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

描述(申请人提供)：基质细胞产生的信号分子参与肿瘤转化，并能直接调节已建立的癌细胞的生长和存活。成纤维细胞激活蛋白α(FAP)是由存在于上皮癌部位的反应性间质细胞选择性地产生的，但在任何其他成人组织的间质中不表达。FAP是一种膜结合型丝氨酸蛋白酶，具有二肽基肽酶、明胶酶和胶原酶活性。因此，这项建议的目的是确定通过FAP激活的前体药物选择性地消除肿瘤相关基质细胞是否对癌症进行有效的靶向治疗。为了实现这一目标，我们提出了以下具体目标：(1)确定FAP蛋白分解活性的选择性多肽底物；(2)通过将FAP多肽与高效的thapsigargin类似物偶联来合成FAP可切割的前药；(3)在体内评价这些FAP激活的前药的药理、毒理和抗肿瘤效果。为了实现目标1，我们已经生成了重组FAP，并生成了重组I型胶原内的切割位点图。基于这些切割位点的多肽将被评估为假定的FAP底物。此外，我们还生成了一个随机噬菌体底物文库，以评估作为FAP底物的更多样化的多肽集合。在目标2中，我们将从这些FAP底物中选择最好的底物来生产FAP激活的thapsigarin前药，以表征FAP在人和小鼠血浆中的水解性和稳定性。此外，这些前药将对一组FAP阴性癌细胞株和一株FAP转染株进行毒性测试，作为阳性对照。在AIM 3中，将进一步评估那些能被FAP有效水解、在血浆中稳定、对FAP阴性细胞系毒性最小的前药，以确定对产生一系列间质的人类癌症异种移植的疗效。这些异种移植物的特征是基质百分比、FAP产生和酶活性。在进行疗效研究之前，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。该提案中描述的研究将确定以癌症内的支持结构(即“间质”)而不是癌细胞本身为目标的疗法是否有效。因此，FAP激活的前药可能代表着一种针对各种人类癌症的新的靶向治疗。

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.