SOMATIC NON-SYNONYMOUS MUTATIONS AS UNIQUE TUMOR ANTIGENS IN MELANOMA

描述（由申请方提供）：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例新发病例。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，因此代表了一个医疗需求未得到满足的疾病领域。黑色素瘤因其与生命早期的紫外线暴露、癌症基因组中最高的突变率（14-30/Mb）和诱导自发T细胞免疫的能力相关而闻名。黑色素瘤中最常见（60%）的体细胞非同义突变是BRAFV 600 E。靶向治疗，如vemurafenib，旨在抑制这种驱动突变，导致高反应率，虽然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较低，而且往往是持久的。新出现的数据表明，BRAFV 600 E抑制，通过矛盾增加MAPK信号，增强抗肿瘤T细胞免疫。总而言之，这些发现支持测试vemurafenib与免疫疗法的组合以改善临床结果。在免疫疗法中，研究性疫苗和过继性T细胞疗法（ACT）开始在早期临床试验中显示出疗效。我们最近的试点1期临床试验使用CD 40 L/IFN-？成熟的树突状细胞（DC）和黑素细胞谱系限制性gp 100抗原揭示了DC产生的IL-12在转移性黑色素瘤患者的疫苗接种中的治疗益处。然而，开发改进的疫苗和ACT的关键障碍是经验证的黑素瘤抗原的性质和缺乏。迄今为止，免疫干预的靶向抗原主要来源于结构未改变的种系/谱系/分化蛋白，并且已经证明了有限的临床益处。需要新的策略来识别患者特异性/独特的肿瘤抗原，以开发下一代个性化癌症免疫疗法。临床前模型中的实验证据支持以下论点：这些独特的肿瘤抗原主要在肿瘤转化期间产生，可以引发能够保护宿主免于癌症进展的T细胞免疫。杂交捕获转录组测序（RNA捕获测序，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已经发现每个肿瘤有超过500个表达的体细胞非同义突变。我们建议使用RNA-cap seq沿着与计算机模拟HLA I类肽结合预测算法和体外T细胞测定来测试以下假设：体细胞非同义突变产生独特的黑素瘤抗原，并且对BRAFV 600 E抑制的抗性的获得伴随着黑素瘤抗原景观的变化。该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的库，并评价其作为独特抗原的潜力，以及（2）评价靶向治疗（BRAFV 600 E，维罗非尼）对黑色素瘤独特抗原库的影响。这项探索性研究应该提供对体细胞突变的有效性和程度的见解，因为独特的肿瘤抗原为查询肿瘤抗原鉴定的基因组数据铺平了新的策略，并可能导致改善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.