Myxoma Virus (MV) Oncolysis for treating human cancer

描述(申请人提供)：粘液瘤病毒(MV)不仅在体内感染兔子，而且在体外和体内也具有感染多种人类癌细胞的天然能力。因此，MV是治疗人类癌症的溶瘤病毒疗法的一个有吸引力的候选者。MV已经成功地用于治疗免疫缺陷小鼠移植的几种不同的人类脑癌和免疫活性小鼠的小鼠转移性黑色素瘤。在这里，MV将被开发用于人类临床试验，方法是开发一种独特的非常适合该病毒的治疗策略：体外清除人骨髓中的癌细胞或动员的PBMC样本，这些样本通常在大剂量化疗后被排除在自体干细胞移植之外。已收集到大量的初步数据支持这一建议：1)MV不干扰或损害移植入正常人骨髓或细胞因子动员的PBMC的免疫缺陷小鼠的多潜能干细胞分化，2)MV可消除体外净化后的多种人类癌细胞，3)MV不仅可有效净化体内许可的白血病/淋巴瘤细胞，而且出人意料地阻止甚至不可许可的人类白血病细胞(如KG1细胞)植入或诱导肿瘤，4)已构建表达各种有用的用于成像的报告蛋白(荧光和生物发光)的MV重组体。具体地说，我们的目标是：1)验证MV在体外治疗正常人类造血干细胞的安全性：将测试MV净化对正常人类干细胞分化的安全性，使用移植的免疫缺陷NOG小鼠来验证完全的造血细胞植入和免疫重建。MV净化将在来自正常骨髓和G-CSF动员的PBMC的原代人类干/祖细胞上进行测试，在体外使用造血细胞集落形成细胞分析，以及在体内进行有效的血细胞植入。2)优化MV体外清除癌细胞：研究两种人类肿瘤，B细胞淋巴瘤和急性髓系白血病的体外MV清除能力，以消除其在NOG小鼠体内的致瘤潜能。癌细胞和病毒将被标记上可区分的荧光素酶，从而可以在体内独立追踪移植的肿瘤细胞和治疗性病毒。来自急性髓系白血病患者的原代细胞也将接受MV的能力测试，以特定地清除受污染的癌细胞，并仅允许选择性地植入非癌症的人类白细胞。3)研究MV清除原代人类白血病细胞的机制：我们最近发现，体外感染MV的人KG1白血病细胞可以阻止这些细胞随后在NOG受体小鼠体内的种植和肿瘤形成，尽管这些细胞在体外完全不允许MV感染。为了评估病毒诱导的细胞信号变化，我们用一系列针对46种不同人类信号磷酸蛋白的抗体来探测MV感染的KG1细胞，并观察到MV感染特异性地诱导KG1细胞中的Stat5和HCK激活。我们将探索这些宿主细胞信号激活对于MV成功清除人类白血病细胞的功能意义。 公共卫生相关性最近，我们发现一种特殊的兔痘病毒，称为粘液瘤病毒(MV)，也可以感染和杀死广泛的人类癌细胞，MV已经在动物模型中成功地治疗了几种类型的癌症。为了促进MV作为一种新的肿瘤溶解疗法在临床前的发展，我们建议利用两种特定的癌症模型(人B细胞淋巴瘤和急性髓系白血病)来建立一种新的治疗性“癌细胞净化”方案的最佳条件。这一战略将使目前被排除在自体骨髓移植之外的白血病/淋巴瘤癌症患者受益，因为它允许他们自己的干细胞样本(从骨髓或血液中)在重新移植之前清除癌细胞，并在大剂量化疗后重建他们的免疫系统。

DESCRIPTION (provided by applicant): Myxoma virus (MV) infects only rabbits in vivo, but also has a natural capacity to infect a wide variety of human cancer cells in vitro and in vivo. Thus, MV is an attractive candidate for oncolytic virotherapy to treat human cancer. MV has been used to successfully treat several diverse human brain cancers in xenografted immunodeficient mice and murine metastatic melanoma in immunocompetent mice. Here, MV will be developed for human clinical trials by exploiting a therapeutic strategy for which the virus is uniquely well-suited: ex vivo purging of cancer cells within human bone marrow or mobilized-PBMC samples from patients who would normally be excluded from autologous stem cell transplantation following high dose chemotherapy. Considerable preliminary data has been collected to support this proposal: 1) MV does not perturb or compromise human multipotent stem cell differentiation in immunodeficient mice engrafted with normal human bone marrow or cytokine-mobilized PBMCs, 2) MV eliminates a wide variety of human cancer cells following ex vivo purging, 3) MV can effectively purge not only permissive leukemia/lymphoma cells in vivo, but also unexpectedly prevents even nonpermissive human leukemia cells (such as KG1 cells) from engraftment or tumor induction, and 4) MV recombinants that express a variety of useful reporter proteins (fluorescent and bioluminescent) for imaging purposes have already been constructed. Specifically, our aims are: 1) Validate MV safety for ex vivo treatment of normal human hematopoietic stem cells: The safety of MV-purging for normal human stem cell differentiation will be tested, using engrafted immunodeficient NOG mice to verify full hematopoietic cell engraftment and immune reconstitution. MV purging will be tested on primary human stem/progenitor cells derived from normal bone marrow and G-CSF-mobilized PBMCs, using hematopoietic colony forming cell assays in vitro as well as for efficient hematologic cell engraftment in vivo. 2) Optimize MV ex vivo cancer cell purging: Two human cancers, B-cell lymphoma and acute myeloid leukemia, will be investigated for the ability of ex vivo MV purging to eliminate their tumorigenic potential in vivo in engrafted NOG mice. The cancer cells and viruses will be tagged with distinguishable luciferases that allow the engrafted tumor cells and the therapeutic virus to be independently tracked in vivo. Primary cells from acute myeloid leukemia patients will also be tested for the ability of MV to specifically eliminate the contaminating cancer cells and allow the selective engraftment of only noncancerous human leukocytes. 3) Investigate the mechanism of MV purging of primary human leukemia cells: We have recently shown that ex vivo infection of human KG1 leukemia cells with MV prevents the subsequent engraftment and tumor formation of these cells into NOG recipient mice, despite the fact that these cells are completely nonpermissive for MV infection in vitro. To assess for virus-induced cell signaling changes, we probed MV-infected KG1 cells with an array of antibodies to 46 different human signaling phosphoproteins, and observed that MV infection specifically induces Stat5 and Hck activation in KG1 cells. We will explore the functional significance of these host cell signaling activations for the successful ex vivo tumor cell purging of human leukemia calls by MV. PUBLIC HEALTH RELEVANCE Recently, we discovered that one particular rabbit-specific poxvirus, called myxoma virus (MV), also infects and kills a wide spectrum of human cancer cells and MV has been used to successfully treat several types of cancers in animal models. In order to facilitate the preclinical development of MV as a new oncolytic therapeutic for cancer in man, we propose to exploit two specific cancer models (human B-cell lymphoma and acute myeloid leukemia) to establish the optimal conditions for a novel therapeutic "cancer cell purging" protocol. This strategy will benefit leukemia/lymphoma cancer patients who are currently excluded from autologous bone marrow transplantation by allowing their own stem cell samples (from bone marrow or blood) to be purged of cancer cells prior to re-engraftment and reconstitution of their immune system following high dose chemotherapy.