Generation and characterization of adduct-specific anti cisplatin DNA antibodies

 描述（由申请人提供）：顺铂和卡铂（铂类药物）是最成功的抗肿瘤药物，用于治疗睾丸癌、乳腺癌、卵巢癌、膀胱癌、颈癌和肺癌。与许多抗肿瘤剂一样，治疗的功效在患者之间可能差异很大，并且耐药性的发生是一个重要的问题。铂的治疗作用是基于不同类型的DNA加合物的形成，主要是链内和链间交联，通过与DNA的一条或两条互补链上的两个嘌呤碱基反应。对铂治疗的耐药性是通过上调或下调几个过程发生的，包括金属转运进出细胞、细胞内代谢和通过DNA修复途径去除顺铂DNA加合物。本申请中提出的工作的主要假设是，通过开发对细胞基因组中的单个铂DNA加合物具有特异性的单克隆抗体，我们将能够1）确定哪种铂加合物是治疗上最相关的，2）理解负责抗性的修复机制，3）在肿瘤活检中测试对铂的敏感性或抗性，以及4）选择最有可能从治疗中受益的患者，并且避免那些可能对与这种类型的治疗相关的严重副作用具有抗性的患者。在该R21应用中，我们建议合成含有1，2-GG-、1，2-AG-、1，3-GNG-链内和1，2-GC-链间铂交联的寡核苷酸，将它们偶联到KLH载体蛋白上，并使用它们免疫小鼠以产生单克隆抗体，该单克隆抗体以高特异性识别各个加合物。这些单克隆抗体将在具有特异性DNA修复缺陷的细胞系中进一步验证。我们预测，这些试剂将使我们能够表明，铂链内交联持续存在于细胞中的核苷酸切除修复（NER）途径的缺陷，而链间交联将持续存在于细胞中的缺陷链间交联（ICL）修复途径。然后，我们将测量NCI-60收集的乳腺癌和卵巢癌细胞系中的铂水平，这些细胞系已被表征为药物敏感性或耐药性。我们将确定是否特定加合物或所有加合物的水平在铂应答细胞系中升高。相反，我们将测试在耐药细胞系中是否不存在特定的加合物。我们预计，在完成本文提出的研究后，我们将开发出一套独特的试剂，这些试剂将对研究和医学界具有巨大的用途，以确定哪些铂加合物在临床上最相关，并致力于开发一种可靠的方法来预测铂治疗的临床结果。

 DESCRIPTION (provided by applicant): Cisplatin and carboplatin (platinum drugs) are among the most successful antitumor drugs and are used to treat testicular, breast, ovarian, bladder, neck and lung cancer. As with many antitumor agents, the efficacy of treatment can vary greatly from patient to patient and the occurrence of resistance is a significant problem. The therapeutic effect of the platinums is based on the formation of different types of DNA adducts, primarily intra- and interstrand crosslinks, by the reaction with two purine bases on one or two complementary strands of DNA. The resistance to platinum treatment occurs by a the up- or down-regulation of several processes, including metal transport in and out of the cell, intracellular metabolism and the removal of the cisplatin DNA adducts by DNA repair pathways. The primary hypothesis of the work proposed in this application is that by developing a monoclonal antibodies with specificity for the individual platinum DNA adducts in the genomes of cells we will be able to 1) determine which platinum adduct is the most therapeutically relevant one, 2) understand the repair mechanisms responsible for resistance, 3) test the sensitivity or resistance to platinum in tumor biopsies and 4) to select patients that are most likely to benefit from therapy and sparing those that are likely to be resistant the severe side effect associated with this type of treatment. In this R21 application, we propose to synthesize oligonucleotides containing the 1,2-GG-, 1,2-AG-, 1,3-GNG-intrastrand and 1,2-GC-interstrand platinum crosslinks, couple them to the KLH carrier protein and use them to immunize mice to generate monoclonal antibodies that recognize the individual adducts with high specificity. These monoclonal antibodies will be then further validated in cell lines with specific DNA repair defects. We predict that these reagents will enable us to show that platinum intrastrand crosslinks persist in cells with defects in the nucleotide excision repair (NER) pathway, while interstrand crosslinks will persist in cells with defects interstrand crosslink (ICL) repair pathwa. We will then measure platinum levels in breast and ovarian cancer cell lines of the NCI-60 collection, which have been characterized for drug sensitivity or resistance. We will determine whether the levels of a specific adduct or all adduct are elevated in platinum-responsive cell lines. Conversely, we will test whether a specific adduct is absent in resistant cell lines. We expect that upon completion of the studies proposed here we will have developed a set of unique reagents that will be of tremendous use for the research and medical community to determine which platinum adducts are clinically most relevant and to work toward developing a robust method to predict clinical outcomes of platinum treatments.