Elucidating the Role of Cancer-Associated FGL1 in Tumor Immunity and Developing FGL1-Guided Anti-LAG-3 Cancer Immunotherapy

项目摘要 大鼠对抗CTLA-4、PD-1/PD-L1(PD)T细胞免疫检查点阻断(ICB)治疗的抵抗 癌症患者的亚群需要寻找其他与肿瘤相关的T细胞免疫检查点 严重影响肿瘤免疫。淋巴细胞激活基因3(LAG-3)是一种T细胞抑制受体， 代表了癌症免疫治疗的一个有希望的靶点，作为阻断LAG-3的单抗 它已知的配体MHC-II与抗PD-1结合在一起在最近的临床中显示出温和但有希望的疗效 审判。FDA最近接受了LAG-3阻断单抗的生物制品许可证申请作为优先审查 Relatlimab和抗pd-1 nivolumab固定剂量联合治疗黑色素瘤。然而，很少有单身人士- 这些MHC-II封闭抗LAG-3单抗的代理活性被观察到，没有对患者有用的生物标志物 到目前为止，人们已经提出了分层的建议。受到大量研究的推动，研究表明 LAG-3的免疫抑制作用在很大程度上不依赖于MHC-II，我们首次发现 纤维蛋白原样蛋白1(FGL1)是LAG-3的高亲和力和主要功能配体，其相互作用界面为 有别于MHC-II。FGL1是一种肝细胞分泌的蛋白质，可以在循环中检测到。我们发现 可溶性FGL1以LAG-3依赖的方式抑制抗原特异性T细胞的激活。FGL1基因消融或 小鼠抗体阻断促进已建立的小鼠肿瘤的抗肿瘤免疫，即使在FGL1阴性的小鼠 肿瘤，提示宿主来源的FGL1的功能作用。与其在正常组织中的最低表达相反 除肝脏外，FGL1在多种癌症中由癌细胞上调，如非小细胞肺癌 (NSCLC)和黑色素瘤；然而，肿瘤相关FGL1在控制肿瘤免疫中的作用仍然是 不清楚。目前主要的抗LAG-3临床方案主要集中在那些仅阻断MHC-II/LAG-3的方案 相互作用，不会引发任何单主体活动。因此，机械地剖析 FGL1/LAG-3轴在肿瘤微环境中的免疫功能设计更好的LAG-3靶向 临床应用途径。在这个项目中，通过两个互补的目标，我们将利用我们的 在癌症免疫学和免疫治疗方面有丰富的经验和专业知识，以1)确定功能作用 肿瘤相关FGL1对肿瘤免疫的调节作用及致癌基因Kras的致癌机制 突变和功能生物标记物在预测免疫治疗反应中的价值；2)发展FGL1引导的， 新一代抗LAG-3癌症免疫疗法。我们将比较我们的内部抗肿瘤药物的抗肿瘤效果 人或鼠LAG-3抗体可不同地阻断FGL1/MHC-II之一或两者并映射LAG-3 通过低温电子显微镜和X射线结晶学研究这些抗体的结合界面。总而言之，我们建议的 研究将显著加深我们对新定义的FGL1/LAG-3轴的理解，并促进新的 治疗人类癌症的免疫疗法设计。

Project Summary Resistance to anti-CTLA-4, PD-1/PD-L1 (PD) T cell-based immune checkpoint blockade (ICB) therapy in large subsets of cancer patients necessitates the search for other tumor-associated T cell immune checkpoints that critically affect tumor immunity. Lymphocyte activating gene 3 (LAG-3) is a T-cell inhibitory receptor and represents a promising target for cancer immunotherapy, as monoclonal antibodies (mAb) blocking LAG-3 with its known ligand, MHC-II, in combination with anti-PD-1 show modest but promising efficacy in recent clinical trials. The FDA recently accepted for priority review the biologics license application for LAG-3 blocking mAb relatlimab and anti-PD-1 nivolumab fixed-dose combination for treating melanoma. However, very few single- agent activities of these MHC-II blocking anti-LAG-3 mAbs were observed, and no useful biomarkers for patient stratification have been suggested thus far. Motivated by numerous studies suggesting that the immunosuppressive effect of LAG-3 is largely independent of MHC-II, we identified for the first time that fibrinogen-like protein 1 (FGL1) is a high affinity and major functional ligand for LAG-3 whose interaction interface is distinct from MHC-II. FGL1 is a hepatocyte-secreted protein that can be detected in circulation. We found that soluble FGL1 inhibits antigen-specific T cell activation in a LAG-3 dependent manner. FGL1 genetic ablation or antibody blockade in mice promotes anti-tumor immunity in established mouse tumors, even in FGL1 negative tumors, suggesting a functional role of host-derived FGL1. In contrast to its minimal expression in normal tissues except the liver, FGL1 is upregulated by cancer cells in multiple cancers, such as non-small cell lung cancer (NSCLC) and melanoma; however, the role of tumor-associated FGL1 in the control of tumor immunity is still unclear. The major anti-LAG-3 clinical programs currently focus on those blocking only the MHC-II/LAG-3 interaction, which do not elicit any single-agent activities. Thus, it is critical to mechanistically dissect the immunological function of the FGL1/LAG-3 axis in the tumor microenvironment to design better LAG-3 targeted approaches for clinical application. In this project and through two complementary aims, we will leverage our extensive experience and expertise in cancer immunology and immunotherapy to 1) determine the functional role of tumor-associated FGL1 in modulating tumor immunity, its cancer induction mechanism by oncogenic Kras mutations and functional biomarker value in predicting responses to immunotherapies; 2) develop FGL1-guided, next-generation anti-LAG-3 cancer immunotherapy. We will compare the anti-tumor efficacy of our in-house anti- human or mouse LAG-3 antibodies that differentially block either or both FGL1/MHC-II and map the LAG-3 binding interfaces of these antibodies via CryoEM and X-ray crystallographic studies. In sum, our proposed studies will significantly deepen our understanding of the newly defined FGL1/LAG-3 axis and foster new immunotherapy designs for treating human cancers.