Imaging Modulation of Immune Phenotype

免疫表型的成像与调控 最重要的是，我们已经创造了TLR递送纳米疗法，并发现TLR NPs与CD40结合， Apd-1和aCTL4(在新兴的胰腺癌研究中缩写为CP4)被有效地回归植入 多部位侵袭性小鼠胰腺肿瘤。我们制定了多种战略，并特别专注于 在18 nm可生物降解的多功能颗粒上结合免疫调节肽、靶向肽 和Toll样受体(TLR)激动剂。我们特别加入了免疫调节肽PADRE(T Helper 调节)和甘露糖(增强巨噬细胞摄取)以及TLR7/8激动剂(Resquimod)。 前期工作。为了最大限度地增加有效载荷，我们通过 高效酯化和无金属点击反应相结合，发现颗粒代谢物 从肾脏中清除。在我们的初步研究中，TLR7/8-纳米颗粒治疗与CP4联合 高转移性、多部位植入性胰腺癌模型(Kras+/LSL-G12D； Trp53+/LSL-R172H；Pdx1-Cre模型：缩写为KPC)。新的初步数据表明，TLR7/8激动剂和 ACD40对胰腺肿瘤细胞均有直接杀伤作用。RNAseq结果表明TLR7/8 激动剂和CD40增强互补途径(CD40的C-凝集素(等)和TLR/干扰素 用于TLR激动剂)。我们发现，这种组合增强了抗肿瘤白细胞，逆转了KPC肿瘤，并对 应答者，100%在重新挑战时不会长出肿瘤。通过监测OX40的表达(T细胞的标志 激活)，我们证明了与其他涉及CD40的免疫调节方法不同，T细胞是 激活了。我们同时开发了使用正电子发射来监测OX40表达的能力 以非侵入性方式进行断层扫描。我们提议的工作的主要目标是将纳米治疗策略 期待人工翻译。因此，我们将在啮齿动物胰腺癌模型中评估疗效。 在更大的动物模型中是安全的。此外，我们将评估接受活组织检查的患者的样本 胰腺癌，以更好地描述免疫环境。我们有两个主要目标：1)发展 系统管理T细胞调节的有效策略以及2)将其与正电子相结合 发射断层成像和RNA测序，以优化多组分方案。在目标1中，我们 将通过1a)调制纳米颗粒来确定最优载体特性以最大化T细胞调节 特征和评估结果的效果，1)使用正电子发射断层扫描(PET)成像 量化系统注射的NP激动剂的累积，以及1c)通过剂量评估毒性 升级和一项大型动物研究，导致IND申请。在目标2中，开发一种成像和体外 利用2a)OX40 PET成像和2b)流式细胞术评估T细胞活化的策略 RNA测序。

Imaging and modulation of immunophenotype Most importantly, we have created TLR delivery nanotherapeutics and find that TLR NPs combined with aCD40, aPD-1 and aCTL4 (abbreviated as CP4 as in emerging pancreatic cancer studies) efficiently regressed implanted multisite invasive murine pancreatic tumors. We have developed multiple strategies and are particularly focused on 18 nm biodegradable, multi-functional particles that combine immune-modulating peptides, targeting peptides and toll-like receptor (TLR) agonists. We specifically included the immune modulating peptide PADRE (T helper modulation) and mannose (to enhance macrophage uptake) in addition to the TLR7/8 agonist (resiquimod) in preliminary work. To maximize payload, we built upon biocompatible unimicellar nanoparticles via the combination of highly efficient esterification and metal-free click reactions and find that the particle metabolites clear through the kidneys. In our preliminary studies, TLR7/8-nanoparticle treatment combined with CP4 enhanced response in a highly metastatic, multi-site implanted pancreatic cancer model (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre model: abbreviated as KPC). New preliminary data indicate that TLR7/8 agonists and aCD40 each have direct efficacy against pancreatic tumor cells. RNAseq results demonstrate that TLR7/8 agonists and CD40 enhance complementary pathways (C-lectin for CD40 (among others) and TLR/interferon for TLR agonists). We find that the combination enhances anti-tumor leukocytes, regresses KPC tumors and for responders, 100% do not grow tumor on re-challenge. By monitoring OX40 expression (a marker of T cell activation), we demonstrated that unlike other immune modulating approaches involving aCD40, T cells were activated. We have simultaneously developed the ability to monitor OX40 expression using positron emission tomography in a noninvasive fashion. Our primary goal in the proposed work is to move the nanotherapy strategy forward to human translation. As a result, we will evaluate efficacy in models of pancreatic cancer in rodents and safety in a larger animal model. Further, we will evaluate samples from patients undergoing biopsy for pancreatic cancer to better characterize the immune environment. We have 2 major goals: 1) the development of an effective strategy for systemically-administered T cell modulation and 2) combining this with positron emission tomographic imaging and RNA sequencing to optimize multi-component protocols. Within Aim 1, we will determine the optimal carrier properties to maximize T cell modulation by 1a) modulating nanoparticle characteristics and evaluating resulting efficacy, 1b) using positron emission tomography (PET) imaging to quantify accumulation of the systemically-injected NP agonists, and 1c) assessing toxicity through dose escalation and a large animal study, leading to IND filing. Within Aim 2, develop an imaging and in vitro assessment strategy for T cell activation by utilizing 2a) OX40 PET imaging and 2b) flow cytometry and RNAsequencing.