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 该提案的最终目标是开发一种新型、有效且安全的溶瘤单纯疱疹病毒（oHSV）- 基于免疫疗法治疗胶质母细胞瘤（GBM），这是一种高度致命且最常见的恶性肿瘤 脑肿瘤。 oHSV 对 GBM 的治疗依赖于病毒的癌症特异性复制，从而导致肿瘤破坏 对邻近非肿瘤组织的毒性最小。它对患者的安全性已得到证实，但疗效仍然存在 有待改进。项目4将专注于通过为oHSV配备强大的抗肿瘤免疫反应 通过测试两种新生成的新型 oHSV（称为 OV-Cmab-CCL5 和 OV- αCD47-IgG1，旨在分别诱导免疫浸润和阻断检查点，作为单一疗法 除了他们的组合。两种 oHSV 预计将相互协同，以最大限度地提高免疫能力 GBM 微环境中的反应，通过针对免疫刺激和免疫抑制来 创建一个整体促炎性肿瘤微环境，已被证明与 项目 2 的 rQNestin34.5 临床试验中的患者存活率。我们生成了 OV-Cmab-CCL5，一种 oHSV 表达表皮生长因子受体（抗 EGFR） IgG1 的可分泌单链可变片段 抗体西妥昔单抗通过 Fc 旋钮入孔系统与 CCL5 连接，产生异二聚体并防止 同型二聚体。为了针对先天免疫细胞的检查点，我们还设计了 oHSV 来表达全长、 可溶性抗 CD47 抗体与人 IgG1 支架（所谓的 OV-αCD47-G1），用于局部区域控制 GBM。病毒感染的GBM细胞分泌的抗体αCD47-IgG1阻断CD47“别吃我”信号 并发挥 Fc 受体介导的抗体依赖性细胞吞噬作用的附加功能 巨噬细胞和 NK 细胞的抗体依赖性细胞毒性。我们的数据显示，在 GBM 小鼠中 模型中，由于抗肿瘤免疫增强，两种 oHSV 都缩小了肿瘤大小并延长了生存期 回应。我们假设 OV-Cmab-CCL5 是一种安全有效的药物，可以改善 GBM 治疗 具有多种作用机制，OV-Cmab-CCL5和OV-αCD47-IgG1的联合治疗将 比单一疗法有更好的疗效。我们制定了良好生产规范 (GMP) 级病毒在我们的内部 GMP 设施中进行，现在提议进行研究性新药 (IND) 开展研究，以便我们的创新 oHSV 为未来的临床试验做好准备。项目4有3个 具体目标：(1) 剖析全身和区域免疫反应，识别外周血中的标记物 项目 2 中与抗肿瘤活性相关的小鼠模型或临床样本，并提高 rQnestin34.5.v2 在 OV-Cmab-CCL5 治疗后 GBM 小鼠模型中的功效； (2) 执行 IND 启用 使用 GMP 级 OV-Cmab-CCL5 进行体内功效和毒性研究； (3) 确定临床前疗效 OV-Cmab-CCL5 联合 OV-αCD47-IgG1 的安全性。我们相信我们的方法将 提供一种新颖、有效且安全的针对 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.