68Ga-FAPI-LM3, a novel FAP and SSTR2 dual targeting peptide heterodimer for PET imaging of nasopharyngeal carcinoma

Introduction: It has been well documented that most non-keratinizing nasopharyngeal carcinoma (NPC) patients express high levels of fibroblast activation protein (FAP) and somatostatin receptor subtype 2 (SSTR2). Radiolabeled fibroblast activation protein inhibitors (FAPIs) and p-Cl-Phe-cyclo(D-Cys-Tyr-D-4-amino-Phe(carbamoyl)-Lys-Thr- Cys)D-Tyr-NH2 (LM3) peptide have been extensively investigated for imaging FAP and SSTR2 positive tumors, respectively. In this study, a FAPI-LM3 heterodimer was labeled with 68Ga and evaluated in tumor xenografts and NPC patients. We hypothesized that the heterodimer, recognizing both FAP and SSTR2, is advantageous over its monomeric counterpart, due to its dual receptor targeting property.

Methods: FAPI-LM3 was synthesized using FAPI-46 and LM3. After radiolabeling with 68Ga (68Ga-FAPI-LM3), its FAP and SSTR2 dual receptor binding ability was evaluated in vitro and in vivo. Preclinical studies, including microPET and biodistribution studies, were performed using HT-1080-FAP+HT-1080-SSTR2, HT-1080-FAP, and C666.1 (NPC cell line) xenografts. The effective dosimetry of 68Ga-FAPI-LM3 was evaluated in three healthy volunteers. The clinical feasibility of 68Ga-FAPI-LM3 PET/CT was evaluated in NPC patients, and the results were compared with those of 68Ga-FAPI-46 and 68Ga-DOTA-LM3.

Results: 68Ga-FAPI-LM3 was stable in phosphate buffered saline and fetal bovine serum for at least 2 h. 68Ga-FAPI-LM3 yielded FAP- and SSTR2-binding affinities comparable to those of monomeric FAPI (IC50, 4.4 nM vs. 11.7 nM) and DOTA-LM3 (IC50, 13.2 nM vs. 1.3 nM), respectively. The representative small-animal PET images of three tracers in HT-1080-FAP+HT-1080-SSTR2 dual target positive xenografts were shown in Figure A. The tumor uptake of 68Ga-FAPI-LM3 was significantly higher than that of 68Ga-FAPI-46 (14.78 ± 0.76 %ID/g vs. 8.08 ± 1.51 %ID/g at 1 h, P < 0.001; 17.68 ± 2.46 %ID/g vs. 7.11 ± 0.99 %ID/g at 4 h, P < 0.001) and 68Ga-DOTA-LM3 (14.78 ± 0.76 %ID/g vs. 7.88 ± 1.10 %ID/g at 1h, P < 0.001; 17.68 ± 2.46 %ID/g vs. 5.46 ± 0.41 %ID/g at 4 h, P < 0.001) in HT-1080-FAP+HT-1080-SSTR2 tumor mice, while the normal organs showed relatively low tracer uptake. The HT-1080-FAP+HT-1080-SSTR2 tumor uptake of 68Ga-FAPI-LM3 at 1 h p.i. (14.78 ± 0.76 %ID/g) was mostly suppressed by FAPI46+DOTA-LM3 (0.86% ± 0.06%, 94% blockade), but was partially inhibited by FAPI-46 (6.51 ± 0.89 %ID/g, 56% blockade) or DOTA-LM3 (6.34 ± 0.77 %ID/g, 57% blockade) (Figure B). In the single-target-positive xenografts, 68Ga-FAPI-LM3 showed comparable tumor uptake as 68Ga-FAPI-46 in HT-1080-FAP xenografts (10.87 ± 1.04 %ID/g vs. 10.47 ± 0.51%ID/g, P = 0.58) and as 68Ga-DOTA-LM3 in C666.1 xenografts (3.30 ± 0.20 %ID/g vs. 3.23 ± 0.25 %ID/g, P = 0.88). 68Ga-FAPI-LM3 was tolerated well, with no adverse events in any of the healthy volunteers or patients. The effective dose from 68Ga-FAPI-LM3 PET/CT was 1.96 × 10-2 mSv/MBq. In clinical investigations with NPC, the radiotracer uptake of primary and metastatic lesions in 68Ga-FAPI-RGD PET/CT was significantly higher than those in 68Ga-FAPI-46 and 68Ga-DOTA-LM3 PET/CT.

Conclusions: ^{68Ga-FAPI-LM3 exhibited FAP and SSTR2 dual receptor targeting properties, resulting in improved tumor uptake and retention compared with monomeric FAPI and LM3. This study demonstrated the safety and clinical feasibility of 68Ga-FAPI-LM3 PET/CT for imaging NPC patients.}

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