Project 4: Awakening immune responses to GBM by enhancing immune cell trafficking and activation with oHSV armed with Cetuximab-CCL5 and anti-CD47 antibody payloads.

项目摘要--项目4 这项建议的最终目标是开发一种新颖、有效、安全的溶瘤单纯疱疹病毒(OHSV)- 恶性胶质母细胞瘤(GBM)的基础免疫治疗 脑瘤。OHSV治疗GBM依赖于癌症特异性病毒的复制，从而导致肿瘤的破坏 对邻近的非肿瘤组织毒性最小。它在患者身上的安全性已经得到证实，但有效性仍然存在。 有待改进。项目4将专注于通过用强大的OHSV武装来增强抗肿瘤免疫反应 通过测试两种新产生的OHSV，命名为OV-CMAB-CCL5和OV-CCL5来测试免疫调制有效载荷 αCD47-Ig1，旨在诱导免疫渗透和阻断检查点，分别作为单一疗法在 除了它们的组合之外。预计这两种OHSV将相互协同，最大限度地提高免疫力 靶向免疫刺激和免疫抑制的GBM微环境中的反应 创造一个全面的促炎肿瘤微环境，这已被证明是正相关的 在项目2的rQNestin34.5临床试验中患者存活。我们产生了OV-CMAB-CCL5，一种OHSV 表达可分泌的表皮生长因子受体(抗EGFR)IgG1单链可变片段 抗体西妥昔单抗与CCL5通过Fc打孔系统连接，产生异源二聚体并防止 同源二聚体。为了针对先天性免疫细胞上的检查点，我们还设计了OHSV来表达全长， 以人α为支架的可溶性抗CD47抗体(所谓OV-CD47-G1)，用于局部区域控制 GBM。病毒感染的肾小球系膜细胞分泌的α抗体阻断CD47CD47Ig_1信号 并通过以下途径发挥Fc受体介导的抗体依赖的细胞吞噬功能 巨噬细胞与NK细胞的抗体依赖性细胞毒作用。我们的数据显示，在GBM小鼠中 由于增强了抗肿瘤免疫，两种OHSV模型都缩小了肿瘤体积并延长了生存期 回应。我们推测OV-CMAB-CCL5是一种安全有效的药物，可以改善GBM的治疗。 具有多种作用机制，OV-CmAB-CCl5和OV-αCD47-Ig G_1联合治疗将 比各自的单一疗法有更好的疗效。我们制造了良好的制造规范 (GMP)级病毒在我们的内部GMP设施，现在建议进行研究新药(IND) 使研究成为可能，这样我们的创新OHSV将为未来的临床试验做好测试准备。项目4有三个 具体目标：(1)解剖全身和局部免疫反应，确定外周血液中的标志物(S) 来自项目2的小鼠模型或临床样本与抗肿瘤活性相关，并改善 RQnestin34.5.v2在OV-CMAB-CCL5治疗GBM小鼠模型中的作用；(2)在 使用GMP级OV-CMAB-CCL5进行体内疗效和毒性研究；以及(3)确定临床前疗效 OV-CMAB-CCL5与OV-αCD47-IgG1二联免疫的安全性。我们相信，我们的方法将 提供一种新颖、有效、安全的针对GBM的溶瘤病毒疗法。

PROJECT SUMMARY – PROJECT 4 The ultimate goal of this proposal is to develop a novel, effective, and safe oncolytic herpes simplex viral (oHSV)- based immunotherapy for the treatment of glioblastoma (GBM), a highly fatal and the most common malignant 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 efficacy remains to be improved. Project 4 will focus on enhancing anti-tumor immune responses by arming oHSVs with powerful immune-modulation payloads by testing two newly generated novel oHSVs, termed OV-Cmab-CCL5 and OV- αCD47-IgG1, designed to induce immune infiltration and block a checkpoint, respectively, as monotherapies in addition to their combination. The two oHSVs are expected to synergize with each other to maximize immune responses in the GBM microenvironment by targeting both immune stimulation and immune suppression to create an overall pro-inflammatory tumor microenvironment, which has been demonstrated to positively correlate with patient survival in the rQNestin34.5 clinical trial of Project 2. We generated OV-Cmab-CCL5, an oHSV expressing a secretable single-chain variable fragment of the epidermal growth factor receptor (anti-EGFR) IgG1 antibody cetuximab linked to CCL5 by an Fc knob-into-hole system that produces heterodimers and prevents homodimers. To target a checkpoint on innate immune cells, we also engineered oHSV to express a full-length, soluble anti-CD47 antibody with a human IgG1 scaffold (so-called OV-αCD47-G1), for locoregional control of GBM. The antibody αCD47-IgG1 secreted by virus-infected GBM cells blocks the CD47 “don't eat me” signal and exerts additional functions of Fc receptor-mediated antibody-dependent cellular phagocytosis by macrophages and antibody-dependent cellular cytotoxicity by NK cells. Our data show that in GBM mouse models, both oHSVs reduced tumor size and prolonged survival, owing to enhanced anti-tumor immune responses. We hypothesize that OV-Cmab-CCL5 is a safe and effective agent that can improve GBM therapy with multiple mechanisms of action, and the combination therapy of OV-Cmab-CCL5 and OV-αCD47-IgG1 will have better efficacy than the respective monotherapies. We have manufactured Good Manufacturing Practice (GMP)-grade viruses at our in-house GMP facility and now propose to conduct Investigational New Drug (IND) enabling studies so that our innovative oHSVs will be ready for testing in future clinical trials. Project 4 has three Specific Aims: (1) dissect systemic and regional immune responses, identify a marker(s) in the peripheral blood of mouse models or clinical samples from Project 2 that correlates with anti-tumor activity, and improve the efficacy of rQnestin34.5.v2 in GBM mouse models after OV-Cmab-CCL5 treatment; (2) perform IND-enabling in vivo efficacy and toxicity studies using GMP-grade OV-Cmab-CCL5; and (3) determine the preclinical efficacy and safety of OV-Cmab-CCL5 combined with OV-αCD47-IgG1. We are confident that our approaches will provide a novel, effective, and safe oncolytic virotherapy against GBM.