The pretargeting strategy has recently emerged in order to overcome the limitations of direct targeting, mainly in the field of radioimmunotherapy (RIT). This strategy is directly dependent on chemical reactions, namely bioorthogonal reactions, which have been developed for their ability to occur under physiological conditions. The Staudinger ligation, the copper catalyzed azide-alkyne cycloaddition (CuAAC) and the strain-promoted [3 + 2] azide–alkyne cycloaddition (SPAAC) were the first bioorthogonal reactions introduced in the literature. However, due to their incomplete biocompatibility and slow kinetics, the inverse-electron demand Diels-Alder (IEDDA) reaction was advanced in 2008 by Blackman et al. as an optimal bioorthogonal reaction. The IEDDA is the fastest bioorthogonal reaction known so far. Its biocompatibility and ideal kinetics are very appealing for pretargeting applications. The use of a trans-cyclooctene (TCO) and a tetrazine (Tz) in the reaction encouraged researchers to study them deeply. It was found that both reagents are sensitive to acidic or basic conditions. Furthermore, TCO is photosensitive and can be isomerized to its cis-conformation via a radical catalyzed reaction. Unfortunately, the cis-conformer is significantly less reactive toward tetrazine than the trans-conformation. Therefore, extensive research has been carried out to optimize both click reagents and to employ the IEDDA bioorthogonal reaction in biomedical applications.

Peptide receptor radionuclide therapy (PRRT) has been applied to the treatment of neuroendocrine tumors (NETs) for over two decades. However, improvement is still needed, and targeted alpha therapy (TAT) with alpha emitters such as lead-212 (²¹²Pb) represents a promising avenue. A series of ligands based on octreotate was developed. Lead-203 was used as an imaging surrogate for the selection of the best candidate for the studies with lead-212. ^203/212Pb radiolabeling and in vitro assays were carried out, followed by SPECT/CT imaging and ex vivo biodistribution in NCI-H69 tumor-bearing mice. High radiochemical yields (≥99%) and purity (≥96%) were obtained for all ligands. [²⁰³Pb]Pb-eSOMA-01 and [²⁰³Pb]Pb-eSOMA-02 showed high stability in PBS and mouse serum up to 24 h, whereas [²⁰³Pb]Pb-eSOMA-03 was unstable in those conditions. All compounds exhibited a nanomolar affinity (2.5–3.1 nM) for SSTR2. SPECT/CT images revealed high tumor uptake at 1, 4, and 24 h post-injection of [²⁰³Pb]Pb-eSOMA-01/02. Ex vivo biodistribution studies confirmed that the highest uptake in tumors was observed with [²¹²Pb]Pb-eSOMA-01. [²¹²Pb]Pb-eESOMA-01 displayed the highest absorbed dose in the tumor (35.49 Gy/MBq) and the lowest absorbed dose in the kidneys (121.73 Gy/MBq) among the three tested radioligands. [²¹²Pb]Pb-eSOMA-01 is a promising candidate for targeted alpha therapy of NETs. Further investigations are required to confirm its potential.

Prostate-specific membrane antigen (PSMA) targeting radiopharmaceuticals have been successfully used for diagnosis and therapy of prostate cancer. Optimization of the available agents is desirable to improve tumor uptake and reduce side effects to non-target organs. This can be achieved, for instance, via linker modifications or multimerization approaches. In this study, we evaluated a small library of PSMA-targeting derivatives with modified linker residues, and selected the best candidate based on its binding affinity to PSMA. The lead compound was coupled to a chelator for radiolabeling, and subject to dimerization. The resulting molecules, 22 and 30, were highly PSMA specific (IC₅₀ = 1.0–1.6 nM) and stable when radiolabeled with indium-111 (>90% stable in PBS and mouse serum up to 24 h). Moreover, [¹¹¹In]In-30 presented a high uptake in PSMA expressing LS174T cells, with 92.6% internalization compared to 34.1% for PSMA-617. Biodistribution studies in LS174T mice xenograft models showed that [¹¹¹In]In-30 had a higher tumor and kidney uptake compared to [¹¹¹In]In-PSMA-617, but increasing T/K and T/M ratios at 24 h p.i. Tumors could be clearly visualized at 1 h p.i. by SPECT/CT after administration of [¹¹¹In]In-22 and [¹¹¹In]In-PSMA-617, while [¹¹¹In]In-30 showed a clear signal at later time-points (e.g., 24 h p.i.).

Small-molecule drug conjugates (SMDCs) are compounds in which a therapeutic payload is conjugated to a targeting vector, for specific delivery to the tumor site. This promising approach can be translated to the treatment of prostate cancer by selecting a targeting vector which binds to the prostate-specific membrane antigen (PSMA). Moreover, the addition of a bifunctional chelator to the molecule allows for the use of both diagnostic and therapeutic radionuclides. In this way, the distribution of the SMDC in the body can be monitored, and combination therapy regimes can be implemented. We combined a glutamate-urea-lysine vector to the cytotoxic agent DM1 and a DOTA chelator via an optimized linker to obtain the theranostic SMDC (T-SMDC) ePSMA-DM1. ePSMA-DM1 retained a high binding affinity to PSMA and demonstrated PSMA-specific uptake in cells. Glutathione stability assays showed that the half-life of the T-SMDC in a reducing environment was 2 h, and full drug release was obtained after 6 h. Moreover, 100 nM of ePSMA-DM1 reduced the cell viability of the human PSMA-positive LS174T cells by >85% after 72 h of incubation, which was comparable to a 10-fold higher dose of free DM1. [¹¹¹In]In-ePSMA-DM1 and [¹⁷⁷Lu]Lu-ePSMA-DM1 were both obtained in high radiochemical yields and purities (>95%), with >90% stability in PBS and >80% stability in mouse serum for up to 24 h post incubation at 37 °C. SPECT/CT imaging studies allowed for a faint tumor visualization of [¹¹¹In]In-ePSMA-DM1 at 1 h p.i., and the ex vivo biodistribution showed tumor uptake (2.39 ± 0.29% ID/g) at 1 h p.i., with the compound retained in the tumor for up to 24 h. Therefore, ePSMA-DM1 is a promising T-SMDC candidate for prostate cancer, and the data obtained so far warrant further investigations, such as therapeutic experiments, after further optimization.

Nuclear and optical dual-modality probes can be of great assistance in prostate cancer localization, providing the means for both preoperative nuclear imaging and intraoperative surgical guidance. We developed a series of probes based on the backbone of the established GRPR-targeting radiotracer NeoB. The inverse electron demand of the Diels–Alder reaction was used to integrate the sulfo-cyanine 5 dye. Indium-111 radiolabeling, stability studies and a competition binding assay were carried out. Pilot biodistribution and imaging studies were performed in PC-3 tumor-bearing mice, using the best two dual-labeled probes. The dual-modality probes were radiolabeled with a high yield (>92%), were proven to be hydrophilic and demonstrated high stability in mouse serum (>94% intact labeled ligand at 4 h). The binding affinity for the GRPR was in the nanomolar range (21.9–118.7 nM). SPECT/CT images at 2 h p.i. clearly visualized the tumor xenograft and biodistribution studies, after scanning confirmed the high tumor uptake (8.47 ± 0.46%ID/g and 6.90 ± 0.81%ID/g for probe [¹¹¹In]In-12 and [¹¹¹In]In-15, respectively). Receptor specificity was illustrated with blocking studies, and co-localization of the radioactive and fluorescent signal was verified by ex vivo fluorescent imaging. Although optimal tumor-to-blood and tumor-to-kidney ratios might not yet have been reached due to the prolonged blood circulation, our probes are promising candidates for the preoperative and intraoperative visualization of GRPR-positive prostate cancer.

Somatostatin receptor subtype 2 (SSTR2) has become an essential target for radionuclide therapy of neuroendocrine tumors (NETs). JR11 was introduced as a promising antagonist peptide to target SSTR2. However, due to its rapid blood clearance, a better pharmacokinetic profile is necessary for more effective treatment. Therefore, two JR11 analogs (8a and 8b), each carrying an albumin binding domain, were designed to prolong the blood residence time of JR11. Both compounds were labeled with lutetium-177 and evaluated via in vitro assays, followed by in vivo SPECT/CT imaging and ex vivo biodistribution studies. [¹⁷⁷Lu]Lu-8a and [¹⁷⁷Lu]Lu-8b were obtained with high radiochemical purity (>97%) and demonstrated excellent stability in PBS and mouse serum (>95%). [¹⁷⁷Lu]Lu-8a showed better affinity towards human albumin compared to [¹⁷⁷Lu]Lu-8b. Further, 8a and 8b exhibited binding affinities 30- and 48-fold lower, respectively, than that of the parent peptide JR11, along with high cell uptake and low internalization rate. SPECT/CT imaging verified high tumor accumulation for [¹⁷⁷Lu]Lu-8a and [¹⁷⁷Lu]Lu-JR11 at 4, 24, 48, and 72 h post-injection, but no tumor uptake was observed for [¹⁷⁷Lu]Lu-8b. Ex vivo biodistribution studies revealed high and increasing tumor uptake for [¹⁷⁷Lu]Lu-8a. However, its extended blood circulation led to an unfavorable biodistribution profile for radionuclide therapy.