A novel peptide-conjugated cytotoxic prodrug selectively activated by Fibroblast Activation Protein alpha (FAP) was designed and demonstrated therapeutic efficacy against breast cancer xenografts. FAP is a membrane-bound serine protease of the dipeptidyl peptidase (DPP) family with both dipeptidase and gelatinase activity. Many cancers, including breast, have been shown to induce a reactive stroma characterized by the recruitment of reactive fibroblasts and various inflammatory cells, increased secretion of growth factors and proteases, induction of angiogenesis, and the formation of an altered extracellular matrix (ECM). FAP has been shown to be up-regulated on the surface of these reactive or carcinoma-associated fibroblasts (CAFs) in the stroma of >90% of solid tumors examined in one series of frozen human tissue specimens, but not on cancerous epithelial or adjacent normal tissue. This proteolytic activity was used to define a FAP-selective peptide substrate based on cleavage sites from the digestion of gelatin derived from human collagen I. The hydrolysis kinetics of these substrates by FAP was evaluated in vitro and the leading candidates were coupled to thapsigargin analogs to generate putative FAP-activated prodrugs. Thapsigargin is a natural plant product that inhibits the SERCA pump present in all cell types and non-selectively induces apoptosis with a mean GI50 of [0.3nM]. These prodrugs were evaluated in vitro on a variety of parameters including activation/proteolysis by FAP, ability to induce the efflux of Ca2+ from the sarcoplasmic/endoplasmic reticulum, solubility, plasma stability and the inhibition of cell growth with an IC50 of ∼[3nM] against the human breast cancer cell line MCF-7. These prodrugs were further evaluated in vivo using MCF-7 xenografts as a model of breast cancer. These drugs were shown to be generally well-tolerated and showed a significant inhibition of tumor growth in this model. Additionally, non-FAP cleavable analogs based on the amino acid sequence of the lead prodrug were generated and tumors treated with these compounds did not regress. Tumors from both treated and control animals were harvested and biodistribution studies were performed demonstrating an accumulation of the active compound in the tumors treated with the FAP-selective prodrugs. Immunohistochemical analysis of these tumors confirmed that significant necrosis was present in the FAP-selective prodrug treated tumors, but not in the tumors from the animals treated with a non-FAP cleavable prodrug nor the untreated tumor controls at 72hrs-post treatment. These studies demonstrate that exploiting the enzymatic activity of FAP is a viable target for chemotherapeutic prodrug activation and that this strategy results in an inhibition of tumor growth in this model of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1424.