Multifunctional Nanoprobe for Early Appraisal of Immunotherapeutic Efficacy

项目摘要 尽管成像技术取得了进步，但准确评估患者的病情需要数周（通常为12-14周）。 免疫治疗的疗效。这种时间滞后对于最终非- 这些药物可能会有反应或复发，因为它们会产生繁重的副作用，并且在替代治疗中会延迟。到 加快与免疫疗法相关的癌症研究，并利用免疫疗法的潜力， 能够早期评估免疫疗法的疗效是至关重要的。拟议努力的目标是 设计、合成和验证多功能纳米探针， 免疫疗法;和（ii）肿瘤内T细胞的细胞毒性潜力的早期检测。考虑到这一点， 具体目标1：设计合成多功能纳米探针 包括作为靶向免疫抑制剂的免疫检查点抑制剂抗体和 荧光团-底物复合物作为预测性生物标志物;和具体目标2： 多功能纳米探针的免疫潜力，以及其快速监测对 免疫疗法我们将利用金纳米颗粒作为载体来递送抗PD-1抗体 （免疫抑制剂）和颗粒酶B的特异性可裂解底物与荧光报告分子 元素到T细胞。抗PD-1抗体将使用免疫印迹法与纳米颗粒化学缀合。 聚乙二醇（PEG）接头。报告者，一种荧光素标记的特异性颗粒酶B底物，也将被 使用另一个PEG连接体将所述载体纳米颗粒偶联到载体纳米颗粒。活化的T细胞与 癌细胞应该导致颗粒酶B的局部分泌和底物的裂解，释放 荧光素等离子体荧光激活的竞争性荧光免疫测定将用于检测 在血浆和随后的尿液中释放报告物（荧光素）。纳米颗粒的尺寸将被选择为 利用增强的渗透和保留作用来改善通常在肿瘤中看到的浸润。 在可活化纳米探针设计的离体优化之后，我们将测试监测纳米探针的能力。 体内免疫治疗诊断功效。在C57 BL/6小鼠中建立的黑色素瘤癌症模型 使用B16-F10黑色素瘤细胞，将用于该目的，尽管该策略是癌症不可知的。到 研究纳米探针的治疗效果和报告能力，肿瘤体积和存活率将是 测量，并将结果与血清和尿液荧光素分析结果进行相关性验证 的治疗效果。显然，这是一项临床前研究，具有最终人类的转化意义。 问题研究总体目标是在早期评估患者免疫治疗的疗效 使患者免于治疗的副作用和经济负担（对保险业和/或 患者持续但不持久的治疗。该策略适用于任何采用 单独免疫治疗或免疫治疗与常规化疗联合

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