Project 4:Targeting M2-like Macrophages and MDSC with Myelolytic-Virotherapy

摘要-项目4： 患有复发性或转移性实体瘤的儿科患者的治愈率仍然低得不可接受。癌 免疫疗法有很大的希望，但令人失望的研究分数突出了我们的相对无知， 了解实体瘤内的免疫抑制微环境。因为他们在 介导免疫抑制的肿瘤相关巨噬细胞（TAM），通常“极化”为所谓的M2- 如免疫抑制表型和髓源性抑制细胞（MDSC），被认为是重要的 治疗目标我们已经发现了一种临床上可行的策略，同时减少TAM/MDSC（我们称之为 “溶髓性”）并极化微环境（通过溶瘤病毒感染），导致显著的抗肿瘤作用。 功效我们假设，通过联合“溶髓”疗法和促骨髓增生治疗，靶向TAM和MDSC， 炎性疗法激活导致癌症消退的先天抗肿瘤机制， 重塑实体瘤微环境，使其更适合细胞免疫疗法。在aim中 1，我们将确定联合骨髓溶解病毒疗法驱动肿瘤的机制， 回归我们将使用荧光纳米金刚石等新技术来确定对先天性 免疫细胞吞噬肿瘤细胞。我们将利用基因组学和免疫监测共享资源 核心B由Elaine Mardis博士指导，使用飞行时间质谱法和单克隆抗体进行流式细胞术。 细胞转录组学以确定对免疫细胞组成和极化的影响。我们还将利用 功能获得和功能丧失的方法，以确定MDSC的丧失是否对肿瘤消退至关重要 用骨髓溶解病毒疗法我们还将在异种移植和免疫活性模型中测试联合疗法 其他癌症类型，以确认其普遍性（骨肉瘤，尤因肉瘤，横纹肌肉瘤， 神经母细胞瘤）。在目标2中，我们将确定髓细胞溶解病毒疗法对T细胞介导的 免疫疗法我们将检查单独的髓细胞溶解和与病毒疗法联合使用对疗效的影响。 抗肿瘤T细胞在T细胞耗竭设置和CAR-T细胞（与项目1负责人Dean Lee和共同 研究者Ruoning Wang，PI-DDN U 01成员）。我们将与核心B一起研究对T细胞的影响， 使用TCR测序的克隆性。在目标3中，我们将确定联合骨髓溶解病毒治疗是否 增强基于NK的细胞疗法的功效。我们将与项目1负责人Dean Lee合作， 项目2负责人Mitch Cairo研究对过继性NK和CAR-NK细胞治疗的影响。总的来说，有了这个 项目，我们将进一步阐明，测试和开发策略，以调节肿瘤微环境，以促进 先天免疫细胞作为癌症治疗。我们的发现可能适用于广泛的儿科癌症 类型，因此非常适合儿科免疫疗法发现和开发网络的目标。

Abstract - Project 4: Cure rates for pediatric patients with relapsed or metastatic solid tumors remain unacceptably low. Cancer immunotherapies hold great promise, but scores of disappointing studies highlight our relative ignorance in understanding the immunosuppressive microenvironment within solid tumors. Because of their central role in mediating immunosuppression, tumor associated macrophages (TAMs), typically “polarized” to a so-called M2- like immunosuppressive phenotype, and myeloid-derived suppressor cells (MDSC), are thought to be important therapeutic targets. We have found a clinically viable strategy that simultaneously reduces TAMs/MDSC (we dub “myelolytic”) and polarizes the microenvironment (via oncolytic virus infection), resulting in significant antitumor efficacy. We hypothesize that targeting TAM and MDSC by combining “myelolytic” therapies with pro- inflammatory therapies activates innate antitumor mechanisms that cause cancer regressions and reshapes the solid tumor microenvironment to be more permissive to cellular immunotherapies. In aim 1, we will determine the mechanism(s) by which combined myelolytic-virotherapy drives tumor regressions. We will use novel technologies such as fluorescent nanodiamonds to determine effects on innate immune cell phagocytosis of tumor cells. We will utilize the Genomics & Immune Monitoring Shared Resource Core B directed by Dr. Elaine Mardis to conduct flow cytometry with time-of-flight mass spectrometry and single cell transcriptomics to determine the effects on immune cell composition and polarization. We will also utilize gain- and loss-of-function approaches to determine if loss of MDSC are critical for enabling tumor regressions with myelolytic-virotherapy. We will also test combination therapies in xenograft and immunocompetent models of other cancer types to confirm its generalizability (osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, neuroblastoma). In aim 2, we will determine the effects of myelolytic-virotherapy on T cell-mediated immunotherapies. We will examine the effect of myelolysis alone and combined with virotherapy on the efficacy of antitumor T cells in a T cell exhaustion setting and with CAR-T cells (with Project 1 Leader Dean Lee and co- investigator Ruoning Wang, PI-DDN U01 member). We will work with Core B to examine the effects on T cell clonality using TCR sequencing. In aim 3, we will determine whether combined myelolytic-virotherapy enhances the efficacy of NK-based cellular therapies. We will work with Project 1 Leader Dean Lee and Project 2 Leader Mitch Cairo to study the effects on adoptive NK and CAR-NK cell therapy. Overall, with this project we will further elucidate, test and develop strategies to modulate the tumor microenvironment to facilitate innate immune cells as cancer therapy. Our findings may be applicable across a broad panel of pediatric cancer types and thus fits well into the aims of the Pediatric Immunotherapy Discovery and Development Network.