Arginine Deprivation: A targeted therapy for Melanoma

描述(申请人提供)：恶性黑色素瘤通常对药物治疗产生抗药性，这种药物治疗在历史上一直是非选择性的，通常毒性很大。一种新的方法是针对黑色素瘤细胞中发现的特定缺陷。我们和其他人已经证明，黑色素瘤细胞暴露在精氨酸脱亚氨酶(ADI)中，精氨酸脱亚胺酶(ADI)是一种催化精氨酸水解为瓜氨酸的酶，会导致细胞凋亡。这种对ADI的独特敏感性主要是由于黑色素瘤细胞与正常细胞不同，不表达精氨酸琥珀酸合成酶(ASS)，因此无法合成精氨酸。ASS基因的表达增强了对AD1的抗性，进一步证实了缺乏ASS的表达是ADI敏感性的关键。我们研制了一种聚乙二醇化的ADI(ADI-PEG20)，以降低免疫原性并延长半衰期。ADI-PEG20具有明显的体内抗肿瘤活性，且毒性较低。我们已经完成了ADI-PEG20治疗晚期黑色素瘤的I期试验。值得注意的是，当治疗剂量为160IU/m2时，5/10的患者有部分反应，这个剂量会使血浆精氨酸消耗到无法检测到的水平，持续7天。未观察到2级毒性。有趣的是，两名没有反应的患者在他们的肿瘤中有ASS表达。在这项应用中，我们计划进行II期试验，以确认特定目标1中概述的晚期黑色素瘤的抗肿瘤活性。在特定目标2中，我们将通过免疫组织化学和RT-PCR检测治疗前后肿瘤标本中的ASS，以评估ASS的表达是否可以预测肿瘤的反应，以及ASS在复发时是否发生抑制。在具体目标3中，我们将探讨ADI-PEG20诱导细胞凋亡的可能机制。此外，将通过使用对ADI-PEG20产生抗药性的体外细胞系和通过使用治疗失败时来自肿瘤的从头抗药性细胞系来研究耐药的可能机制(S)。为了优化ADI-PEG20的未来应用，我们将研究药物操作是否可以诱导/抑制ASS的表达。我们的目标是通过针对黑色素瘤细胞的特定缺陷来改善黑色素瘤的治疗结果，同时将毒性降至最低。

DESCRIPTION (provided by applicant): Malignant melanoma is usually resistant to drug therapy which historically has been non-selective in action and often very toxic. A novel approach is to target a specific defect found in melanoma cells. We and others have shown that exposure of melanoma cells to arginine deiminase (ADI), an enzyme that catalyzes the hydrolysis of arginine to citrulline, results in apoptotic cell death. This unique sensitivity to ADI is primarily due to the fact that melanoma cells, unlike normal cells, do not express argininosuccinate synthetase (ASS) and hence are unable to synthesize arginine. Transfection of ASS cDNA confers resistance to AD1, further confirming that lack of ASS expression is critical for ADI sensitivity. We formulated a pegylated form of ADI (ADI-PEG20) to reduce immunogenicity and to increase the half-life. ADI-PEG20 has shown significant antitumor activity in vivo with low toxicity. We have completed a Phase I trial of ADI-PEG20 in advanced melanoma. Remarkably, 5/10 patients had partial response when treated at a dose >160 IU/m2, a dose that depleted plasma arginine to non detectable levels for >7 days. No > grade 2 toxicity was observed. Interestingly, two patients who did not respond had ASS expression in their tumors. In this application, we plan to conduct a Phase II trial to confirm the antitumor activity in advanced melanoma as outlined in specific aim 1. In specific aim 2, we will assay ASS in tumor samples by immunohistochemistry and RT-PCR prior and after treatment to assess whether ASS expression can be a predictor for tumor response, and whether de-repression of ASS occurs at relapse. In specific aim 3, we will investigate the possible mechanism of apoptotic cell death by ADI-PEG20. In addition, the possible mechanism(s) of resistance will be examined by using an in-vitro cell line made resistant to ADI-PEG20 and by using de-novo resistant cell lines derived from tumors at time of treatment failure. In order to optimize future use of ADI-PEG20, we will investigate whether pharmacological manipulation can induce/repress ASS expression. Our goal is to improve the treatment outcome of melanoma while minimizing toxicity by targeting a specific defect in melanoma cells.