Nanobodies are the recombinant variable domains of heavy-chain-only antibodies, with many unique properties such as small size, excellent solubility, superior stability, quick clearance from blood, and deep tissue penetration. As a result, nanobodies have become a promising tool for the diagnosis and therapy of diseases. As imaging tracers, nanobodies allow an early acquisition of high-quality images, provide a comprehensive evaluation of the disease, and subsequently enable a personalized precision therapy. As therapeutic agents, nanobodies enable a targeted therapy by lesion-specific delivery of drugs and effector domains, thereby improving the specificity and efficacy of the therapy. Up to date, a wide variety of nanobodies have been developed for a broad range of molecular targets and have played a significant role in patients with a broad spectrum of diseases. In this review, we aim to outline the current state-of-the-art research on the nanobodies for medical applications and then discuss the challenges and strategies for their further clinical translation.

Background Adnectin is a protein family derived from the 10th type III domain of human fibronectin (10Fn3) with high-affinity targeting capabilities. Positron emission tomography (PET) probes derived from anti-programmed death ligand-1 (PD-L1) Adnectins, including 18F- and 68Ga-labeled BMS-986192, are recently developed for the prediction of patient response to immune checkpoint blockade. The 68Ga-labeled BMS-986192, in particular, is an attractive probe for under-developed regions due to the broader availability of 68Ga. However, the pharmacokinetics and biocompatibility of 68Ga-labeled BMS-986192 are still unknown, especially in non-human primates, impeding its further clinical translation. Methods We developed a variant of 68Ga-labeled BMS-986192 using 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) as the radionuclide–chelator. The resultant probe, 68Ga-NODAGA-BMS986192, was evaluated in terms of targeting specificity using a bilateral mouse tumor model inoculated with wild-type B16F10 and B16F10 transduced with human PD-L1 (hPD-L1-B16F10). The dynamic biodistribution and radiation dosimetry of this probe were also investigated in non-human primate cynomolgus. Results 68Ga-NODAGA-BMS986192 was prepared with a radiochemical purity above 99%. PET imaging with 68Ga-NODAGA-BMS986192 efficiently delineated the hPD-L1-B16F10 tumor at 1 h post-injection. The PD-L1-targeting capability of this probe was further confirmed using in vivo blocking assay and ex vivo biodistribution studies. PET dynamic imaging in both mouse and cynomolgus models revealed a rapid clearance of the probe via the renal route, which corresponded to the low background signals of the PET images. The probe also exhibited a favorable radiation dosimetry profile with a total-body effective dose of 6.34E-03 mSv/MBq in male cynomolgus. Conclusions 68Ga-NODAGA-BMS986192 was a feasible and safe tool for the visualization of human PD-L1. Our study also provided valuable information on the potential of targeted PET imaging using Adnectin-based probes.

Fibroblast activation protein (FAP) is highly expressed in many tumor types and constitutes a promising target for tumor-specific delivery of therapeutic radionuclides. [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 is a novel radiopharmaceutical based on a novel bidentate inhibitor of FAP that is excreted more slowly than its monomeric counterparts. Still, the efficacy of radiotherapy is mitigated by cascades of DNA damage repair signaling in tumor cells including those via Poly(ADP-ribose) polymerase (PARP). We hereby aimed to evaluate the efficacy of [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 in combination with a PARP inhibitor, Olaparib, in the 4T1 murine triple negative breast cancer (TNBC) model. The therapeutic efficacy was visualized using 18 F-FDG and [ 68 Ga]Ga-FAPI-04 positron emission imaging/computer tomography (PET/CT). Our results demonstrated that Olaparib suppressed BALB/3T3 fibroblasts in vitro and sensitized the efficacy of [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 in mice bearing 4T1 tumors via enhancement of DNA damage. Treatment-associated toxicity was tolerable with only mild leukopenia. Therefore, the combination of [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 and Olaparib is a feasible treatment against TNBC.