Non-invasive imaging of the anti-tumor immune response

项目概要 使用检查点封锁的免疫疗法彻底改变了癌症治疗。直接影响治疗效果 由于检查点封锁对肿瘤微环境（TME）造成的变化。然而，只有一个 部分患者有反应。人们对是什么控制了这种差异知之甚少。我们建议开发和应用 很快就能帮助区分不同临床前肿瘤模型中的反应者和非反应者的工具 治疗的开始。我们已经证明可以使用免疫正电子发射断层扫描（Immuno-PET） 监测特定免疫细胞亚群（即 T 细胞和骨髓细胞）的浸润状态。我们用小 (~15 kDa) 骆驼衍生的单域抗体（纳米抗体），其靶标具有 nM 到 pM 的亲和力 进行免疫 PET 成像。我们独特的化学方法提供了前所未有的显像剂 质量和灵敏度。即使是表现出丰度相对较低的蛋白质（例如 CTLA-4）的细胞也可以清楚地 成像。我们已经在几个（同基因）肿瘤模型中展示了监测细胞毒性 T 细胞动态的方法 TME 中的信息可用于区分早期响应者和非响应者。这一观察使我们 将动物分为有反应者和无反应者，然后切除肿瘤，分离免疫浸润 细胞，并对它们进行单细胞 RNA 测序。这些数据表明，骨髓室和 它产生的细胞因子和趋化因子在决定抗 PD-1 治疗的结果中起着重要作用。 考虑到不同的细胞来源，我们建议将这些初步发现扩展到其他小鼠肿瘤模型 这导致了它们以及它们对免疫干预的易感性差异。该项目旨在 使用免疫 PET 揭示 TME 早期发生的关键变化。我们的互补 分子分析将有助于设计更有效的疗法。应答者 TME 中的巨噬细胞和 DC 产生 CXCL9，一种细胞毒性 T 细胞的化学引诱剂，有助于维持其激活状态。这种趋化因子 因此，它是抗 PD-1 治疗结果的关键因素。因此，我们建议通过使用来重新设计 TME 化学来制造新型 CXCL9 融合蛋白并将其递送至 TME。单域抗体是 这种融合的完美候选者。它们尺寸小，具有出色的组织渗透性和高亲和力 确保高效地交付到 TME 并保留在 TME 中。 CXCL9或其受体的分布成像会脱落 进一步阐明抗肿瘤免疫状态。因此，我们将生成纳米抗体作为特异性显像剂 对于此类细胞因子及其受体。

Project Summary Immunotherapy using checkpoint blockade has revolutionized cancer treatment. The outcome of therapy directly results from changes imposed on the tumor microenvironment (TME) by checkpoint blockade. However, only a subset of patients respond. What controls this disparity is poorly understood. We propose to develop and apply tools that can help differentiate responders from non-responders in different pre-clinical tumor models soon after the start of treatment. We have shown that immuno-positron emission tomography (Immuno-PET) can be used to monitor infiltration status of specific subsets of immune cells, namely T cells and myeloid cells. We use small (~15 kDa) camelid-derived single domain antibodies (nanobodies) that have nM to pM affinity for their targets to perform immuno-PET imaging. Our unique chemical approaches provide imaging agents of unprecedented quality and sensitivity. Even cells that display proteins of relatively low abundance such as CTLA-4 can be clealrly imaged. We have shown in several (syngeneic) tumor models that monitoring the dynamics of cytotoxic T cells in the TME can be used to distinguish early responders from non-responders. This observation has allowed us to stratify animals into responders and non-responders, then excise their tumors, isolate the immune infiltrating cells, and subject these to single-cell RNA sequencing. These data show that the myeloid compartment and the cytokines and chemokines it produces, plays a major role in determining the outcome of anti PD-1 treatment. We propose to expand these initial findings to additional mouse tumor models, given the distinct cells of origin that give rise to them and their differences in susceptibility to immune intervention. This project is aimed at bringing to light key changes that take place in the TME early on, using immuno-PET. Our complementary molecular analyses will help design more effective therapies. Macrophages and DCs in the TME of responders produce CXCL9, a chemoattractant for cytotoxic T cells that helps maintain their activated state. This chemokine is therefore a key player in the outcome of anti-PD-1 therapy. We thus propose to re-engineer the TME by using chemistry to make novel CXCL9-fusion proteins and deliver them to the TME. Single-domain antibodies are perfect candidates for such fusions. Their small size allows excellent tissue penetration and their high affinity ensures efficient delivery to, and retention in, the TME. Imaging the distribution of CXCL9 or its receptor will shed further light on the anti-tumor immune status. We will therefore generate nanobodies as imaging agents specific for such cytokines and their receptors.