Fibroblast Activation Protein Triggers Release of Drug Payload from Non-internalizing Small Molecule Drug Conjugates in Solid Tumors

Abstract

Purpose: Small Molecule-Drug Conjugates (SMDCs) are modular anti-cancer pro-drugs that include a tumor-targeting small organic ligand, a cleavable linker and a potent cytotoxic agent. Most of the SMDC products that have been developed for clinical applications target internalizing tumor-associated antigens on the surface of tumor cells. We have recently described a novel non-internalizing small organic ligand (named OncoFAP) of Fibroblast Activation Protein (FAP), a tumor-associated antigen highly expressed in the stroma of most of solid human malignancies. Experimental Design: In this article, we describe a new series of OncoFAP-Drug derivatives based on monomethyl auristatin E (MMAE, a potent cytotoxic tubulin poison) and dipeptide linkers that are selectively cleaved by FAP in the tumor microenvironment. Results: The tumor targeting potential of OncoFAP was confirmed in cancer patients using Nuclear Medicine procedures. We used mass spectrometry methodologies in order to quantify the amount of pro-drug delivered to tumors and normal organs, as well as the efficiency of the drug release process. Linkers previously exploited for anticancer drug conjugates were used as benchmark. We identified OncoFAP-Gly-Pro-MMAE as the best performing SMDC, which has now been prioritized for further clinical development. OncoFAP-Gly-Pro-MMAE selectively delivered more than 10% injected dose per gram of MMAE to FAP-positive tumors, with a tumor-to-kidney ratio of 16:1 at 24-hours post-injection. Conclusions: The FAP-specific drug conjugates described in this article promise to be efficacious for the targeting of human malignancies. The extracellular release of potent anticancer payloads mediates durable complete remission in difficult-to-treat animal models of cancer.

A Comparative Analysis of Fibroblast Activation Protein-Targeted Small Molecule–Drug, Antibody–Drug, and Peptide–Drug Conjugates

“…OncoFAP-Fluo (5), 7NP2-Fluo (7), and FAP2286-Fluo (9) exhibited high affinity toward hFAP with apparent K D values of 4.2, 19.5, and 8.3 nM, respectively. Alb-Fluo (11) and the negative controls Neg-Fluo ( 6), KSF-Fluo ( 8), and Neg2286-Fluo (10) did not show any significant interaction with the target antigen (Figure 2B). The fluorescein conjugates were also evaluated by FACS analysis for their ability to bind hFAP when expressed on the surface of cancer cells.…”

“…4 The therapeutic performance of ADCs is hampered by their slow extravasation, heterogeneous diffusion in solid tumors, and significant liver uptake at early and late time points after systemic administration. 5−7 Peptide−drug conjugates (PDCs) 8−10 and small molecule− drug conjugates (SMDCs) 11,12 have emerged as promising drug prototypes with low molecular weight (i.e., between 1 and 5 kDa) that may overcome some ADC limitations. PDCs and SMDCs are characterized by a deeper, more homogeneous, and rapid penetration of solid cancer lesions.…”

“…25−27 In this Communication, we have generated tumor-targeting drug conjugates based on antibodies, peptides, and small organic ligands, directed against fibroblast activation protein, a serine protease overexpressed by cancer associated fibroblasts in the microenvironment of most of solid human malignancies. 28,29 We compared the in vitro and in vivo properties of fibroblast activation protein (FAP)targeted ADCs, PDCs, and SMDCs equipped with "Gly-Pro- MMAE", a novel FAP-cleavable linker-payload module 11 for the selective release of MMAE in tumor lesions.…”

“…Peptide–drug conjugates (PDCs) − and small molecule–drug conjugates (SMDCs) , have emerged as promising drug prototypes with low molecular weight (i.e., between 1 and 5 kDa) that may overcome some ADC limitations. PDCs and SMDCs are characterized by a deeper, more homogeneous, and rapid penetration of solid cancer lesions. , …”

“…Bicycle Therapeutics Inc. has recently developed a number of PDCs that are currently being investigated in Phase I and II clinical trials , with encouraging reports of early signs of efficacy in patients with urothelial cancer. , Many linear and cyclic peptide products may suffer from a durable kidney retention after systemic administration. − This feature could have a negative impact on the delivery of certain highly active therapeutic payloads (e.g., toxic drugs and radionuclides). In contrast, with the exception of certain specific structures (e.g., folate-targeted drugs), small molecule–drug conjugates with high affinity to cognate specific targets rapidly localize to tumor lesions with low and transient accumulation in healthy organs, including kidney. − In this Communication, we have generated tumor-targeting drug conjugates based on antibodies, peptides, and small organic ligands, directed against fibroblast activation protein, a serine protease overexpressed by cancer associated fibroblasts in the microenvironment of most of solid human malignancies. , We compared the in vitro and in vivo properties of fibroblast activation protein (FAP)-targeted ADCs, PDCs, and SMDCs equipped with “Gly-Pro-MMAE”, a novel FAP-cleavable linker-payload module for the selective release of MMAE in tumor lesions.…”