Preclinical Evaluation of a Radiotheranostic Single-Domain Antibody Against Fibroblast Activation Protein α
Yana Dekempeneer, Sam Massa, Francis Santens, Laurent Navarro, Marion Berdal, Melissa Miranda Lucero, Ana Rita Pombo Antunes, Tony Lahoutte, Jo A. Van Ginderachter, Nick Devoogdt, Matthias D’Huyvetter
Abstract
Fibroblast activation protein α (FAP) is highly expressed on cancer-associated fibroblasts of epithelial-derived cancers. Breast, colon, and pancreatic tumors often show strong desmoplastic reactions, which result in a dominant presence of stromal cells. FAP has gained interest as a target for molecular imaging and targeted therapies. Single-domain antibodies (sdAbs) are the smallest antibody-derived fragments with beneficial pharmacokinetic properties for molecular imaging and targeted therapy. <b>Methods:</b> We describe the generation, selection, and characterization of a sdAb against FAP. In mice, we assessed its imaging and therapeutic potential after radiolabeling with tracer-dose <sup>131</sup>I and <sup>68</sup>Ga for SPECT and PET imaging, respectively, and with <sup>131</sup>I and <sup>225</sup>Ac for targeted radionuclide therapy. <b>Results:</b> The lead sdAb, 4AH29, exhibiting picomolar affinity for a distinct FAP epitope, recognized both purified and membrane-bound FAP protein. Radiolabeled versions, including [<sup>68</sup>Ga]Ga-DOTA-4AH29, [<sup>225</sup>Ac]Ac-DOTA-4AH29, and [<sup>131</sup>I]I-guanidinomethyl iodobenzoate (GMIB)-4AH29, displayed radiochemical purities exceeding 95% and effectively bound to recombinant human FAP protein and FAP-positive GM05389 human fibroblasts. These radiolabeled compounds exhibited rapid and specific accumulation in human FAP–positive U87-MG glioblastoma tumors, with low but specific uptake in lymph nodes, uterus, bone, and skin (∼2–3 percentage injected activity per gram of tissue [%IA/g]). Kidney clearance of unbound [<sup>131</sup>I]I-GMIB-4AH29 was fast (<1 %IA/g after 24 h), whereas [<sup>225</sup>Ac]Ac-DOTA-4AH29 exhibited slower clearance (8.07 ± 1.39 %IA/g after 24 h and 2.47 ± 0.18 %IA/g after 96 h). Mice treated with [<sup>225</sup>Ac]Ac-DOTA-4AH29 and [<sup>131</sup>I]I-GMIB-4AH29 demonstrated prolonged survival compared with those receiving vehicle solution. <b>Conclusion:</b> [<sup>68</sup>Ga]Ga-DOTA-4AH29 and [<sup>131</sup>I]I-GMIB-4AH29 enable precise FAP-positive tumor detection in mice. Therapeutic [<sup>225</sup>Ac]Ac-DOTA-4AH29 and [<sup>131</sup>I]I-GMIB-4AH29 exhibit strong and sustained tumor targeting, resulting in dose-dependent therapeutic effects in FAP-positive tumor-bearing mice, albeit with kidney toxicity observed later for [<sup>225</sup>Ac]Ac-DOTA-4AH29. This study confirms the potential of radiolabeled sdAb 4AH29 as a radiotheranostic agent for FAP-positive cancers, warranting clinical evaluation.