Development of Fibroblast Activation Protein-Targeted Radiotracers with Improved Tumor Retention

Abstract

Cancer-associated fibroblasts constitute a vital subpopulation of the tumor stroma and are present in more than 90% of epithelial carcinomas. The overexpression of the serine protease fibroblast activation protein (FAP) allows a selective targeting of a variety of tumors by inhibitor-based radiopharmaceuticals (FAPIs). Of these compounds, FAPI-04 has been recently introduced as a theranostic radiotracer and demonstrated high uptake into different FAP-positive tumors in cancer patients. To enable the delivery of higher doses, thereby improving the outcome of a therapeutic application, several FAPI variants were designed to further increase tumor uptake and retention of these tracers. Methods: Novel quinoline-based radiotracers were synthesized by organic chemistry and evaluated in radioligand binding assays using FAP-expressing HT-1080 cells. Depending on their in vitro performance, small-animal PET imaging and biodistribution studies were performed on HT-1080-FAP tumor-bearing mice. The most promising compounds were used for clinical PET imaging in 8 cancer patients. Results: Compared with FAPI-04, 11 of 15 FAPI derivatives showed improved FAP binding in vitro. Of these, 7 compounds demonstrated increased tumor uptake in tumor-bearing mice. Moreover, tumor-to-normal-organ ratios were improved for most of the compounds, resulting in images with higher contrast. Notably two of the radiotracers, FAPI-21 and -46, displayed substantially improved ratios of tumor to blood, liver, muscle, and intestinal uptake. A first diagnostic application in cancer patients revealed high intratumoral uptake of both radiotracers already 10 min after administration but a higher uptake in oral mucosa, salivary glands, and thyroid for FAPI-21. Conclusion: Chemical modification of the FAPI framework enabled enhanced FAP binding and improved pharmacokinetics in most of the derivatives, resulting in high-contrast images. Moreover, higher doses of radioactivity can be delivered while minimizing damage to healthy tissue, which may improve therapeutic outcome.

Keywords: FAP inhibitor; PET/CT; fibroblast activation protein; theranostics; tracer development.

FIGURE 1.

Relative binding rates of ¹⁷⁷Lu-labeled FAPI derivatives compared with FAPI-04 (set to 100%) using FAP-expressing HT-1080 cells (n = 3).

FIGURE 2.

Organ SUV_max of ⁶⁸Ga-labeled FAPI derivatives in HT-1080-FAP tumor–bearing mice determined by small-animal PET imaging (n = 1).

FIGURE 3.

Tumor–to–normal-organ ratios of ⁶⁸Ga-labeled FAPI derivatives, calculated from AUCs 0–2 h after intravenous administration of radiotracers (n = 1).

FIGURE 4.

Organ uptake of ¹⁷⁷Lu-labeled FAPI derivatives in HT-1080-FAP tumor–bearing mice (n = 3). *P < 0.05. **P < 0.01. ***P < 0.001.

FIGURE 5.

Tumor–to–normal-organ ratios (calculated from %ID/g 0–24 h after intravenous administration) of ¹⁷⁷Lu-labeled FAPI derivatives in HT-1080-FAP tumor–bearing mice (n = 3).

FIGURE 6.

Whole-body PET/CT imaging of tumor patients. (A–F) Maximum-intensity projections 1 h after intravenous administration of ⁶⁸Ga-labeled FAPI-21 (A–C) and FAPI-46 (D–F). (G and H) Maximum (G) and mean (H) tracer uptake of ⁶⁸Ga-labeled FAPI-21 and -46 in tumor and healthy organs as compared with FAPI-04 (n = 2–25) (Supplemental Table 4).