Micellar formulation provides potent and tolerable TLR7 agonist treatment for anti-cancer immunotherapy

Data file for manuscript

This study aimed at improving the therapeutic index for TLR7 agonists by formulating the agonist in nanoparticles. Initially, we investigated the optimal nanoparticle formulation with regards to anti-PEG antibody response and accelerated blood clearance in vivo. Following this, we determined biodistribution as well as anti-tumor activity of 1V270-micelles against a commonly used TLR7 agonist using syngeneic cancer models. The immunological mechanism of action was characterized for immediate and adaptive immunological changes on a single cell level using flow cytometry and on mRNA level using Nanostring. Anti-tumor activity was evaluated in additional subcutaneous syngeneic cancer models in combination with checkpoint blockade. The immediate effects were then confirmed in cancer models having showed differential efficacy. Finally, tolerability was evaluated in cynomolgus monkeys in a dose escalating study.
All experiments were based on protocols with specific research questions, with a clear design and plan for analysis. Treatment of tumor-bearing animals were only conducted on established tumors (mean volume of ≈100 mm3), which for most models occurred between 7-14 days post inoculation, and after being subjected to a stratified block randomization based on tumor volume. Group sizes were determined based on prior experience. Experimental groups were typically composed of five to ten animals per group to ensure statistical power. Lower number of animals were used with larger animals for ethical reasons. In non-tumor bearing mouse studies, mice were subjected to a stratified block randomization based on weight before treatments. No exclusion criteria were defined. Animals were kept under controlled circumstances (light conditions and air temperature and humidity) and acclimatized for at least 5 days prior to study conduct. Investigators were blinded during some of the efficacy studies and treatment and measurements order was random in some experiments. Laboratory animals’ care was in accordance with institutional guidelines. No outliers were removed from data.

手稿数据文件

本研究旨在通过将TLR7激动剂（TLR7 agonist）制备成纳米颗粒以提高其治疗指数。首先，我们针对体内抗PEG抗体反应及加速血液清除效应，探究了纳米颗粒的最优制剂配方。随后，我们利用同基因癌症模型，测定了1V270胶束相对于常用TLR7激动剂的体内分布及抗肿瘤活性。我们通过流式细胞术在单细胞水平、以及通过Nanostring技术在mRNA水平，表征了其作用的免疫学机制——包括即时和适应性免疫变化。我们在额外的皮下同基因癌症模型中，结合检查点阻断（checkpoint blockade）评估了其抗肿瘤活性。

所有实验均基于具有明确研究问题、清晰设计及分析计划的方案开展。对荷瘤动物的治疗仅在肿瘤确立后进行（平均体积≈100 mm³），大多数模型中这一时间点为接种后7-14天，且动物已根据肿瘤体积进行分层区组随机化。每组动物数量基于既往经验确定；实验组通常每组5至10只动物以确保统计效力；出于伦理考虑，大型动物实验中使用的动物数量较少。在无瘤小鼠研究中，治疗前小鼠根据体重进行分层区组随机化；未设定排除标准。动物饲养于受控环境（光照、温度及湿度）中，且在实验开始前至少适应5天；在部分疗效研究（efficacy studies）中研究者采用盲法，部分实验中治疗及测量顺序随机化。实验动物的护理遵循机构指南；数据未剔除异常值。