Project 4: Modulating the natural killer cell response to oHSV1 in recurrent human GBM

项目概要-项目4 这项建议的最终目标是了解先天免疫在溶瘤背景下的作用。 单纯疱疹病毒（oHSV）治疗胶质母细胞瘤（GBM），一种高度致命的脑肿瘤。GBM的oHSV治疗 依赖于病毒的癌症特异性复制，导致肿瘤破坏，对邻近的肿瘤具有最小的毒性。 非肿瘤组织。它在患者中的安全性已被证明，但显着疗效的证据仍有待证实。 确立了习项目4将关注oHSV给药后宿主的自然杀伤（NK）细胞反应。 由于其强大的抗病毒特性，NK细胞代表了oHSV治疗的潜在屏障。或者， 抗肿瘤NK细胞应答具有增强oHSV治疗的肿瘤清除性质的潜力。我们 先前发表的研究表明：1）NK细胞迅速响应oHSV并在病毒感染前消除病毒， 它可以有效地复制和传播整个GBM; 2）短暂的免疫调节， NK细胞对oHSV的应答，即使单次注射TGF-β，也显著增强oHSV复制， 扩散和抗肿瘤功效。因此，我们假设NK细胞协调一个细胞因子。 初始oHSV给药后的强烈炎症反应限制了oHSV的复制、扩散和 肿瘤溶解，从而为GBM的有效oHSV治疗创造屏障。这个假设的一个推论是 通过了解NK细胞如何识别oHSV感染的GBM，我们可以最好地调节这一过程， 优化这种针对GBM的高选择性疗法。因此，我们最近开发了一种新的测定方法， 将每个HSV 1基因克隆到载体中，使我们能够确定其诱导或抑制NK细胞的能力， activation.我们发现了NK细胞识别和破坏HSV感染靶点的机制， 对初次免疫反应的发展有很大影响我们称这种基本的新发现为被动抗体 依赖性细胞毒性或被动ADCC，由此IgG的Fc片段（IgGFc）形成桥 在NK细胞Fc受体（称为CD 16）和表达HSV Us 8的GBM细胞之间， Fc结合蛋白糖蛋白E（gE）。CD 16-IgGFC-gE三元复合物激活NK细胞， oHSV感染的GBM，而不需要IgG的抗原特异性Fab部分。在这份报告中，项目 4将：（1）进一步研究被动ADCC，以最好地了解如何调节该过程，以提高 用项目1观察oHSV治疗GBM的疗效;（2）确定TGF-β信号转导在调节GBM中的作用。 NK细胞与oHSV编码的病毒蛋白的相互作用，特别是与被动ADCC和NOTCH相关的相互作用 （3）评估复发性GBM患者的NK细胞免疫应答， 在项目2中接收我们的oHSV，rQNestin34.5。通过阐明NK细胞如何识别和破坏oHSV- 感染的肿瘤在其他促炎和抗炎过程的背景下，我们将最好地了解如何 优化GBM的神经选择性治疗，并识别导致NK的关键机制信号， 细胞介导的病毒感染清除。

PROJECT SUMMARY – PROJECT 4 The ultimate goal of this proposal is to understand the role of innate immunity within the context of oncolytic herpes simplex viral (oHSV) therapy for glioblastoma (GBM), a highly fatal brain tumor. oHSV treatment of GBM relies on cancer-specific replication of the virus leading to tumor destruction with minimal toxicity to adjacent non-neoplastic tissue. Its safety in patients has been proven, yet evidence for significant efficacy remains to be established. Project 4 will focus on the host's natural killer (NK) cell response following oHSV administration. Due to their strong antiviral properties, NK cells represent a potential barrier to oHSV therapy. Alternatively, the anti-tumor NK cell response has the potential of augmenting the tumor clearing properties of oHSV therapy. Our previously published studies demonstrated that: 1) NK cells rapidly respond oHSV and eliminate the virus before it can effectively replicate and disseminate throughout the GBM; 2) transient immune modulation delaying the NK cell response to oHSV, even with a single injection of TGF-beta, significantly enhances oHSV replication, spread and anti-tumor efficacy following inoculation. As a result, we hypothesize that NK cells coordinate a robust inflammatory response following initial oHSV administration that limits oHSV replication, spread, and tumor lysis, thereby creating a barrier to effective oHSV therapy for GBM. A corollary to this hypothesis is that by understanding how NK cells recognize oHSV-infected GBM we can best modulate this process so as to optimize this highly selective therapy for GBM. We therefore recently developed a novel assay whereby we cloned each HSV1 gene into a vector that allowed us to determine its ability to either induce or inhibit NK cell activation. We discovered the mechanism by which NK cells recognize and destroy HSV-infected targets prior to the development of a primary immune response. We term this fundamental, novel discovery passive antibody dependent cellular cytotoxicity or passive ADCC, whereby the Fc fragment of IgG (IgGFc) forms a bridge between the NK cell Fc receptor for IgGFc, called CD16, and the GBM cell expressing HSV Us8, which encodes the Fc binding protein glycoprotein E (gE). The CD16-IgGFc-gE ternary complex activates NK cells to destroy oHSV-infected GBM without the requirement for the antigen-specific Fab portion of IgG. In this proposal, Project 4 will: (1) further investigate passive ADCC to best understand how to modulate the process so as to enhance the efficacy of oHSV therapy for GBM with Project 1; (2) determine the role of TGF-β signaling in regulating the NK cell interaction with oHSV-encoded viral proteins, specifically as relates to passive ADCC and to NOTCH signaling with Project 3; (3) To assess the NK cell immune response in patients with recurrent GBM who will be receiving our oHSV, rQNestin34.5 in Project 2. By elucidating how NK cells recognize and destroy oHSV- infected tumors in the context of other pro- and anti-inflammatory processes, we will best understand how to optimize this neuro-selective treatment for GBM as well as discern the key mechanistic signals leading to NK cell-mediated clearance of viral infection.