Arginine Deprivation: A targeted therapy for Melanoma

描述（由申请人提供）：恶性黑色素瘤通常对药物治疗具有耐药性，而药物治疗在历史上一直是非选择性的，并且通常毒性很大。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，将黑色素瘤细胞暴露于精氨酸脱亚胺酶（ADI）（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），因此不能合成精氨酸。ASS cDNA的转染赋予了对AD 1的抗性，进一步证实了ASS表达的缺乏对于ADI敏感性至关重要。我们配制了ADI的聚乙二醇化形式（ADI-PEG 20），以降低免疫原性并延长半衰期。ADI-PEG 20在体内显示出显著的抗肿瘤活性，且毒性低。我们已经完成了ADI-PEG 20治疗晚期黑色素瘤的I期试验。值得注意的是，当以>160 IU/m2的剂量治疗时，5/10的患者具有部分响应，该剂量将血浆精氨酸消耗至不可检测的水平>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。在本申请中，我们计划进行一项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。在具体目标2中，我们将在治疗之前和之后通过免疫组织化学和RT-PCR测定肿瘤样品中的ASS，以评估ASS表达是否可以是肿瘤应答的预测因子，以及复发时是否发生ASS的去抑制。在具体目标3中，我们将研究ADI-PEG 20引起凋亡性细胞死亡的可能机制。此外，将通过使用对ADI-PEG 20具有抗性的体外细胞系和通过使用在治疗失败时来源于肿瘤的从头抗性细胞系来检查可能的抗性机制。为了优化ADI-PEG 20的未来使用，我们将研究药理学操作是否可以诱导/抑制ASS表达。我们的目标是通过靶向黑色素瘤细胞中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.