Multifunctional Nanoprobe for Early Appraisal of Immunotherapeutic Efficacy

项目摘要 尽管成像技术取得了进步，但仍需要数周(通常为12-14周)才能准确评估 免疫治疗的疗效。这一时间延迟对于那些最终不是 有反应或复发，因为它们会产生繁重的副作用，并在替代治疗中延迟。至 加快与免疫治疗相关的癌症研究，利用免疫治疗这一技术的潜力 能够及早评估免疫治疗的疗效至关重要。拟议工作的目标是 设计、合成和验证一种多功能纳米探针，以(I)有效地给药 免疫治疗；以及(Ii)早期检测肿瘤内T细胞的细胞毒性潜力。考虑到这一点， 提出了以下具体目标：具体目标1：设计和合成多功能纳米探针 包括作为靶向免疫治疗剂的免疫检查点抑制抗体和 作为预测生物标记物的荧光团-底物复合体；以及特定目标2：体内验证 多功能纳米探针的免疫治疗潜力，以及快速监测对 免疫疗法。我们将利用金纳米颗粒作为载体来传递抗PD-1抗体 (免疫治疗剂)和具有荧光报告的颗粒酶B的特定可切割底物 元素转化为T细胞。抗PD-1抗体将通过化学方法连接到纳米颗粒上 聚乙二醇链接剂。记者，一种荧光素标记的特定颗粒酶B底物，也将被 使用另一种聚乙二醇链接物连接到载体纳米颗粒上。活化的T细胞与T细胞的相互作用 癌细胞应导致颗粒酶B的局部分泌和底物的裂解，释放 荧光素。将使用血浆-荧光启用的竞争性荧光免疫分析来检测 在血浆和随后的尿液中释放报告(荧光素)。纳米颗粒的大小将被选择为 利用增强的渗透和滞留效应来改善肿瘤中常见的渗透。 在对可激活纳米探针的设计进行体外优化后，我们将测试监控 体内免疫热疗效果。C57BL/6小鼠黑色素瘤模型的建立 使用B16-F10黑色素瘤细胞，将用于这一目的，尽管这一策略是癌症不可知的。至 调查纳米探针的治疗效果和报告能力，肿瘤体积和存活率将 测量，并将结果与血清和尿液荧光素分析的结果相关联以进行验证 对疗效的影响。显然，这是一项临床前研究，对最终的人类具有翻译意义。 学习。总体目标是在早期评估免疫疗法对患者的疗效。 使患者避免治疗的副作用和经济负担(给保险业和/或 患者)持续但不持久的治疗。这一策略适用于任何治疗方案 单用免疫疗法或免疫疗法联合常规化疗

Project Summary Despite advances in imaging technologies, it takes weeks (typically 12-14 weeks) to accurately assess the efficacy of immunotherapy treatment. This time lag is highly undesirable for patients who are ultimately non- responsive or relapse, as they are subject to onerous side effects and are delayed in alternate treatments. To expedite immunotherapy related cancer research and harness the potential of immunotherapy, a technology capable of early assessment of immunotherapy efficacy is critically important. The goal of the proposed effort is to design, synthesize and validate a multifunctional nanoprobe for (i) efficiently administering immunotherapy; and (ii) early detection of the cytotoxic potential of the intra-tumor T cells. With this in mind, the following Specific Aims are proposed: Specific Aim 1: Design and synthesize a multifunctional nanoprobe comprised of an immune checkpoint inhibitor antibody as a targeting immunotherapeutic agent and a fluorophore-substrate complex as a predictive biomarker; and Specific Aim 2: In vivo validation of the multifunctional nanoprobe’s immunotherapeutic potential, and its ability to rapidly monitor the response to immunotherapy. We will utilize gold nanoparticles as a carrier for delivering anti-PD-1 antibody (immunotherapeutic agent) and a specific cleavable substrate for granzyme B with a fluorescent reporter element to T cells. An anti-PD-1 antibody will be chemically conjugated to the nanoparticle using a polyethylene glycol (PEG) linker. The reporter, a fluorescein-tagged specific granzyme B substrate, will also be conjugated to the carrier nanoparticle using another PEG linker. The interaction between activated T cells and cancer cells should lead to localized granzyme B secretion and cleavage of the substrate, releasing fluorescein. Plasmonic-fluor enabled competitive fluoroimmunoassay will be used for the detection of the released reporter (fluorescein) in plasma and subsequently urine. The size of the nanoparticle will be chosen to exploit the enhanced permeation and retention effect for improved infiltration typically seen in tumors. Following ex vivo optimization of the activatable nanoprobe design, we will test the ability to monitor the immuno-theranostic efficacy in vivo. An established melanoma cancer model, developed in C57BL/6 mice using B16-F10 melanoma cells, will be utilized for this purpose although this strategy is cancer agnostic. To investigate the therapeutic efficacy and reporting ability of the nanoprobes, tumor volume and survival will be measured, and the results correlated with those from the serum and urine analysis for fluorescein for validation of the therapeutic efficacy. Clearly, this is a pre-clinical study with translational implications to eventual human studies. The overall goal is to assess the efficacy of immunotherapy treatment in patients at an early time sparing patients the side effects of the therapy and the financial burden (to the insurance industry and/or patient) of continued but non-durable treatment. This strategy is applicable to any therapeutic option employing immunotherapy alone or immunotherapy in combination with conventional chemotherapy