New Approaches to the Selective Targeting of Cancer-associated Fibroblasts

描述(由申请人提供)：这项研究致力于开发基于蛋白质的疗法，用于癌症的治疗，建立在消除实体肿瘤支持网络的关键组成部分的原则上。特别是，这项工作将集中在癌症相关成纤维细胞(CAF)的选择性靶向上。肿瘤相关的成纤维细胞在实体瘤的发生和发展中起着重要的作用，但这些具有治疗效果的基质细胞的选择性靶向已被证明是困难的。最广泛研究的选择性靶向CAF的方法是利用位于实体瘤部位的CAF表面几乎唯一表达细胞表面蛋白酶成纤维细胞激活蛋白(FAP)的优势。这些努力的重点是开发针对表达FAP的细胞的抗体和小分子。然而，这些方法并没有产生有效的癌症治疗方法。通过FAP靶向CAF的治疗发展中出现问题的一个潜在原因是，FAP在肿瘤发生和发展中的潜在生物学作用仍然知之甚少。这里描述的工作采取了一种结合的方法来研究FAP的生物学和开发新的癌症治疗方法。本项目将致力于三个具体目标：1)开发与活化的成纤维细胞表面蛋白的多个表位结合的蛋白，并利用这些结合蛋白构建基于抗体的多价试剂。2)使用AIM 1中开发的多价抗体试剂、针对成纤维细胞激活蛋白的蛋白酶抑制剂或两者的组合，以研究这些试剂在成纤维细胞和癌细胞共培养中对成纤维细胞激活蛋白介导的功能的影响。3)用小鼠肿瘤模型检测AIM 2中确定的试剂对肿瘤生长和转移的影响。将作为AIM 1的一部分产生的结合蛋白将使用酵母表面展示提供的蛋白质工程技术来开发。多价结合蛋白将在抗体形式中结合已识别的结合子。体外研究将主要集中在肿瘤细胞和CAF混合物的集体侵袭行为，以及分别使用侵袭分析和微孔阵列技术分离的肿瘤细胞和CAF的行为。使用在AIM 1中开发的结合剂，这些研究将有助于阐明FAP的哪些部分负责侵袭性表型，并检查选择性下调CAF上的FAP表达是否会抑制这些行为。小鼠癌症模型将被用来研究以多价结合蛋白为靶点的FAP的治疗效果，通过实验测试这些蛋白是否可以防止肿瘤生长或防止免疫系统对肿瘤的耐受性的形成。这些研究将为科学界研究和干预FAP的生物学功能提供新的工具，并为癌症的治疗提供新的治疗线索。

DESCRIPTION (provided by applicant): This research strives to develop protein-based therapeutics useful for the treatment of cancer built on the principle of eliminating key components of the solid tumor support network. In particular, this work will focus on the selective targeting of cancer-associated fibroblasts (CAFs). Cancer-associated fibroblasts have been implicated in a number of important roles in the initiation and progression of solid tumors, but selective targeting of these stromal cells with therapeutic effect has proven difficult. The most widely investigated approaches to selectively target CAFs take advantage of the nearly exclusive expression of the cell surface protease fibroblast activation protein-¿ (FAP) on the surfaces of CAFs located at the site of solid tumors. These efforts have focused on the development of antibodies and small molecules that target cells expressing FAP. However, these approaches have not yielded effective therapeutic cancer treatments. One potential reason for problems in the development of therapeutics that target CAFs via FAP is that the underlying biological role of FAP in tumor initiation and progression is still poorly understood. The work described here takes a combined approach to studying the biology of FAP and developing new cancer therapies. Three specific aims will be pursued in this project: 1) Develop binding proteins to multiple epitopes of the activated fibroblast cell surface protease fibroblast activation protein-¿ and use these binding proteins to construct multivalent antibody-based reagents. 2) Use multivalent antibody-based reagents developed in Aim 1, protease inhibitors targeting fibroblast activation protein-¿, or combinations of the two, in order to study the effect these reagents may have on fibroblast activation protein-¿-mediated functions in co-cultures of fibroblasts and cancer cells. 3) Use mouse models of cancer to examine the effects of reagents identified in Aim 2 on tumor growth and metastasis. The binding proteins to be generated as a part of Aim 1 will be developed using the protein engineering techniques afforded by yeast surface display. Multivalent binding proteins will combine identified binders within an antibody format. In vitro studies will primarily focus on the collective invasive behaviors of mixtures of tumor cells and CAFs and the behaviors of isolated pairs of tumor cells and CAFs using invasion assays and microwell array technology, respectively. Using binders developed in Aim 1, these studies will help elucidate which portions of FAP are responsible for invasive phenotypes and examine whether the selective downregulation of FAP expression on CAFs will inhibit these behaviors. Murine models of cancer will be used to study the therapeutic benefits of targeting FAP with multivalent binding proteins using experiments to test whether the proteins prevent tumor growth or prevent the development of immune system tolerance to a tumor. These studies will give the scientific community new tools for studying and interfering with the biological functions of FAP and new therapeutic leads for the treatment of cancer.