Non-invasive imaging of the anti-tumor immune response

项目摘要 使用检查站封锁的免疫疗法使癌症治疗发生了革命性的变化。直接治疗的结果 通过检查点封锁对肿瘤微环境(TME)施加改变的结果。然而，只有一个 部分患者有反应。是什么控制了这种差距，人们知之甚少。我们建议开发和应用 工具可以帮助区分不同临床前肿瘤模型中的应答者和非应答者 治疗的开始。我们已经证明，免疫正电子发射断层扫描(免疫正电子发射断层扫描)可以使用 监测特定免疫细胞亚群，即T细胞和髓系细胞的渗透状态。我们用小的 (~15 kDa)驼源性单域抗体(纳米体)，对其靶标具有Nm-Pm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 品质和敏感度。即使是显示相对较低丰度的蛋白质的细胞，如CTLA-4，也可以清楚地 已成像。我们已经在几个(同基因)肿瘤模型中表明，监测细胞毒性T细胞的动态 在TME中可用于区分早期应答者和无应答者。这一观察使我们能够 将动物分为反应者和无反应者，然后切除它们的肿瘤，分离免疫渗透 细胞，并对其进行单细胞RNA测序。这些数据表明，髓系间室和 其产生的细胞因子和趋化因子在决定抗PD-1治疗结果中起着重要作用。 我们建议将这些初步发现扩展到其他小鼠肿瘤模型，因为有不同的起源细胞。 这导致了它们以及它们对免疫干预的敏感度的差异。这个项目的目标是 使用免疫PET揭示TME早期发生的关键变化。我们的互补性 分子分析将有助于设计更有效的治疗方法。应答者TME中的巨噬细胞和DC 产生CXCL9，这是一种细胞毒性T细胞的趋化剂，有助于维持其激活状态。这种趋化因子 因此，在抗PD-1治疗的结果中起着关键作用。因此，我们建议通过使用以下方式重新设计TME 化学制造新型CXCL9-融合蛋白，并将它们输送到TME。单域抗体是 这种融合的完美候选者。它们的体积小，具有良好的组织渗透性和高亲和力 确保向TME高效交付，并在TME中保留。成像CXCL9或其受体的分布将脱落 进一步阐明抗肿瘤免疫状态。因此，我们将产生纳米体作为特定的显像剂 这些细胞因子和它们的受体。

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.