Tailoring Fibroblast-Activation Protein Targeting for Theranostics: A Comparative Preclinical Evaluation of the 68Ga- and 177Lu-Labeled Monomeric and Dimeric Fibroblast-Activation Protein Inhibitors DOTA.SA.FAPi and DOTAGA.(SA.FAPi)2.

BACKGROUND: FAP radiopharmaceuticals show promise for cancer diagnosis; however, their limited tumor residency hinders treatment. This study compared two FAPi derivatives, DOTA.SA.FAPi and DOTAGA.(SA.FAPi)2, labeled with gallium-68 and lutetium-177, aiming to determine an optimum combination for creating theranostic pairs. METHODS: The radiotracers were studied for lipophilicity, binding to human serum proteins, and binding to human cancer-associated fibroblasts (CAFs) in vitro, including saturation and internalization/externalization studies. PET/SPECT/CT and biodistribution studies were conducted in PC3 and U87MG xenografts for [68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DOTAGA.(SA.FAPi)2. [177Lu]Lu-DOTA.SA.FAPi and [177Lu]Lu-DOTAGA.(SA.FAPi)2, were evaluated in PC3 xenografts. Biodistribution studies of [68Ga]Ga-DOTA.SA.FAPi were performed in healthy male and female mice. RESULTS: All radiotracers exhibited strong binding to FAP. Their internalization rate was fast while only [177Lu]Lu-DOTAGA.(SA.FAPi)2 was retained longer in CAFs. [68Ga]Ga-DOTAGA.(SA.FAPi)2 and [177Lu]Lu-DOTAGA.(SA.FAPi)2 displayed elevated lipophilicity and affinity for human serum proteins compared to [68Ga]Ga-DOTA.SA.FAPi and [177Lu]Lu-DOTA.SA.FAPi. In vivo studies revealed slower washout of [68Ga]Ga-DOTAGA.(SA.FAPi)2 within 3 h compared to [68Ga]Ga-DOTA.SA.FAPi. The tumor-to-tissue ratios of [68Ga]Ga-DOTAGA.(SA.FAPi)2 versus [68Ga]Ga-DOTA.SA.FAPi did not exhibit any significant differences. [177Lu]Lu-DOTAGA.(SA.FAPi)2 maintained a significant tumor uptake even after 96 h p.i. compared to [177Lu]Lu-DOTA.SA.FAPi. CONCLUSIONS: Dimeric compounds hold promise for therapy, while monomers are better suited for diagnostics. Finding the right combination is essential for effective disease management.

Therapeutic potential of [177Lu]Lu-DOTAGA-FAPi dimers in metastatic breast cancer patients with limited treatment options: efficacy and safety assessment.

PURPOSE: The upregulation of fibroblast activation protein (FAP) expression has been observed in various cancers, including metastatic breast carcinoma, prompting research into small molecule inhibitors for both diagnostic and therapeutic purposes. While the diagnostic value of PET/CT imaging using 68 Ga- or 18F-labelled FAPi-monomers in breast cancer diagnosis is well-established, there is a significant need for therapeutic analogs. This retrospective study aimed to assess the safety and effectiveness of [177Lu]Lu-DOTAGA.FAPi dimer radionuclide therapy in patients with advanced-stage breast cancer who had previously undergone [68 Ga]Ga-DOTA.SA.FAPi PET/CT scans to confirm the expression of FAP. MATERIALS AND METHODS: Between November 2020 and March 2023, a compassionate treatment approach was utilized to administer [177Lu]Lu-DOTAGA.FAPi dimer radionuclide therapy to heavily pretreated patients with advanced breast cancer. Nineteen patients (18 females, 1 male) with metastatic breast cancer participated in the study, with an average age of 44.6 ± 10.7 years. The therapy was administered at intervals of 8 to 12 weeks, and the median follow-up duration was 14 months. The primary objective of the study was to assess molecular response using [68 Ga]Ga-DOTA.SA.FAPi PET/CT scans, with response evaluation based on the PERCIST criteria. Secondary endpoints included overall survival (OS), progression-free survival (PFS), clinical response assessment, and safety evaluation using CTCAE v5.0 guidelines. RESULTS: A total of 65 cycles were administered, with a mean cumulative activity of 19 ± 5.7 GBq (510 ± 154 mCi) ranging from 11 to 33.3 GBq (300 to 900 mCi) of [177Lu]Lu-DOTAGA.FAPi dimer. The number of cycles ranged from 2 to 6, with a median of 3 cycles. The treatment protocol consisted of different numbers of cycles administered to the patients: specifically, two cycles were given to five patients, three cycles to nine patients, four cycles to one patient, and six cycles to four patients. Most patients had invasive/infiltrative ductal carcinoma (94.7%), while a small percentage had invasive lobular carcinoma (5.3%). All patients had bone metastases, and five of them also had liver involvement, while seven had brain metastases. Response assessment using [68 Ga]Ga-DOTA.SA.FAPi PET/CT scans showed that 25% of the 16 patients evaluated had partial remission, while 37.5% exhibited disease progression. According to the VAS response criteria, 26.3% achieved complete response, 15.7% had partial response, 42% showed minimal response, 11% had stable disease, and 5% had no response. The clinical disease control rate was promising, with 95% of patients achieving disease control. The clinical objective response rate was 84%. The median follow-up period was 14 months. At the time of analysis, the median overall survival was 12 months, and the median progression-free survival was 8.5 months. Notably, no severe hematological, renal, or hepatic toxicities, electrolyte imbalances, or adverse events of grade 3 or 4 were observed during the study. CONCLUSION: The findings suggest that [177Lu]Lu-DOTAGA.FAPi dimer therapy is well-tolerated, safe, and effective for treating end-stage metastatic breast cancer patients. [177Lu]Lu-DOTAGA.FAPi dimer treatment demonstrated promising efficacy in patients with advanced breast cancer, as indicated by high disease control rates, favorable response outcomes, and acceptable safety profile. Further research and longer follow-up are warranted to assess long-term outcomes and validate these findings.

Development of Fibroblast Activation Protein Inhibitor-Based Dimeric Radiotracers with Improved Tumor Retention and Antitumor Efficacy.

Fibroblast activation protein (FAP), a fundamental component of the tumor stroma, is overexpressed in cancer-associated fibroblasts (CAFs). As a promising theranostic probe, we evaluated whether the FAP inhibitor (FAPI) dimer (DOTA-2P[FAPI]2) is more effective than its monomeric analogs for FAP-targeted radionuclide therapy. [68Ga]Ga/[177Lu]Lu-DOTA-2P(FAPI)2 were assayed in a stability study, small-animal positron emission tomography (PET) and single-photon emission computed tomography (SPECT), biodistribution, and radionuclide therapy to comprehensively evaluate their preclinical pharmacokinetics. The pharmacokinetics of [68Ga]Ga-DOTA-2P(FAPI)2 and [177Lu]Lu-DOTA-2P(FAPI)2 were determined in FAP-positive hepatocellular carcinoma patient-derived xenografts (PDXs) and HT-1080-FAP cell-derived xenografts (CDXs). [68Ga]Ga-DOTA-2P(FAPI)2 and [177Lu]Lu-DOTA-2P(FAPI)2 were stable in phosphate-buffered saline for 4 h. The tumor retention of [68Ga]Ga-DOTA-2P(FAPI)2 was better than that of [68Ga]Ga-FAPI-46 in HT-1080-FAP CDXs, while healthy organs showed low tracer uptake and fast body clearance. In single-photon emission computed tomography, [177Lu]Lu-DOTA-2P(FAPI)2 showed a higher uptake and longer retention for tumors in both PDXs and CDXs from 1-48 h. [177Lu]Lu-DOTA-2P(FAPI)2 showed the best inhibition of tumor growth in PDXs and CDXs. DOTA-2P(FAPI)2 has increased tumor uptake and retention properties compared to FAPI-46, which significantly improves the use of FAPI-based vectors for PET imaging and radionuclide therapy. [177Lu]Lu-DOTA-2P(FAPI)2 may be safe and effective for the treatment of FAP-positive malignant tumors.

Synthesis, Preclinical Evaluation, and a Pilot Clinical PET Imaging Study of 68Ga-Labeled FAPI Dimer.

Cancer-associated fibroblasts (CAFs) are crucial components of the tumor microenvironment. Fibroblast activation protein (FAP) is overexpressed in CAFs. FAP-targeted molecular imaging agents, including the FAP inhibitors (FAPIs) 04 and 46, have shown promising results in tumor diagnosis. However, these molecules have a relatively short tumor-retention time for peptide-targeted radionuclide therapy applications. We aimed to design a 68Ga-labeled FAPI dimer, 68Ga-DOTA-2P(FAPI)2, to optimize the pharmacokinetics and evaluate whether this form is more effective than its monomeric analogs. Methods:68Ga-DOTA-2P(FAPI)2 was synthesized on the basis of the quinoline-based FAPI variant (FAPI-46), and its binding properties were assayed in CAFs. Preclinical pharmacokinetics were determined in FAP-positive patient-derived xenografts using small-animal PET and biodistribution experiments. The effective dosimetry of 68Ga-DOTA-2P(FAPI)2 was evaluated in 3 healthy volunteers, and PET/CT imaging of 68Ga-FAPI-46 and 68Ga-DOTA-2P(FAPI)2 was performed on 3 cancer patients. Results:68Ga-DOTA-2P(FAPI)2 was stable in phosphate-buffered saline and fetal bovine serum for 4 h. The FAPI dimer showed high affinity and specificity for FAP in vitro and in vivo. The tumor uptake of 68Ga-DOTA-2P(FAPI)2 was approximately 2-fold stronger than that of 68Ga-FAPI-46 in patient-derived xenografts, whereas healthy organs showed low tracer uptake and fast body clearance. The effective dose of 68Ga-DOTA-2P(FAPI)2 was 1.19E-02 mSv/MBq, calculated using OLINDA. Finally, the PET/CT scans of the 3 cancer patients revealed higher intratumoral uptake of 68Ga-DOTA-2P(FAPI)2 than of 68Ga-FAPI-46 in all tumor lesions (SUVmax, 8.1-39.0 vs. 1.7-24.0, respectively; P < 0.001). Conclusion:68Ga-DOTA-2P(FAPI)2 has increased tumor uptake and retention properties compared with 68Ga-FAPI-46, and it could be a promising tracer for both diagnostic imaging and targeted therapy of malignant tumors with positive expression of FAP.