Fibroblast Activation Protein-Targeted Radioligand Therapy for Treatment of Solid Tumors.

Fibroblast activation protein (FAP) has received increasing attention as an oncologic target because of its prominent expression in solid tumors but virtual absence from healthy tissues. Most radioligand therapies (RLTs) targeting FAP, however, suffer from inadequate tumor retention or clearance from healthy tissues. Herein we report a FAP-targeted RLT comprising an FAP6 ligand conjugated to DOTA and an albumin binder (4-p-iodophenylbutyric acid, or IP) for enhanced pharmacokinetics. We evaluated the performance of the resulting FAP6-IP-DOTA conjugate in 4 tumor models, 3 of which express FAP only on cancer-associated fibroblasts, that is, analogously to human tumors. Methods: Single-cell RNA-sequencing data were analyzed from 34 human breast, ovarian, colorectal, and lung cancers to quantify FAP-overexpressing cells. FAP6-DOTA conjugates were synthesized with or without an albumin binder (IP) and investigated for binding to human FAP-expressing cells. Accumulation of 111In- or 177Lu-labeled conjugates in KB, HT29, U87MG, and 4T1 murine tumors was also assessed by radioimaging or biodistribution analyses. Radiotherapeutic potency was quantitated by measuring tumor volumes versus time. Results: Approximately 5% of all cells in human tumors overexpressed FAP (cancer-associated fibroblasts comprised ∼77% of this FAP-positive subpopulation, whereas ∼2% were cancer cells). FAP6 conjugates bound to FAP-expressing cells with high affinity (dissociation constant, ∼1 nM). 177Lu-FAP6-IP-DOTA achieved an 88-fold higher tumor dose than 177Lu-FAP6-DOTA and improved all tumor-to-healthy-organ ratios. Single doses of 177Lu-FAP6-IP-DOTA suppressed tumor growth by about 45% in all tested tumor models without causing reproducible toxicities. Conclusion: We conclude that 177Lu-FAP6-IP-DOTA constitutes a promising candidate for FAP-targeted RLT of solid tumors.

Design and characterization of fibroblast activation protein targeted pan-cancer imaging agent for fluorescence-guided surgery of solid tumors.

Tumor-targeted fluorescent dyes have been shown to significantly improve a surgeon's ability to locate and resect occult malignant lesions, thereby enhancing a patient's chances of long term survival. Although several tumor-targeted fluorescent dyes have been developed for imaging specific subsets of human cancers, no tumor-targeted dye has been designed that can image all cancer types. Based on observations that fibroblast activation protein (FAP) is upregulated on cancer-associated fibroblasts (CAFs) that infiltrate essentially all solid tumors, we have undertaken to develop a FAP-targeted fluorescent dye that can image CAFs without accumulating in healthy cells or fibroblasts. We report here that FTL-S-S0456, a novel FAP-targeted near infrared dye that binds FAP with high affinity (∼12 nM) and specificity (>5000-fold over PREP and DPP-IV), concentrates in all seven solid tumor types examined, yielding fluorescence images with high tumor to background ratios that persist for several days. We conclude that FTL-S-S0456 constitutes an excellent ligand-targeted near infrared dye that enables intra-operative imaging of most if not all solid tumors.

Targeted inhibition of PI3 kinase/mTOR specifically in fibrotic lung fibroblasts suppresses pulmonary fibrosis in experimental models.

Idiopathic pulmonary fibrosis (IPF) is a lethal disease with an average life expectancy of 3 to 5 years. IPF is characterized by progressive stiffening of the lung parenchyma due to excessive deposition of collagen, leading to gradual failure of gas exchange. Although two therapeutic agents have been approved from the FDA for IPF, they only slow disease progression with little impact on outcome. To develop a more effective therapy, we have exploited the fact that collagen-producing myofibroblasts express a membrane-spanning protein, fibroblast activation protein (FAP), that exhibits limited if any expression on other cell types. Because collagen-producing myofibroblasts are only found in fibrotic tissues, solid tumors, and healing wounds, FAP constitutes an excellent marker for targeted delivery of drugs to tissues undergoing pathologic fibrosis. We demonstrate here that a low-molecular weight FAP ligand can be used to deliver imaging and therapeutic agents selectively to FAP-expressing cells. Because induction of collagen synthesis is associated with phosphatidylinositol 3-kinase (PI3K) activation, we designed a FAP-targeted PI3K inhibitor that selectively targets FAP-expressing human IPF lung fibroblasts and potently inhibited collagen synthesis. Moreover, we showed that administration of the inhibitor in a mouse model of IPF inhibited PI3K activation in fibrotic lungs, suppressed production of hydroxyproline (major building block of collagen), reduced collagen deposition, and increased mouse survival. Collectively, these studies suggest that a FAP-targeted PI3K inhibitor might be promising for treating IPF.

Reprogramming of profibrotic macrophages for treatment of bleomycin-induced pulmonary fibrosis.

Fibrotic diseases cause organ failure that lead to ~45% of all deaths in the United States. Activated macrophages stimulate fibrosis by secreting cytokines that induce fibroblasts to synthesize collagen and extracellular matrix proteins. Although suppression of macrophage-derived cytokine production can halt progression of fibrosis, therapeutic agents that prevent release of these cytokines (e.g., TLR7 agonists) have proven too toxic to administer systemically. Based on the expression of folate receptor ß solely on activated myeloid cells, we have created a folate-targeted TLR7 agonist (FA-TLR7-54) that selectively accumulates in profibrotic macrophages and suppresses fibrosis-inducing cytokine production. We demonstrate that FA-TLR7-54 reprograms M2-like fibrosis-inducing macrophages into fibrosis-suppressing macrophages, resulting in dramatic declines in profibrotic cytokine release, hydroxyproline biosynthesis, and collagen deposition, with concomitant increases in alveolar airspaces. Although nontargeted TLR7-54 is lethal at fibrosis-suppressing doses, FA-TLR7-54 halts fibrosis without evidence of toxicity. Taken together, FA-TLR7-54 is shown to constitute a novel and potent approach for treating fibrosis without causing dose-limiting systemic toxicities.