Non-invasive imaging of the anti-tumor immune response

项目摘要 使用检查点阻断的免疫疗法彻底改变了癌症治疗。治疗的结果直接 由检查点阻断对肿瘤微环境（TME）施加的变化引起。但只有 部分患者有反应。控制这种差异的因素尚不清楚。我们建议开发和应用 这些工具可以帮助在不同的临床前肿瘤模型中区分反应者和非反应者， 治疗的开始。我们已经证明，免疫正电子发射断层扫描（免疫PET）可以用于 监测免疫细胞的特定亚群，即T细胞和骨髓细胞的浸润状态。我们使用小 （~15 kDa）骆驼衍生的单结构域抗体（纳米抗体），其对其靶具有nM至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成像剂 质量和灵敏度。即使是显示相对低丰度的蛋白质（如CTLA-4）的细胞也可以清楚地表达。 成像。我们已经在几个（同源）肿瘤模型中表明，监测细胞毒性T细胞的动态 可以用来区分早期反应者和无反应者。这一观察使我们能够 将动物分为应答者和非应答者，然后切除肿瘤，分离免疫浸润， 细胞，并对这些细胞进行单细胞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确定抗PD-1治疗的结果中起主要作用。 我们建议将这些初步的发现扩展到其他小鼠肿瘤模型，考虑到不同的细胞来源 以及它们对免疫干预敏感性的差异。该项目旨在 使用免疫PET，揭示TME早期发生的关键变化。我们互补的 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CXCL 9，一种细胞毒性T细胞的化学引诱物，有助于维持其激活状态。该趋化因子 因此，它是抗PD-1治疗结果的关键因素。因此，我们建议重新设计TME， 通过化学方法制备新型CXCL 9融合蛋白并将其递送至TME。单结构域抗体是 完美的融合体它们的小尺寸允许优异的组织渗透和它们的高亲和力 确保有效地向TME交付和保留TME。对CXCL 9或其受体的分布进行成像， 进一步阐明抗肿瘤免疫状态。因此，我们将产生作为显像剂特异性的纳米抗体 这些细胞因子及其受体。

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.