Evaluation of the Effect of Bifunctional Chelator Choice on the Biodistribution of [Pb-203]Pb-FAPI Bioconjugates

Introduction: Fibroblast activation protein (FAP) is a membrane-bound serine protease that plays a role in the remodeling of the extracellular matrix and protein degradation. FAP stands out as a promising target for theranostic applications as it is highly overexpressed in over 90% of common epithelial cancers. There is minimal to no expression observed in normal fibroblasts or healthy tissues, with the exception of fibrotic diseases, lessening concerns of off-target effects. FAP inhibitor (FAPI) tracers incorporating DOTA as the chelator of choice have been shown to exhibit rapid tumour accumulation and excellent image quality with ⁶⁸Ga. However, the short blood half-life and poor tumour retention of these tracers limits the potential for effective radionuclide therapy. Three novel FAPI bioconjugates were developed to evaluate the effect of chelator hydrophobicity on tumour uptake and retention in HEK293T:hFAP models for applications with the ²⁰³Pband ²¹²Pb theranostic pair.

Methods: The novel bifunctional chelator Cyclen-4Py-NCS (2-(4-isothiocyanatobenzyl)-1,4,7,10-tetrakis(pyridin-2-ylmethyl)-1,4,7,10-tetraazacyclododecane) was synthesized from the p-NO₂-Bn-cyclen (S-2-(4-Nitrobenzyl)-1,4,7,10-tetraazacyclododecane) backbone and characterized using ¹H and ¹³C NMR and high-resolution mass spectrometry (HR-MS). Crypt-FAPI, TCMC-FAPI, and Cyclen-4Py-FAPI were synthesized by coupling the NCS-containing bifunctional chelators to the primary amine of the FAPI pharmacophore of FAPI-04. The bioconjugates were purified using high performance liquid chromatography and characterized by HR-MS. Concentration-dependent (10^-4­ - 10^-7 M) ²⁰³Pb and ²¹²Pb radiolabeling studies were performed at room temperature and 80^oC (0.1 M NH₄OAc, pH 7) and radiochemical yields quantified using iTLC. The stability of the ²⁰³Pbcomplexes was evaluated using human serum at 37 ^oC. LogD_7.4 studies of the ²⁰³Pb complexes were performed and quantified using gamma spectroscopy. In vitroFAP binding affinity assays, as well as in vivo SPECT imaging and biodistribution studies in HEK293T: hFAP tumour bearing NRG mice with the ²⁰³Pb complexes of the bioconjugates are underway.

Results: At room temperature, in both ²⁰³Pb and ²¹²Pb concentration-dependent labeling studies, cyclen-4Py-FAPI showed superior radiometal complexation abilities as it displayed greater, quantitative radiolabeling yields compared to both other bioconjugates at concentrations of 10^-4and 10^-5 M. At 80^oC, cyclen-4Py-FAPI and TCMC-FAPI were superior to Crypt-FAPI and had non-significant differences in radiolabeling yields until 10^-7 M, where TCMC-FAPI was superior. All complexes were >94% stable in human serum at 72 hours (²⁰³Pb) and 24 hours (²¹²Pb). [²⁰³Pb][Pb(TCMC-FAPI)]²⁺ was found to be fairly hydrophilic with a logD_7.4 value of -2.18 ± 0.08 while [²⁰³Pb][Pb(Cyclen-4Py-FAPI)]²⁺was found to be less hydrophilic at -0.76 ± 0.03 and [²⁰³Pb][Pb(Crypt-FAPI)]²⁺ the most hydrophobic at 0.072 ± 0.011 (n = 3).

Conclusions: All tested bioconjugates showed promise for use with the ²⁰³Pb and ²¹²Pb theranostic pair with excellent radiochemical yields and stability in human serum. However, it was found that the choice of chelator drastically affected the logD_7.4 value of the ²⁰³Pb complexes and it is expected that this will translate to differences in tumour accumulation and clearance rates, as well as retention of the tracers in the bloodstream prior to tumour interaction. To progress FAPI-based radiopharmaceuticals towards therapy, the incorporation of novel chelators needs to be investigated in order to balance high tumour uptake with hematological toxicity and this will be explored in the forthcoming experiments of this study to identify a suitable tracer for therapeutic applications with ²¹²Pb.

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