Design and evaluation of a FAP/αvβ3 dual-targeted 68Ga/177Lu heterodimeric radioligand for cancer theranostics

Abstract Background Although the unique features of the tumor microenvironment (TME) present compelling targets for cancer theranostics, monovalent targeting often yields suboptimal outcomes due to heterogeneity and inadequate retention of the theranostic agents. To overcome these limitations, we designed a novel series of heterodimeric radiotracers (designated INN01−INN03) by conjugating FAP- and integrin αvβ3-targeting motifs. Methods A series of heterodimeric radiotracers was developed by integrating gallium-68 ( 68 Ga)/lutetium-177 ( 177 Lu) with a cyclic RGD peptide (RGDyK or RGDfK) and a potent boronic acid-based FAP-targeting motif using a DOTA chelator. The cellular internalization, uptake, efflux, and blockade of the radiotracers were evaluated using the U87MG cell line. Competitive binding experiments were conducted using the A549-FAP cell line. The biodistribution and small-animal PET/CT imaging characteristics of the novel radiotracers were compared to that of [ 68 Ga]Ga-PNT6555 in the U87MG tumor-bearing mouse mode. The efficacy of radionuclide therapy was evaluated in the same model (single dose; n = 8–10/group). In addition, a pilot prospective clinical study of [ 68 Ga]Ga-INN02 PET/CT was conducted ( n = 6). Results INN01, INN02, and INN03 showed high binding affinities for FAP, with IC 50 values of 1.28 ± 0.14 nM, 2.86 ± 0.83 nM, and 1.07 ± 0.35 nM, respectively. [ 68 Ga]Ga-INN02 demonstrated the highest tumor uptake at 60 min (11.65 ± 2.21% I.A./g), compared with [ 68 Ga]Ga-PNT6555 (1.91 ± 1.10% I.A./g), and exhibited predominantly renal clearance. [ 177 Lu]Lu-INN02 achieved higher early tumor uptake than [ 177 Lu]Lu-PNT6555 (21.96 ± 3.09% I.A./g vs. 12.98 ± 5.19% I.A./g at 1 h), and showed lower liver and blood exposure. Treatment with 37 MBq [ 177 Lu]Lu-INN02 led to marked tumor growth inhibition (day 14: 583 ± 338 mm 3 vs. 1130 ± 424 mm 3 for saline; p = 0.030) and prolonged median survival (20 vs. 16 days) without overt acute toxicity under the conditions tested. Furthermore, [ 68 Ga]Ga-INN02 demonstrated high uptake and low background in clinical PET imaging across multiple cancer types, allowing clear visualization of primary and metastatic lesions. Conclusion 68 Ga/ 177 Lu-INN02 shows favorable pharmacokinetics, enhanced tumor uptake and imaging contrast, and promising therapeutic efficacy, supporting further clinical evaluation. Trial registration chictr.org.cn, ChiCTR2200058108, registered 29 March 2022.