Hiroyuki Watanabe

Neurotensin receptor 1 (NTSR1) is overexpressed in various types of cancer and known as a promising target for cancer therapy. Targeted alpha therapy (TAT) using α-emitting radionuclides with high linear energy transfer is expected to show potent therapeutic efficacy. In this study, we aimed to develop an 211At-labeled NTSR1-TAT agent. To achieve this, [125I]IB-NTS, [125I]IB-DOTA-NTS, and [125I]IB-In-DOTA-NTS were newly designed, synthesized, and evaluated using 125I as a surrogate radionuclide. These 125I-labeled compounds showed high stability in murine plasma and NTSR1-binding affinity. In the biodistribution study, [125I]IB-In-DOTA-NTS demonstrated the most favorable tumor uptake among the three compounds and in vivo NTSR1-specificity. Therefore, [211At]AB-In-DOTA-NTS was synthesized and its utility as an NTSR1-TAT agent was evaluated. [211At]AB-In-DOTA-NTS exhibited dose-dependent antitumor effects without sustained or dose-dependent body weight loss in HT-29 tumor-bearing mice. This study demonstrated that [211At]AB-In-DOTA-NTS is a promising candidate for TAT and represents the first report of an 211At-labeled NTSR1-TAT agent.

Radiolabeled heterodimers targeting prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) have been extensively investigated to overcome heterogeneous target expression in prostate cancer. However, low tumor retention and high kidney uptake remain major challenges. In this study, we designed and synthesized a novel albumin binder (ALB)-modified PSMA/GRPR-targeting heterodimer, PADA, to improve tumor retention and tumor-to-kidney ratios. [111In]In-PADA exhibited marked binding ability to albumin, PSMA, and GRPR in vitro. In biodistribution studies, the tumor retention of [111In]In-PADA was improved by the introduction of ALB in both PSMA-positive and GRPR-positive tumor-bearing mice. Overall, the tumor AUC values and tumor-to-kidney AUC value ratios of [111In]In-PADA were higher than those of a control agent without an ALB moiety in both model mice. In single-photon emission computed tomography (SPECT) imaging studies, [111In]In-PADA could be used to successfully visualize both PSMA-positive and GRPR-positive tumors and exhibited enhanced tumor-to-kidney contrast due to the introduction of ALB. These results suggest that [111In]In-PADA exhibits favorable pharmacokinetics as a PSMA/GRPR-targeting heterodimer.

Amyloid β (Aβ) aggregates are primary targets for the diagnosis and curative treatment of Alzheimer’s disease (AD). While various theranostic agents targeting Aβ aggregates have been developed, most rely on fluorescent imaging, which has limited clinical translation. In contrast, nuclear medicine imaging, particularly positron emission tomography (PET), offers high sensitivity and deep tissue permeability suitable for clinical settings. Moreover, photosensitization has emerged as a promising strategy to attenuate Aβ toxicity. In this study, we designed and synthesized novel bithiophene derivatives as PET/photosensitization theranostic agents. Western blotting analysis and MALDI-TOF MS spectrometry demonstrated that FABT-2 selectively oxidized Aβ aggregates under light irradiation, leading to a significant reduction in Aβ-induced cytotoxicity, as demonstrated by CCK8 and LDH assays. Furthermore, ¹⁸F-labeled FABT-2 showed blood–brain barrier permeability in normal mice. These results suggest that FABT-2 may be a promising lead compound for the development of theranostic agents targeting Aβ aggregates.

In the development of radiopharmaceuticals, high tumor accumulation is required for their diagnostic and therapeutic effectiveness. A key strategy to enhance such tumor accumulation is the incorporation of small-molecule albumin binders (ALBs) into radioligands, extending the blood circulation time through albumin interaction for efficient delivery to tumors. In this study, we systematically investigated the potential use of fatty acids (FAs) with various carbon-chain lengths (C12, C14, C16, and C18) as ALBs (FA12, FA14, FA16, and FA18). First, we synthesized four radiolabeled compounds composed of a macrocyclic radiometal chelator and various FAs ([¹¹¹In]In-DFA12, [¹¹¹In]In-DFA14, [¹¹¹In]In-DFA16, and [¹¹¹In]In-DFA18). In vitro albumin-binding assays and biodistribution studies using normal mice revealed a chain-length dependence: longer carbon chains led to higher albumin affinity and prolonged blood retention, with [¹¹¹In]In-DFA18 exhibiting the most favorable pharmacokinetic profile. Based on these results, we developed [¹¹¹In]In-PDFA18, a prostate-specific membrane antigen (PSMA)-targeted radioligand conjugated with FA18. Tumor accumulation of [¹¹¹In]In-PDFA18 at 24 and 96 h postinjection (p.i.) (43.70 and 52.59% injected dose (ID)/g, respectively) was markedly higher and more prolonged compared with that of the non-FA18-conjugated control radioligand, [¹¹¹In]In-PSMA-617 (14.86 and 5.36% ID/g). These findings suggest that FA18 is a promising ALB to improve the tumor accumulation of radioligands.

Theranostics, an integrated approach combining cancer diagnosis and therapy, has gained increasing attention, particularly in the field of radiotheranostics using radioligands. The cholecystokinin 2 receptor (CCK2R), which is overexpressed in various neuroendocrine tumors (NETs), is considered a promising target for radiotheranostic applications. However, the currently available CCK2R-targeted ligand, DOTA-MGS5, exhibits limited tumor accumulation. Incorporation of an albumin binder (ALB) unit, which is capable of interacting with serum albumin, has been reported to enhance tumor accumulation of radioligands by prolonging their biological half-lives. To establish a more efficient CCK2R-targeted radiotheranostic strategy, we developed a novel CCK2R-targeted ALB-containing radioligand, [¹¹¹In]In/[¹⁷⁷Lu]Lu-MGS5-DA, based on DOTA-MGS5. In biodistribution studies, [¹¹¹In]In-MGS5-DA showed markedly higher tumor accumulation compared with [¹¹¹In]In-DOTA-MGS5. Moreover, single-photon emission computed tomography/computed tomography (SPECT/CT) imaging confirmed that [¹¹¹In]In-MGS5-DA could be used to clearly visualize CCK2R-expressing tumors. In a peptide receptor radionuclide therapy (PRRT) study, [¹⁷⁷Lu]Lu-MGS5-DA led to greater tumor growth inhibition than [¹⁷⁷Lu]Lu-DOTA-MGS5. These findings suggest that [¹¹¹In]In/[¹⁷⁷Lu]Lu-MGS5-DA is a promising radiotheranostic pair for the treatment of CCK2R-expressing cancers.

Fibroblast activation protein (FAP) has been identified as a promising biomarker for tumor-targeting radioligands. Among these, a small molecule FAP inhibitor (FAPI), [⁶⁸Ga]Ga-FAPI-46, and a cyclic peptide-based FAP-targeting radioligand, [¹⁷⁷Lu]Lu-FAP-2286, are recognized as promising agents. To date, multiple strategies to further improve the potential of FAPI-46 and FAP-2286 have been employed for the development of FAP-targeting radioligands. Multimerization of FAP-targeting moieties is widely utilized as a promising strategy to improve tumor accumulation. In this study, we newly designed and synthesized three types of FAP-targeting dimers based on FAPI-46 and FAP-2286: heterodimer FAP-PID, small molecule-based homodimer FAP-ID, and cyclic peptide-based homodimer FAP-PD. To identify preferable dimeric structures, we conducted a head-to-head comparison of the three dimers and their monomeric counterparts (FAPI-46 and FAP-2286). All compounds were successfully radiolabeled with ¹¹¹In. Each dimer exhibited comparable stability in murine plasma and in vitro FAP-selectivity and specificity. FAP-binding affinities of [^natIn]In-FAP-PID and [^natIn]In-FAP-ID were comparable to or higher than those of the monomeric counterparts. In the biodistribution assays, [¹¹¹In]In-FAP-PID demonstrated the highest tumor accumulation and tumor/blood ratio among the five radioligands. Furthermore, [¹¹¹In]In-FAP-PID allowed clear visualization of tumors in the SPECT/CT studies. In conclusion, [¹¹¹In]In-FAP-PID achieved a favorable tumor-to-background ratio and higher tumor accumulation than homodimers, suggesting the utility of the heterodimeric structure based on both small molecule FAPI and cyclic peptide for FAP-targeting radioligands in tumor imaging and therapy.

Biparatopic antibodies (BpAbs) are an attractive format of engineered therapeutic antibodies that bind to two distinct epitopes of a single antigen. Accelerated cell internalization is anticipated in many developmental campaigns, and both bivalent and tetravalent forms have been developed for this purpose. However, their pharmacokinetic properties have not been understood systematically; thus, optimization approaches for BpAbs have been limited. In this study, we conducted a comparative biodistribution analysis of bivalent and tetravalent BpAbs using the same variable fragments. Two different pairs of epitopes of human epidermal growth factor receptor 2 (HER2) were targeted, and their distribution was evaluated in a tumor xenograft model by ¹¹¹In-labeling. Bivalent BpAbs showed higher accumulation in tumors than tetravalent BpAbs in both cases. However, epitope-dependent differences in biodistribution did not correlate with those of the original monoclonal antibodies. In addition, cell internalization during the early stages of incubation was higher for tetravalent BpAbs. These results suggest the advantage of bivalent BpAbs over tetravalent BpAbs in pharmacokinetics; however, the design may require optimization depending on the mechanism of action of the BpAb of interest.

A novel strategy using dual pharmacokinetic modifiers, one for improved tumor delivery and another for increased tumor retention, synergistically enhanced tumor accumulation of a PSMA-targeting radioligand.

Somatostatin receptor 2 (SSTR2) is a valuable target for the treatment of patients with neuroendocrine tumors (NETs). [¹⁷⁷Lu]-Lu-DOTA-TATE, an SSTR2-targeted ligand, is clinically used for therapy in patients with NETs; however, a low-absorbed dose in tumors remains a limitation. Albumin binders (ALBs) are commonly used to enhance the tumor accumulation of radioligands, but the effect of modulating ALB affinity has not been thoroughly investigated for DOTA-TATE-based SSTR2-targeted radioligands. In this study, we newly developed three DOTA-TATE-based radioligands with 4-(p-iodophenyl)-butyric acid ([¹¹¹In]-In-TATE-DA-I), 4-(p-bromophenyl)-butyric acid ([¹¹¹In]-In-TATE-DA-Br), and 4-(p-tolyl)-butyric acid ([¹¹¹In]-In-TATE-DA-CH₃) as ALB moieties. In the albumin-binding assay, the order of albumin-binding potency of each radioligand was consistent with that of the corresponding ALB moieties alone. In the biodistribution study, the radioligands with stronger albumin-binding potencies showed higher area under the curve (AUC) values in the blood. All ALB-containing radioligands exhibited higher tumor AUC values than [¹¹¹In]-In-DOTA-TATE, and the order of tumor AUC values corresponded with those of blood AUC values and albumin-binding potencies. Among them, [¹¹¹In]-In-TATE-DA-I showed the highest tumor AUC value and clearly visualized the SSTR2-positive tumor in SPECT/CT imaging. These findings highlight the importance of structural modification in the ALB moiety to enhance tumor accumulation of DOTA-TATE-based radioligands.

A novel bivalent albumin binder composed of two molecules of Lys-4-( p -iodophenyl)butyric acid markedly enhanced tumor retention of a FAP-targeted radioligand, suggesting its promising potential as a versatile pharmacokinetic modifier of radioligands.