Myxoma Virus (MV) Oncolysis for treating human cancer

描述（由申请人提供）：黏液瘤病毒（MV）在体内仅感染兔子，但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是一个有吸引力的候选溶瘤病毒治疗人类癌症。MV已被成功用于治疗多种移植免疫缺陷小鼠的人类脑癌和免疫正常小鼠的小鼠转移性黑色素瘤。在这里，MV将被开发用于人体临床试验，利用一种独特的治疗策略，该病毒非常适合：体外清除人骨髓内的癌细胞或动员pbmc样本，这些患者通常在高剂量化疗后被排除在自体干细胞移植之外。已经收集了相当多的初步数据来支持这项建议：1)在移植了正常人骨髓或细胞因子动员的PBMCs的免疫缺陷小鼠中，MV不会干扰或损害人多能干细胞的分化，2)MV在体外清除后可以清除多种人类癌细胞，3)MV不仅可以有效清除体内的许可白血病/淋巴瘤细胞，而且出人意料地阻止非许可的人类白血病细胞（如KG1细胞）的植入或肿瘤诱导。4)表达多种有用的报告蛋白（荧光和生物发光）用于成像目的的MV重组已经构建。具体而言，我们的目标是：1)验证MV对正常人造血干细胞体外治疗的安全性：我们将通过植入免疫缺陷的NOG小鼠来验证MV清除对正常人干细胞分化的安全性，以验证完全造血细胞植入和免疫重建。MV清除将在来自正常骨髓和g - csf动员的PBMCs的原代人干细胞/祖细胞上进行测试，使用体外造血集落形成细胞试验以及体内有效的血液细胞植入。2)优化MV体外清除癌细胞：在移植NOG小鼠体内，研究体外清除MV清除b细胞淋巴瘤和急性髓系白血病两种人类癌症的致瘤潜能的能力。癌细胞和病毒将被不同的荧光素酶标记，使植入的肿瘤细胞和治疗病毒能够在体内独立追踪。来自急性髓性白血病患者的原代细胞也将被测试MV特异性消除污染癌细胞的能力，并允许选择性地只植入非癌性人类白细胞。3)研究MV清除原代人白血病细胞的机制：我们最近的研究表明，体外感染MV的人KG1白血病细胞可以阻止这些细胞随后移植到NOG受体小鼠体内并形成肿瘤，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化蛋白的抗体探测了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。我们将探讨这些宿主细胞信号激活在体外肿瘤细胞清除人白血病细胞中的功能意义。

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.