. LP-induced immune responses were mediated by CD8 T cells and interferon-γ, but not by CD4 T cells and CD25 T cells. LP simultaneously activated TLR2 and TLR3 signaling, thereby extensively changing the immune-related gene signatures within the tumor microenvironment (TME). Moreover, intratumoral LP treatment led to systemic abscopal antitumor effects in non-injected distant tumors. Notably, LP treatment combined with ɑPD-1 and ɑCTLA-4 further enhanced the efficacy of monotherapy, resulting in complete tumor regression and prolonged overall survival. Furthermore, LP-based combination immunotherapy elicited durable antitumor immunity with tumor-specific immune memory in colon cancer and melanoma. CONCLUSIONS: Our study demonstrated that intratumoral LP treatment improves the innate and adaptive antitumor immunity within the TME and enhances the efficacy of αPD-1 and αCTLA-4 immune checkpoint blockade.

背景：Toll 样受体 (TLRs) 是关键的先天免疫传感器，可在癌症免疫治疗中引发抗肿瘤免疫反应。虽然有几种 TLR 激动剂已被批准用于治疗早期局限性癌症患者，但它们在晚期侵袭性癌症患者中的治疗效果有限。在这里，我们确定了 TLR2/3 激动剂 L-pampo（LP）的治疗作用，它可促进抗肿瘤免疫并增强免疫检查点阻断。

方法：我们通过将 TLR2 激动剂 Pam3CSK4 与 TLR3 激动剂 Poly（I：C）结合来生成 LP。比较了对各种 TLR 激动剂刺激的免疫反应。用 LP 对荷瘤小鼠进行肿瘤内治疗，并测量其肿瘤大小。通过流式细胞术、多重成像和 NanoString nCounter 免疫分析对 LP 治疗的抗肿瘤作用进行了测定。在同源 MC38 结肠癌细胞和 B16F10 黑色素瘤中评估了 LP 与 α-程序性细胞死亡蛋白 1（PD-1）或 α-细胞毒性 T 淋巴细胞相关蛋白 4（CTLA-4）联合的免疫治疗潜力。

结果：LP 治疗可强烈激活 Th1 和 Th2 介导的免疫、肿瘤细胞凋亡和免疫原性肿瘤细胞死亡。肿瘤内 LP 治疗通过激活肿瘤特异性 T 细胞免疫有效抑制肿瘤进展。LP 诱导的免疫反应是由 CD8 T 细胞和干扰素-γ介导的，而不是由 CD4 T 细胞和 CD25 T 细胞介导的。LP 同时激活 TLR2 和 TLR3 信号通路，从而广泛改变肿瘤微环境（TME）中的免疫相关基因特征。此外，肿瘤内 LP 治疗导致非注射远处肿瘤的系统抗肿瘤作用。值得注意的是，LP 治疗联合 αPD-1 和 αCTLA-4 进一步增强了单药治疗的疗效，导致完全肿瘤消退和总生存期延长。此外，基于 LP 的联合免疫疗法在结肠直肠癌和黑色素瘤中引发了具有肿瘤特异性免疫记忆的持久抗肿瘤免疫。

结论：我们的研究表明，肿瘤内 LP 治疗可改善 TME 中的先天和适应性抗肿瘤免疫，并增强 αPD-1 和 αCTLA-4 免疫检查点阻断的疗效。