New Approaches to the Selective Targeting of Cancer-associated Fibroblasts

描述（由申请人提供）：本研究致力于开发可用于治疗癌症的基于蛋白质的疗法，其原理是消除实体瘤支持网络的关键组分。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。癌症相关的成纤维细胞在实体瘤的发生和发展中起着重要作用，但选择性靶向这些基质细胞的治疗效果已被证明是困难的。最广泛研究的选择性靶向CAF的方法利用了位于实体瘤部位的CAF表面上的细胞表面蛋白酶成纤维细胞活化蛋白（FAP）的几乎排他性表达。这些努力集中在开发靶向表达FAP的细胞的抗体和小分子。然而，这些方法尚未产生有效的治疗性癌症治疗。在开发通过FAP靶向CAF的治疗方法中存在问题的一个潜在原因是，FAP在肿瘤起始和进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综合方法来研究FAP的生物学和开发新的癌症疗法。本项目将实现三个具体目标：1）开发活化的成纤维细胞表面蛋白酶成纤维细胞活化蛋白-<$的多个表位的结合蛋白，并使用这些结合蛋白构建基于多价抗体的试剂。2)使用目标1中开发的基于多价抗体的试剂、靶向成纤维细胞活化蛋白-<$的蛋白酶抑制剂或两者的组合，以研究这些试剂可能对成纤维细胞和癌细胞共培养物中成纤维细胞活化蛋白-<$介导的功能的影响。3)使用小鼠癌症模型检查目标2中确定的试剂对肿瘤生长和转移的影响。作为目标1的一部分，将使用酵母表面展示提供的蛋白质工程技术开发待产生的结合蛋白。多价结合蛋白将在抗体形式内结合联合收割机鉴定的结合剂。体外研究将主要集中在肿瘤细胞和CAF的混合物的集体侵入行为和孤立的对肿瘤细胞和CAF的行为，分别使用侵入测定和微孔阵列技术。使用目标1中开发的粘合剂，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CAFs上FAP表达的选择性下调是否会抑制这些行为。癌症的小鼠模型将用于研究用多价结合蛋白靶向FAP的治疗益处，使用实验来测试蛋白质是否阻止肿瘤生长或阻止免疫系统对肿瘤耐受性的发展。这些研究将为科学界提供研究和干预FAP生物学功能的新工具，以及治疗癌症的新治疗线索。

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.