Engineering therapeutic and diagnostic proteins for tumor biomarker mesothelin

 描述（由申请人提供）：迫切需要为目前不存在的肿瘤开发新的靶向治疗，并开发伴随诊断以确定哪些患者将对靶向治疗产生反应。靶向治疗正在对癌症治疗产生重大影响，但许多卵巢癌、三阴性乳腺癌、胰腺癌或肺癌患者尚未有可靠的靶向治疗选择。在来自这四种癌症类型中的每一种的临床样品中，间皮素（MSLN）是一种肿瘤生物标志物，其经常在肿瘤细胞表面上以高水平表达并与不良预后相关。MSLN在健康组织中的表达有限。因此，MSLN具有作为诊断和治疗肿瘤的新靶点的广泛潜力。因此，迫切需要MSLN靶向治疗和分子诊断来鉴定最可能对MSLN靶向治疗有反应的患者，尽管目前不存在这样的FDA批准的药剂。我们建议使用纤连蛋白非抗体蛋白质支架来设计高度稳定的间皮素靶向剂，以用作靶向治疗和分子诊断。可以起到诊断和治疗作用或“治疗诊断”作用的靶向剂具有特定的临床前景，允许诊断性鉴定最有可能对伴侣治疗剂应答的患者。使用治疗诊断学的策略尚未进入常规临床使用，主要是由于缺乏合适的靶向分子。为了开发候选治疗诊断剂，我们将设计一种高度稳定的蛋白质支架，以高特异性结合MSLN。 我们将使用的蛋白质支架是基于人纤连蛋白的结构域，其生物物理特性使其对诊断和治疗应用都具有吸引力。MSLN与肿瘤细胞表面生物标志物MUC 16的相互作用导致肿瘤侵袭和转移增加，因此阻断这种相互作用可以具有直接的治疗效果。我们将专门设计蛋白质来阻断MSLN和MUC 16的结合相互作用。在初步研究中，我们已经使用定向进化和酵母表面展示来鉴定以中等亲和力结合到介导其与MUC 16结合的MSLN结构域的蛋白质变体库。在目标1中，我们将在我们的蛋白质文库中引入更多的遗传多样性，并使用定向进化来设计以高亲和力结合MSLN并阻断MUC 16激活的蛋白质。在目标2中，我们将测量工程蛋白的临床相关生物物理特性，包括与表达MSLN的肿瘤细胞的结合。在目标3中，我们将测量工程蛋白的体外生物活性，评估它们抑制细胞迁移和激活细胞死亡的能力。我们将测试工程蛋白作为体内分子成像剂，测量小鼠异种移植模型中的生物分布和肿瘤对比度。在完成这项提案后，我们将确定靶向MSLN的治疗/诊断蛋白。我们提出的工作的一个广泛的结果是验证纤连蛋白蛋白支架用于开发其他靶向治疗/诊断分子。

 DESCRIPTION (provided by applicant): There is an urgent need for developing new targeted therapies for tumors where none currently exist, and for developing companion diagnostics to identify which patients will respond to the targeted therapy. Targeted therapeutics are making a significant impact in cancer treatment, but many patients with ovarian, triple- negative breast, pancreatic, or lung cancer do not yet have a reliable targeted therapeutic option. In clinical samples from each of these four cancer types, mesothelin (MSLN) is a tumor biomarker frequently expressed at high levels on the surface of tumor cells and correlating with poor prognosis. MSLN has limited expression in healthy tissues. Consequently, MSLN has broad potential as a novel tumor target for diagnosis and therapy. Thus, there is a critical need for MSLN-targeted therapeutics and molecular diagnostics to identify patients who are most likely to respond to MSLN-targeting therapies, although currently no such FDA-approved agents exist. We propose to engineer highly stable mesothelin targeting agents using the fibronectin non-antibody protein scaffold, for use as both a targeted therapeutic and molecular diagnostic. Targeting agents that can serve both diagnostic and therapeutic, or "theranostic," roles have particular clinical promise, allowing diagnostic identification of patients most likely to respond o the partner therapeutic. The strategy of using theranostics has not yet entered routine clinical use, largely due to the lack of appropriate targeting molecules. To develop candidate theranostics, we will engineer a highly stable protein scaffold to bind MSLN with high specificity. The protein scaffold we will use is based on a domain of human fibronectin, whose biophysical properties make it attractive for both diagnostic and therapeutic applications. The interaction of MSLN with tumor cell surface biomarker MUC16 leads to increased tumor invasiveness and metastasis, so that blocking this interaction can have a direct therapeutic effect. We will engineer proteins specifically to block the binding interaction of MSLN and MUC16. In preliminary studies, we have used directed evolution and yeast-surface display to identify a pool of protein variants that bind with moderate affinity to the domain of MSLN that mediates its binding to MUC16. In Aim 1, we will introduce further genetic diversity into our library of proteins, and use directed evolution to engineer proteins that bind to MSLN with high affinity and block its activation by MUC16. In Aim 2, we will measure clinically relevant biophysical properties of engineered proteins, including binding to tumor cells expressing MSLN. In Aim 3, we will measure in vitro bioactivity of engineered proteins, assessing their ability to inhibit cel migration and activate cell death. We will test the engineered proteins as in vivo molecular imaging agents, measuring biodistribution and tumor contrast in mouse xenograft models. Upon completion of this proposal, we will have identified therapeutic/diagnostic proteins targeting MSLN. A broad outcome of our proposed work is to validate the fibronectin protein scaffold for development of other targeted therapeutic/diagnostic molecules.