E-64

Fibroblast activation protein (FAP) is highly upregulated in the stroma of >90 % of epithelial cancers with restricted expression in normal tissues, making it an attractive drug target. Efforts by numerous laboratories developing FAP-targeted radiopharmaceuticals have been significant and led to the development of promising diagnostic agents (e.g., [⁶⁸Ga]Ga-FAPI-46). However, the poor tumor residence times of many of these low-molecular-weight agents remain a hurdle to radiotherapeutic clinical translation. Our laboratory has been exploring an endolysosomal trapping approach to increase the tumor residence time of radiopharmaceuticals through irreversible adduct formation using epoxysuccinyl peptide inhibitors. Herein, we explore this approach by incorporating an irreversible inhibitor into a FAP-targeted agent, [¹⁷⁷Lu]Lu-FAPI-ET1. Along with controls ([¹⁷⁷Lu]Lu-FAPI-SHAM and [¹⁷⁷Lu]Lu-FAPI-46), the biological performance of [¹⁷⁷Lu]Lu-FAPI-ET1 was examined through a battery of in vitro and in vivo studies using the FAP-positive human U-87MG glioblastoma cell line as our model.

FAPI-ET1, FAPI-SHAM and FAPI-46 were obtained by multi-step synthesis and radiolabeled using [¹⁷⁷Lu]LuCl₃. [¹⁷⁷Lu]Lu-FAPI-ET1 contains the incorporated epoxysuccinyl peptide inhibitor, while [¹⁷⁷Lu]Lu-FAPI-SHAM is a nearly identical physiochemical control incapable of adduct formation, and [¹⁷⁷Lu]Lu-FAPI-46 was used as a clinical benchmark. Western Blot confirmed the FAP expression of U-87MG cells. Kinetics assays of FAPI-ET1 and positive control (E-64) were conducted using human cysteine cathepsin B. In vitro IC₅₀, cellular uptake and adduct formation analysis of the three radioconstructs were performed using U-87MG cells. In vivo biodistribution studies were conducted in U-87MG xenograft mouse models, evaluating the constructs at 3-, 24-, 72- and 168-h post-injection time points. FAPI-ET1 and FAPI-SHAM (1.85 ± 0.04 and 0.8 ± 0.7 nM, respectively) exhibited similar FAP binding affinities compared to FAPI-46 (0.9 ± 0.7 nM). FAPI-ET1 demonstrated significantly higher cathepsin B inhibition kinetics (120,000 ± 14,000 M^-1 s^-1) than the E-64 inhibitor (42,000 ± 4000 M^-1 s^-1). The cellular uptake, including surface-bound and internalized fraction, of [¹⁷⁷Lu]Lu-FAPI-ET1 and [¹⁷⁷Lu]Lu-FAPI-SHAM were significantly lower than [¹⁷⁷Lu]Lu-FAPI-46 (P ≤ 0.001). By 240 min, the internalized fraction for [¹⁷⁷Lu]Lu-FAPI-46 was 9.9 ± 1.7 %, while [¹⁷⁷Lu]Lu-FAPI-ET1 and [¹⁷⁷Lu]Lu-FAPI-SHAM were 2.7 ± 0.8 % and 2.7 ± 1.1 %, respectively. When the radioconjugates were incubated with U-87MG cells, two adduct bands were observed at 30-35 and 100 kDa for [¹⁷⁷Lu]Lu-FAPI-ET1, while only a single adduct band of 100 kDa was observed for [¹⁷⁷Lu]Lu-FAPI-SHAM and [¹⁷⁷Lu]Lu-FAPI-46. The 30-35 kDa band for [¹⁷⁷Lu]Lu-FAPI-ET1 is anticipated due to adduct formation with endolysosomal proteases (e.g., cysteine cathepsins). However, the 100 kDa band for all FAP-targeted agents is attributed to adduct formation between the cyanopyrrolidine moiety of the FAPI-targeted vector and FAP. In vivo biodistribution studies revealed higher initial blood retention for [¹⁷⁷Lu]Lu-FAPI-ET1 and [¹⁷⁷Lu]Lu-FAPI-SHAM (7 ± 2 and 5.3 ± 1.3 %ID/g, P < 0.0001 and P = 0.02) compared to [¹⁷⁷Lu]Lu-FAPI-46 (2.0 ± 0.5 %ID/g) at 3 h but all radioconjugates cleared from the blood by 72 h. On average, [¹⁷⁷Lu]Lu-FAPI-ET1 and [¹⁷⁷Lu]Lu-FAPI-SHAM exhibited higher liver and kidney uptake (P > 0.05) than [¹⁷⁷Lu]Lu-FAPI-46 at 3 h. At later time points, [¹⁷⁷Lu]Lu-FAPI-ET1 retained significantly higher levels of activity in the liver and kidneys (P < 0.05) compared to [¹⁷⁷Lu]Lu-FAPI-SHAM, indicating that adduct formation increased tissue retention. [¹⁷⁷Lu]Lu-FAPI-46 (15 ± 8 %ID/g) demonstrated significantly higher uptake (P < 0.0001) in U-87MG tumors, compared to [¹⁷⁷Lu]Lu-FAPI-ET1 (7 ± 4 %ID/g) or [¹⁷⁷Lu]Lu-FAPI-SHAM (3.5 ± 0.8 %ID/g). However, [¹⁷⁷Lu]Lu-FAPI-ET1 demonstrated significantly prolonged tumor retention in U-87MG xenograft tumors at 168 h, with a five-fold increase compared to [¹⁷⁷Lu]Lu-FAPI-46.

[¹⁷⁷Lu]Lu-FAPI-ET1 demonstrates that the endolysosomal trapping approach can be successfully implemented in FAP-targeted agents and achieve enhanced FAP-positive U-87MG tumor residualization. Although the decreased internalization rate and in vivo targeting of [¹⁷⁷Lu]Lu-FAPI-ET1 compared to [¹⁷⁷Lu]Lu-FAPI-46 and the ambiguity concerning FAP-mediated internalization efficacy due to targeting vector adduct formation are hurdles to the current implementation. Future approaches are focused on better characterizing FAP adduct formation and cellular trafficking to reveal new pathways to optimize the endolysosomal trapping approach for FAP-targeted agents.