Fibroblast activation protein (FAP) is abundantly expressed in the stroma of most human solid tumors. Clinical-stage radiolabeled FAP ligands are increasingly used as tools for the detection of various cancer lesions. To unleash the full therapeutic potential of FAP-targeting agents, ligands need to remain at the tumor site for several days after administration. We recently described the discovery of OncoFAP, a high-affinity small organic ligand of FAP with a rapid accumulation in tumors and low uptake in healthy tissues in cancer patients. Trimerization of OncoFAP provided a derivative (named TriOncoFAP, or OncoFAP-23) with improved FAP affinity. In this work, we evaluated the tissue biodistribution profile and the therapeutic performance of OncoFAP-23 in tumor-bearing mice. Methods: OncoFAP-23 was radiolabeled with the theranostic radionuclide ¹⁷⁷Lu. Preclinical experiments were conducted on mice bearing SK-RC-52.hFAP (BALB/c nude mice) or CT-26.hFAP (BALB/c mice) tumors. ¹⁷⁷Lu-OncoFAP and ¹⁷⁷Lu-FAP-2286 were included in the biodistribution study as controls. Toxicologic evaluation was performed on Wistar rats and CD1 mice by injecting high doses of OncoFAP-23 or its cold-labeled counterpart, respectively. Results: ¹⁷⁷Lu-OncoFAP-23 emerged for its best-in-class biodistribution profile, high and prolonged tumor uptake (i.e., ∼16 percentage injected dose/g at 96 h), and low accumulation in healthy organs, which correlates well with its potent single-agent anticancer activity at low levels of administered radioactivity. Combination treatment with the tumor-targeted interleukin 2 (L19-IL2, a clinical-stage immunocytokine) further expands the therapeutic window of ¹⁷⁷Lu-OncoFAP-23 by potentiating its in vivo antitumor activity. Proteomics studies revealed a potent tumor-directed immune response on treatment with the combination. OncoFAP-23 and ^natLu-OncoFAP-23 exhibited a favorable toxicologic profile, without showing any side effects or signs of toxicity. Conclusion: OncoFAP-23 presents enhanced tumor uptake and tumor retention and low accumulation in healthy organs, findings that correspond to a strongly improved in vivo antitumor efficacy. The data presented in this work support the clinical development of ¹⁷⁷Lu-OncoFAP-23 for the treatment of FAP-positive solid tumors.

成纤维细胞活化蛋白 （FAP） 在大多数人类实体瘤的基质中大量表达。临床阶段放射性标记的 FAP 配体越来越多地用作检测各种癌症病变的工具。为了释放 FAP 靶向药物的全部治疗潜力，配体在给药后需要在肿瘤部位停留数天。我们最近描述了 OncoFAP 的发现，这是一种高亲和力的 FAP 小有机配体，在癌症患者的肿瘤中快速积累，在健康组织中摄取率低。OncoFAP 的三聚化提供了一种衍生物（命名为 TriOncoFAP 或 OncoFAP-23），具有改进的 FAP 亲和力。在这项工作中，我们评估了 OncoFAP-23 在荷瘤小鼠中的组织生物分布概况和治疗性能。方法：OncoFAP-23 用治疗诊断放射性核素 ¹⁷⁷Lu 进行放射性标记。在携带 SK-RC-52.hFAP （BALB/c 裸鼠） 或 CT-26.hFAP （BALB/c 小鼠） 肿瘤的小鼠上进行临床前实验。¹⁷⁷ 元Lu-OncoFAP 和 ¹⁷⁷个 Lu-FAP-2286 作为对照纳入生物分布研究。通过分别注射高剂量的 OncoFAP-23 或其冷标记的对应物，对 Wistar 大鼠和 CD1 小鼠进行毒理学评价。结果： ¹⁷⁷Lu-OncoFAP-23 因其一流的生物分布谱、高且持久的肿瘤摄取 （即 96 小时注射剂量/g ∼16% ）和在健康器官中的低积累而出现，这与它在低水平施用放射性下有效的单药抗癌活性密切相关。 与肿瘤靶向白细胞介素 2 （L19-IL2，一种临床阶段的免疫细胞因子） 联合治疗通过增强 ¹⁷⁷Lu-OncoFAP-23 的体内抗肿瘤活性进一步扩大了其治疗窗。蛋白质组学研究显示，联合治疗会产生有效的肿瘤定向免疫反应。OncoFAP-23 和 ^natLu-OncoFAP-23 表现出良好的毒理学特征，未显示任何副作用或毒性迹象。结论：OncoFAP-23 表现出增强的肿瘤摄取和肿瘤保留以及健康器官中的低积累，这些发现对应于体内抗肿瘤疗效的强烈改善。本研究中提供的数据支持 ¹⁷⁷个 Lu-OncoFAP-23 治疗 FAP 阳性实体瘤的临床开发。