Fibroblast Activation Protein-alpha Activated Anti-Stromal Prodrug Therpay for Ca

描述（由申请人提供）：基质细胞产生的信号分子参与肿瘤转化，并可直接调节已建立的癌细胞的生长和存活。成纤维细胞活化蛋白α（FAP）由上皮癌部位内存在的反应性基质细胞选择性产生，但在任何其他成人组织的基质内不表达。FAP是具有二肽基肽酶、明胶酶和胶原酶酶活性的膜结合丝氨酸蛋白酶。因此，本提案的目的是确定通过FAP活化的前药选择性消除肿瘤相关基质细胞是否是有效的癌症靶向治疗。为了实现这一目的，提出了以下具体目的：（1）确定蛋白水解活性FAP的选择性肽底物;（2）通过将FAP肽偶联至毒胡萝卜素的高效类似物来合成FAP可裂解的前药;（3）评价这些FAP活化的前药的体内药理学、毒理学和抗肿瘤功效。为了实现目标1，我们已经产生了重组FAP并产生了重组胶原I内的切割位点的图谱。基于这些切割位点的肽将作为推定的FAP底物进行评价。此外，我们已经产生了一个随机噬菌体底物库，以评估作为FAP底物的肽的更多样化的集合。在目标2中，我们将选择这些FAP底物中最好的以产生FAP活化的毒胡萝卜素前药，其将表征人和小鼠血浆中的FAP水解和稳定性。此外，将测试前药对一组FAP阴性癌细胞系和作为阳性对照的FAP转染系的毒性。被FAP有效水解、在血浆中稳定并且对FAP阴性细胞系具有最低毒性的前药将在Aim 3中进一步体内评估，以确定针对产生一系列基质的人癌症异种移植物的功效。将表征这些异种移植物的基质%、FAP产生和酶活性。在疗效研究之前，将进行药代动力学分析和毒理学研究，以确定最佳给药方案。本提案中描述的研究将确定靶向癌症内的支持结构（即“基质”）而不是癌细胞本身的疗法是否可以是有效的疗法。因此，FAP激活的前药可能代表了多种人类癌症的新靶向治疗。

DESCRIPTION (provided by applicant): Stromal cell production of signaling molecules is involved in neoplastic transformation and can directly regulate growth and survival of established cancer cells. Fibroblast activation protein alpha (FAP) is selectively produced by reactive stromal cells present within sites of epithelial cancers but is not expressed within stroma of any other adult tissues. FAP is a membrane bound serine protease with dipeptidyl peptidase, gelatinase and collagenase enzymatic activities. Therefore, the objective of this proposal is to determine if selective elimination of tumor associated stromal cells by a FAP activated prodrug would be effective, targeted treatment for cancer. To achieve this objective the following Specific Aims are proposed: (1) to define selective peptide substrates for the proteolytic activity FAP; (2) to synthesize FAP-cleavable prodrugs by coupling the FAP peptide to highly potent analogs of thapsigargin; (3) to evaluate pharmacology, toxicology and antitumor efficacy of these FAP-activated prodrugs in vivo. To accomplish Aim 1 we have generated recombinant FAP and generated a map of cleavage sites within recombinant collagen I. Peptides based on these cleavage sites will be evaluated as putative FAP substrates. In addition, we have generated a random phage substrate library to evaluate a more diverse collection of peptides as FAP substrates. In Aim 2, we will select the best of these FAP substrates to produce FAP activated thapsigargin prodrugs which will be characterized for FAP hydrolysis and stability in human and mouse plasma. In addition, the prodrugs will be tested for toxicity against a panel of FAP negative cancer cell lines and a FAP transfected line as a positive control. Prodrugs that are efficiently hydrolyzed by FAP, stable in plasma, and minimally toxic to FAP negative cell lines will be further evaluated in Aim 3 in vivo to determine efficacy against human cancer xenografts producing a range of stroma. These xenografts will be characterized for % stroma, for FAP production and enzymatic activity. Prior to efficacy studies, pharmacokinetic analysis and toxicology studies will be performed to determine optimal dosing regimen. The studies described in this proposal will define whether therapies that target the supporting structures (i.e. "stroma") within cancers rather than the cancer cells themselves can be effective therapy. The FAP- activated prodrug, therefore, could represent a new targeted therapy for a variety of human cancers.