SOMATIC NON-SYNONYMOUS MUTATIONS AS UNIQUE TUMOR ANTIGENS IN MELANOMA

描述（由申请人提供）：黑色素瘤的发病率在全球范围内持续上升，2012年美国估计有76,250例新病例。尽管出现了新的治疗方法，但黑色素瘤仍然是一种无法治愈的恶性肿瘤，因此，它代表了一个医疗需求未得到满足的疾病领域。值得注意的是，黑色素瘤与生命早期的紫外线照射、癌症基因组中最高的突变率（每Mb 14-30个）和诱导自发T细胞免疫的能力有关。黑色素瘤中最常见（60%）的体细胞非同义突变是BRAFV600E。靶向治疗，如vemurafenib，旨在抑制这种驱动突变，导致高反应率，尽管由于获得耐药性的持续时间有限。相比之下，基于免疫的疗法产生的反应率很低，而且往往是持久的。新出现的数据表明，BRAFV600E抑制，通过矛盾的增加MAPK信号传导，增强抗肿瘤T细胞免疫。总之，这些发现支持vemurafenib联合免疫疗法来改善临床结果。在免疫疗法中，研究性疫苗和过继性T细胞疗法（ACT）在早期临床试验中开始显示出疗效。我们最近的试点1期临床试验使用CD40L/IFN-？成熟树突状细胞（DC）和黑素细胞谱系限制的gp100抗原已经揭示了由DC产生的IL-12在转移性黑色素瘤患者疫苗接种中的治疗益处。然而，开发改进疫苗和ACT的一个关键障碍是经过验证的黑色素瘤抗原的性质和缺乏。迄今为止，针对免疫干预的抗原主要来源于结构未改变的种系/谱系/分化蛋白，并且已证明有限的临床益处。为了开发下一代个性化的癌症免疫疗法，需要新的策略来识别患者特异性/独特的肿瘤抗原。临床前模型的实验证据支持这一论点，即这些独特的肿瘤抗原，主要是在肿瘤转化过程中产生的，可以引发能够保护宿主免受癌症进展的T细胞免疫。杂交捕获转录组测序（RNA-capture sequencing, RNA-cap seq）的出现提供了一种灵敏的方法来询问癌症基因组中表达的体细胞突变并评估其表达水平。我们正在进行的研究黑色素瘤转录组的实验，发现每个肿瘤有超过500个体细胞非同义突变。我们建议使用RNA-cap序列以及HLA I类肽结合预测算法和体外T细胞分析来验证体细胞非同义突变产生独特黑色素瘤抗原的假设，以及对BRAFV600E抑制的抗性的获得伴随着黑色素瘤抗原环境的变化。这一假设将在以下特定目的的实验中得到解决：(1)表征黑色素瘤表达的体细胞突变库并评估其作为独特抗原的潜力；(2)评估靶向治疗（BRAFV600E, vemurafenib）对黑色素瘤独特抗原库的影响。这项探索性研究将提供体细胞突变作为独特肿瘤抗原的有效性和程度的见解，为查询肿瘤抗原鉴定的基因组数据提供新的策略，并可能导致改进黑色素瘤和其他癌症的患者特异性治疗。

DESCRIPTION (provided by applicant): The incidence of melanoma continues to rise worldwide with an estimated 76,250 new cases in the US for 2012. Despite the advent of new therapies, melanoma remains an incurable malignancy and thus, represents a disease area of unmet medical need. Melanoma is notable for its association with early-in-life UV-light exposure, highest mutational rate (14-30 per Mb) among cancer genomes and ability to induce spontaneous T cell immunity. The most common (60%) somatic non-synonymous mutation in melanoma is BRAFV600E. Targeted therapies, such as vemurafenib, directed at inhibiting this driver mutation have resulted in high responses rates, albeit of limited duration due to acquisition of drug resistance. In contrast, immune-based therapies have yielded low response rates that are often durable. Emerging data suggest that BRAFV600E inhibition, through paradoxical increased MAPK signaling, potentiates anti-tumor T cell immunity. Altogether, these findings support testing vemurafenib in combination with immunotherapies to improved clinical outcomes. Among immunotherapies, investigational vaccines and adoptive T cell therapies (ACT) are beginning to show efficacy in early phase clinical trials. Our recent pilot phase 1 clinical trial using CD40L/IFN-? matured dendritic cells (DC) and melanocyte lineage-restricted gp100 antigen have revealed the therapeutic benefit of IL-12, produced by DC, in vaccination of patients with metastatic melanoma. However, a critical barrier to development of improved vaccines and ACT is the nature and paucity of validated melanoma antigens. To date, antigens targeted for immune intervention are predominantly derived from structurally unaltered germline/ lineage/differentiation proteins and have demonstrated limited clinical benefit. Novel strategies are needed to identify patient-specific/unique tumor antigens in order to develop the next generation of personalized cancer immunotherapies. Experimental evidence in pre-clinical models supports the thesis that these unique tumor antigens, primarily arising during neoplastic transformation, can elicit T cell immunity capable of protecting the host from cancer progression. The emergence of hybrid capture transcriptome sequencing (RNA-capture sequencing, RNA-cap seq) offers a sensitive method to interrogate cancer genomes for expressed somatic mutations and assess their level of expression. Our ongoing experiments studying the melanoma transcriptome, have found over 500 expressed somatic non-synonymous mutations per tumor. We propose to use RNA-cap seq along with in-silico HLA class I peptide binding prediction algorithms and in vitro T cell assays to test the hypothesis that somatic non- synonymous mutations give rise to unique melanoma antigens and that acquisition of resistance to BRAFV600E inhibition is accompanied by changes in the melanoma antigenic landscape. This hypothesis will be addressed in the experiments of the following Specific Aims: (1) Characterize the repertoire of melanoma expressed somatic mutations and evaluate their potential as unique antigens and (2) Evaluate the effect of targeted therapy (BRAFV600E, vemurafenib) on the repertoire of melanoma unique antigens. This exploratory study should provide insights into the validity and extent of somatic mutations as unique tumor antigens pave a new strategy to query genomic data for tumor antigen identification and potentially lead to improved patient-specific therapies in melanoma as well as other cancers.