Improving Vaccinia for Peritoneal Tumors: Enhanced Distribution & Immune Evasion

描述(由申请人提供)：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。我们为腹膜表面恶性肿瘤患者的临床管理制定了全面的计划，并正在探索新的治疗方案。我们对用于溶瘤病毒治疗的痘病毒也有长期的兴趣，我们以前开发了一种用于临床的肿瘤选择性、复制型溶瘤痘苗病毒(VvDD)。虽然vvDD已被证明是一种成功的直接注射到患者肿瘤中的方法，但它受到患者过早免疫介导的病毒清除的限制。在这个项目中，我们建议加强这种痘苗病毒用于腹膜肿瘤的治疗，解决病毒过早免疫清除的局限性。我们最近发现，趋化因子CCL5或CCL19的表达会导致肿瘤微环境中长时间的选择性复制，而在正常组织中，表达趋化因子的病毒会被迅速清除。我们推测，肿瘤中痘病毒感染的癌细胞分泌的CKs(CCL5或CCL19)已经从循环中吸引了大量幼稚的白细胞，包括单核细胞和淋巴细胞。肿瘤微环境中的这些细胞启动并维持强大的2型免疫反应，使表达CCL5(或CCL19)的病毒能够在肿瘤中持续存在。既往的抗痘病毒免疫也影响vvDD的疗效。我们最近证明，A34R突变可以促进病毒包膜形式(EEV)的释放，这种形式可能不被中和抗体识别。A34R突变还有一个额外的优势，即从细胞中释放大量病毒，加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。因此，我们假设，当我们的肿瘤选择性vvDD与A34R突变和适当的趋化因子表达相结合时，将导致改善腹膜表面恶性肿瘤的治疗，并克服我们临床试验中遇到的重要限制。我们的目标是：(1)确定表达CCL5和/或CCL19趋化因子时vvDD在体内延长复制的程度和机制；(2)通过体外建模和基因敲除小鼠模型来确定病毒延长复制的机制；(3)通过突变A34R基因，使vDD具有逃避预先存在的抗痘病毒免疫并在整个腹膜腔广泛传播的能力。有了这些目标，我们有信心克服病毒治疗成功的一些障碍，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所有病毒治疗都将是重要的。A34R缺失和vvDD释放增强的意义对所有痘病毒的临床应用都将是重要的。

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.