Improving Vaccinia for Peritoneal Tumors: Enhanced Distribution & Immune Evasion

描述（由申请方提供）：腹膜表面恶性肿瘤（包括结肠、卵巢、阑尾和间皮瘤）需要新的治疗方法。我们对腹膜表面恶性肿瘤患者的临床管理有一个全面的计划，并正在探索新的治疗方案。我们也对痘病毒用于溶瘤病毒治疗有着长期的兴趣，我们以前开发了一种肿瘤选择性、复制型溶瘤痘苗病毒用于临床（vvDD）。虽然vvDD已被证明成功地直接注射到患者的肿瘤中，但它受到患者过早免疫介导的病毒清除的限制。我们建议在这个项目中，以提高这种牛痘病毒治疗腹膜肿瘤，解决病毒的过早免疫清除的限制。我们最近发现，趋化因子CCL 5或CCL 19的表达导致肿瘤微环境中延长的选择性复制，然而，在正常组织中，表达趋化因子的病毒被迅速清除。我们假设，从痘病毒感染的癌细胞分泌的CKs（CCL 5或CCL 19）在肿瘤中吸引了大量的幼稚白细胞，包括单核细胞和淋巴细胞从循环。肿瘤微环境中的这些细胞启动并维持强烈的2型免疫应答，这使得表达CCL 5（或CCL 19）的病毒能够在肿瘤中持续存在。预先存在的抗痘病毒免疫也影响vvDD的疗效。我们最近证明，A34 R突变增强了病毒包膜形式（EEV）的释放，这可能不被中和抗体识别。A34 R突变的另一个优点是从细胞中释放大量病毒，增强病毒向远处的传播。这在腹膜扩散的情况下尤其重要，在腹膜扩散的情况下，通常有数千个单独的肿瘤沉积物。因此，我们假设我们的肿瘤选择性vvDD，当用A34 R突变和适当的趋化因子表达进行工程化时，将导致腹膜表面恶性肿瘤的治疗改善，并克服我们的临床试验中遇到的重要限制。我们的目标是：（1）表征当表达CCL 5和/或CCL 19趋化因子时体内延长的vvDD复制的程度和机制，（2）使用体外建模和基因敲除小鼠模型定义延长的病毒复制的机制，和（3）通过突变A34 R基因，使vvDD具有逃避预先免疫的抗痘病毒免疫并广泛传播遍及腹膜腔的能力。有了这些目标，我们相信我们将克服成功病毒治疗的一些障碍，并能够推进腹膜表面恶性肿瘤患者的新临床试验。所获得的关于肿瘤微环境中趋化因子表达的免疫后果的信息对于所有病毒疗法都将是重要的。A34 R缺失与vvDD释放增强的意义对于所有痘病毒临床应用将是重要的。 公共卫生相关性：该提案的总体目标是使用A34 R突变和趋化因子（CCL 5、CCL 19）表达来增强溶瘤的复制选择性牛痘病毒治疗腹膜表面恶性肿瘤的应用。这些肿瘤患者的生存期以月为单位，并且没有有效的治疗策略。病人痛苦不堪，腹痛、呕吐、肠梗阻、腹水、呼吸困难、食欲不振，最终死亡。成功的腹腔接种疫苗治疗有可能提高生活质量和持续时间，并有机会获得长期生存。了解A34 R突变的作用机制和意义以及趋化因子表达的影响将有利于所有类型的病毒治疗。

DESCRIPTION (provided by applicant): Novel treatments are warranted for peritoneal surface malignancies (including colon, ovarian, appendiceal, and mesothelioma). We have a comprehensive program for the clinical management of patients with peritoneal surface malignancies and are exploring new therapeutic options. We also have a long standing interest in poxviruses for oncolytic viral therapy, and we previously developed a tumor-selective, replicating oncolytic vaccinia virus for clinical use (vvDD). While vvDD has demonstrated success as a direct injection into patients' tumors, it is limited by the patient's premature immune mediated clearance of the virus. We propose in this project to enhance this vaccinia virus for the treatment of peritoneal tumors, addressing the limitation of premature immune clearance of the virus. We recently discovered that the expression of chemokines, CCL5 or CCL19, leads to prolonged, selective replication in the tumor microenvironment, however, in the normal tissues the chemokine-expressing virus is cleared rapidly. We hypothesize that the CKs (CCL5 or CCL19) secreted from poxvirus-infected cancer cells in the tumor have attracted from circulation a large amount of naive leukocytes including monocytes and lymphocytes. These cells in the tumor microenvironment initiate and sustain strong type 2 immune responses which enable the CCL5 (or CCL19)-expressing virus to persist in the tumor. Pre-existing anti-poxviral immunity also affects the efficacy of vvDD. We have recently demonstrated that an A34R mutation enhances the release of an enveloped form (EEV) of the virus, which may not be recognized by neutralizing antibodies. The A34R mutation has the additional advantage of releasing large quantities of virus from the cell, enhancing the spread of the virus to distant sites. This is especially important in the setting of peritoneal spread, where it is common to have thousands of individual tumor deposits. Thus, we hypothesize that our tumor-selective vvDD, when engineered with an A34R mutation and the appropriate chemokine expression, will result in improved treatment of peritoneal surface malignancies, and overcome important limitations encountered in our clinical trial. Our aims are: (1) Characterize the extent and mechanism of prolonged vvDD replication in vivo when expressing CCL5 and/or CCL19 chemokines, (2) Define the mechanism of prolonged viral replication using in vitro modeling, and gene knockout mouse models, and (3) Arming vvDD with the ability to evade the pre-exisiting anti-poxviral immunity and spread widely throughout the peritoneal cavity, by mutating the A34R gene. With these aims we are confident that we will overcome some of the obstacles to successful viral therapy and be able to move forward with a new clinical trial for patients with peritoneal surface malignancies. The information obtained regarding the immune consequences of chemokine expression in the tumor microenvironment will be important for all viral therapies. The significance of the A34R deletion with enhanced release of the vvDD will be important for all poxvirus clinical applications. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to enhance the application of oncolytic, replication- selective vaccinia virus to treat peritoneal surface malignancies, using an A34R mutation and chemokine (CCL5, CCL19) expression. The survival of patients with these tumors is measured in months, and no effective strategies for treatment exist. Patients suffer greatly, with abdominal pain, vomiting, bowel obstruction, ascites, difficulty breathing, inanition and ultimately death. Successful intraperitoneal vaccinia therapy has the potential to improve quality and duration of life, with the chance for long term survival. Understanding the mechanism of action and significance of A34R mutation and the effects of chemokine expression will benefit all types of viral therapies.