Engineering a multispecific cell receptor antagonist to treat metastatic cancer

描述(申请人提供)：癌症是美国第二大最常见的死亡原因，据估计，仅在2013年就导致580,350人死亡。大约90%的死亡是由癌症进展到转移阶段引起的，在转移阶段，来自原发肿瘤的细胞开始进入并通过血液流动，在全身形成新的肿瘤。这一转移过程是由某些称为受体的蛋白质驱动的，这些蛋白质存在于癌细胞表面，数量较多，活性相对较高。 转化为正常细胞。这些受体的丰富使癌症能够迅速生长、增殖并在体内迁移，因此许多受体已被证实是癌症治疗的靶点。例如，20多年来，臭名昭著的尿激酶受体(UPAR)一直是已知的癌症靶点。UPAR协调转移所必需的多个过程；它在几乎所有癌症中都存在于高水平，并且高水平与肿瘤侵袭性增加、对化疗的耐药性、转移以及总体较短的患者生存时间有关。然而，尽管它很重要，但还没有FDA批准的分子可以针对这种受体。这是由于uPAR极其复杂；它有多种功能位点，可以独立协调肿瘤的生长和转移。开发一种有效阻断所有功能性uPAR位点的治疗方法仍然是转移癌治疗进展的关键障碍。在这里，我们概述了一组 目的：1)利用两种已有的uPAR拮抗剂，研究同时靶向两个主要功能uPAR位点对体外肿瘤进展的影响。这一结果将提高对驱动肿瘤生长和转移的关键uPAR功能的科学认识，并将表明是否存在相加或协同效应。 同时瞄准多种功能。目的2)改进和化学连接来自AIM 1的两个拮抗剂，以产生一种拮抗剂，该拮抗剂能以超高的亲和力(皮摩尔到毫微摩尔)结合uPAR，并阻断所有协调癌症进展的功能。每个拮抗剂的107个突变体库将使用容易出错的PCR生成，在酵母表面表达，并使用高通量(每秒10,000个细胞)流式细胞仪筛选与uPAR的结合。这项强大的技术在实验室里施加选择性压力，将数百万年的进化浓缩成短短几个月的时间。随后，亲和力最高的两个拮抗剂将使用107个不同的多肽连接物的文库进行偶联，并进行类似的筛选以结合uPAR。具有最高亲和力的关联对手将被测试其 在体外抑制或防止癌症进展的能力。目的3)AIM 2的最佳候选者将接受体内动物模型治疗转移性癌症的测试--这是FDA要求的一个步骤，然后才能进行人体试验。我们的最终目标是继续与斯坦福大学的内科科学家合作，开发一种针对uPAR的治疗方法，并将其转化为临床治疗转移性癌症。

DESCRIPTION (provided by applicant): Cancer is the second most common cause of death in the United States, and is estimated to cause 580,350 deaths in 2013 alone. Approximately 90% of these mortalities will result from cancers progressing to the metastatic stage, where cells from the primary tumor begin to enter and travel through the blood stream to form new tumors throughout the body. This process of metastasis is driven by certain proteins called receptors that are present on the surface of cancer cells at high numbers and/or elevated activities relative to normal cells. The abundance of these receptors allows the cancer to rapidly grow, proliferate, and migrate through the body, and thus many have been validated as therapeutic targets for cancer treatment. For example, the notorious urokinase receptor (uPAR) has been a known cancer target for over two decades. uPAR coordinates multiple processes that are essential for metastasis; it is present at elevated levels in virtually all cancers, and high levels are associatd with increased tumor aggressiveness, resistance to chemotherapy, metastasis, and overall low patient survival time. However, despite its importance, no FDA- approved molecules have become available that target this receptor. This is due to the overwhelming complexity of uPAR; it has multiple functional sites that independently coordinate cancer growth and metastasis. The development of a therapeutic that efficiently blocks all functional uPAR sites still represents a critical barrier to progress in the treatment of metastatic cancer. Here, we have outlined a set of objectives aimed to overcome this barrier: Aim 1) use two pre-existing uPAR antagonists to determine the effect of simultaneously targeting the two major functional uPAR sites on cancer progression in vitro. The results will improve scientific knowledge of key uPAR functions that drive cancer growth and metastasis, and will indicate if there is an additive or synergistic effect of targeting multiple functions simultaneously. Aim 2) Improve and chemically link the two antagonists from Aim 1 to generate an antagonist that binds uPAR with ultra-high (picomolar to femtomolar) affinity and blocks all functions that coordinate cancer progression. Libraries of 107 mutants of each antagonist will be generated using error-prone PCR, expressed on the surface of yeast, and screened for binding to uPAR using high throughput (10,000 cells per second) flow cytometry. This powerful technique applies selective pressure to condense millions of years of evolution into just a few months at the lab bench. Subsequently, the two antagonists with the highest affinity will be coupled using a library of 107 different peptide linkers and similarly screened for binding to uPAR. Linked antagonists with the highest affinity will be tested for their ability to inhibit or prevent cancer progression in vitro. Aim 3) The top candidates from Aim 2 wil be tested for treating metastatic cancer in vivo using animal models - a step required by the FDA before human trials can ensue. Our ultimate objective is to continue our collaborations with physician-scientists at Stanford to develop a uPAR-targeted therapy and translate it into the clinic for treatment of metastatic cancer.