抗 PD-1 增加了由疫苗和节拍式环磷酰胺组成的强大免疫疗法诱导的肿瘤浸润抗原特异性 T 细胞的克隆性和活性。

BACKGROUND: Future cancer immunotherapies will combine multiple treatments to generate functional immune responses to cancer antigens through synergistic, multi-modal mechanisms. In this study we explored the combination of three distinct immunotherapies: a class I restricted peptide-based cancer vaccine, metronomic cyclophosphamide (mCPA) and anti-PD-1 treatment in a murine tumor model expressing HPV16 E7 (C3). METHODS: Mice were implanted with C3 tumors subcutaneously. Tumor bearing mice were treated with mCPA (20 mg/kg/day PO) for seven continuous days on alternating weeks, vaccinated with HPV16 E7 peptide antigen formulated in the DepoVax (DPX) adjuvanting platform every second week, and administered anti-PD-1 (200 μg/dose IP) after each vaccination. Efficacy was measured by following tumor growth and survival. Immunogenicity was measured by IFN-γ ELISpot of spleen, vaccine draining lymph nodes and tumor draining lymph nodes. Tumor infiltration was measured by flow cytometry for CD8α peptide-specific T cells and RT-qPCR for cytotoxic proteins. The clonality of tumor infiltrating T cells was measured by TCRβ sequencing using genomic DNA. RESULTS: Untreated C3 tumors had low expression of PD-L1 in vivo and anti-PD-1 therapy alone provided no protection from tumor growth. Treatment with DPX/mCPA could delay tumor growth, and tri-therapy with DPX/mCPA/anti-PD-1 provided long-term control of tumors. We found that treatment with DPX/mCPA/anti-PD-1 enhanced systemic antigen-specific immune responses detected in the spleen as determined by IFN-γ ELISpot compared to those in the DPX/mCPA group, but immune responses in tumor-draining lymph nodes were not increased. Although no increases in antigen-specific CD8α TILs could be detected, there was a trend for increased expression of cytotoxic genes within the tumor microenvironment as well as an increase in clonality in mice treated with DPX/mCPA/anti-PD-1 compared to those with anti-PD-1 alone or DPX/mCPA. Using a library of antigen-specific CD8α T cell clones, we found that antigen-specific clones were more frequently expanded in the DPX/mCPA/anti-PD-1 treated group. CONCLUSIONS: These results demonstrate how the efficacy of anti-PD-1 may be improved by combination with a potent and targeted T cell activating immune therapy.

背景：未来的癌症免疫疗法将结合多种治疗方法，通过协同的多模式机制，产生针对癌症抗原的功能性免疫反应。在这项研究中，我们探索了三种不同免疫疗法的联合应用：一种基于 I 类限制肽的癌症疫苗、节拍式环磷酰胺（mCPA）和抗 PD-1 治疗，在表达 HPV16 E7（C3）的鼠肿瘤模型中。

方法：将 C3 肿瘤皮下植入小鼠。荷瘤小鼠连续 7 天每天口服 mCPA（20mg/kg），交替周进行，每两周用 DPX 佐剂平台配制的 HPV16 E7 肽抗原接种疫苗，每次接种后给予抗 PD-1（200μg/剂量 IP）。通过监测肿瘤生长和存活来衡量疗效。通过 IFN-γ ELISpot 检测脾、疫苗引流淋巴结和肿瘤引流淋巴结的免疫原性。通过流式细胞术检测 CD8α 肽特异性 T 细胞和 RT-qPCR 检测细胞毒性蛋白来测量肿瘤浸润。使用基因组 DNA 通过 TCRβ 测序测量肿瘤浸润 T 细胞的克隆性。

结果：未经处理的 C3 肿瘤在体内低表达 PD-L1，单独使用抗 PD-1 治疗不能防止肿瘤生长。DPX/mCPA 治疗可延迟肿瘤生长，而 DPX/mCPA/抗 PD-1 三联疗法可长期控制肿瘤。我们发现，与 DPX/mCPA 组相比，DPX/mCPA/抗 PD-1 治疗可增强系统中的抗原特异性免疫反应，这是通过 IFN-γ ELISpot 检测到的，但肿瘤引流淋巴结中的免疫反应没有增加。尽管无法检测到抗原特异性 CD8α TIL 的增加，但在 DPX/mCPA/抗 PD-1 治疗的小鼠中，肿瘤微环境中的细胞毒性基因表达增加，并且与单独使用抗 PD-1 或 DPX/mCPA 相比，克隆性增加。使用抗原特异性 CD8α T 细胞克隆文库，我们发现 DPX/mCPA/抗 PD-1 治疗组中抗原特异性克隆的扩增更为频繁。

结论：这些结果表明，通过与有效的、靶向 T 细胞激活免疫疗法联合使用，抗 PD-1 的疗效可以得到提高。