Engineering a multispecific cell receptor antagonist to treat metastatic cancer

描述（由申请人提供）：癌症是美国第二大常见的死亡原因，仅在2013年估计就造成580，350人死亡。这些死亡率中的大约90%将由进展到转移阶段的癌症引起，其中来自原发性肿瘤的细胞开始进入并穿过血流以在全身形成新的肿瘤。这种转移过程是由某些称为受体的蛋白质驱动的，这些蛋白质以相对高的数量和/或升高的活性存在于癌细胞表面。 正常细胞。这些受体的丰度允许癌症快速生长，增殖和迁移通过身体，因此许多已被验证为癌症治疗的治疗靶点。例如，臭名昭著的尿激酶受体（uPAR）已经成为已知的癌症靶点超过二十年。uPAR协调转移所必需的多个过程;它在几乎所有癌症中以升高的水平存在，并且高水平与增加的肿瘤侵袭性、对化疗的抗性、转移和总体低患者存活时间相关。然而，尽管它的重要性，没有FDA批准的分子已经成为可用的目标，这种受体。这是由于uPAR的压倒性复杂性;它具有多个独立协调癌症生长和转移的功能位点。有效阻断所有功能性uPAR位点的治疗剂的开发仍然是转移性癌症治疗进展的关键障碍。在这里，我们概述了一套 目的1）使用两种预先存在的uPAR拮抗剂来确定同时靶向两个主要功能性uPAR位点对体外癌症进展的影响。这些结果将提高对驱动癌症生长和转移的关键uPAR功能的科学认识，并将表明是否存在累加或协同效应。 同时针对多个功能。目的2）改进和化学连接来自目的1的两种拮抗剂以产生以超高（皮摩尔至飞摩尔）亲和力结合uPAR并阻断协调癌症进展的所有功能的拮抗剂。将使用易错PCR产生每种拮抗剂的107个突变体的文库，在酵母表面上表达，并使用高通量（每秒10，000个细胞）流式细胞术筛选与uPAR的结合。这种强大的技术应用选择性压力，将数百万年的进化浓缩到实验室工作台上的几个月内。随后，将具有最高亲和力的两种拮抗剂使用107种不同肽接头的文库偶联，并类似地筛选与uPAR的结合。将测试具有最高亲和力的连接拮抗剂的活性 在体外抑制或预防癌症进展的能力。目标3）目标2中的最佳候选药物将使用动物模型进行体内转移性癌症治疗测试-这是FDA在人体试验之前要求的步骤。我们的最终目标是继续与斯坦福大学的医生-科学家合作，开发uPAR靶向治疗，并将其转化为转移性癌症的临床治疗。

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.