Preclinical Evaluation of a Radiotheranostic Single-Domain Antibody Against Fibroblast Activation Protein α

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. Methods: 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 ¹³¹I and ⁶⁸Ga for SPECT and PET imaging, respectively, and with ¹³¹I and ²²⁵Ac for targeted radionuclide therapy. Results: The lead sdAb, 4AH29, exhibiting picomolar affinity for a distinct FAP epitope, recognized both purified and membrane-bound FAP protein. Radiolabeled versions, including [⁶⁸Ga]Ga-DOTA-4AH29, [²²⁵Ac]Ac-DOTA-4AH29, and [¹³¹I]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 [¹³¹I]I-GMIB-4AH29 was fast (<1 %IA/g after 24 h), whereas [²²⁵Ac]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 [²²⁵Ac]Ac-DOTA-4AH29 and [¹³¹I]I-GMIB-4AH29 demonstrated prolonged survival compared with those receiving vehicle solution. Conclusion: [⁶⁸Ga]Ga-DOTA-4AH29 and [¹³¹I]I-GMIB-4AH29 enable precise FAP-positive tumor detection in mice. Therapeutic [²²⁵Ac]Ac-DOTA-4AH29 and [¹³¹I]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 [²²⁵Ac]Ac-DOTA-4AH29. This study confirms the potential of radiolabeled sdAb 4AH29 as a radiotheranostic agent for FAP-positive cancers, warranting clinical evaluation.

Keywords: fibroblast activation protein α; radiotheranostics; single-domain antibodies; targeted radionuclide therapy.