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Background: Integrating radioactive and optical imaging techniques can facilitate the prognosis and surgical guidance for cancer patients. Using a single dual-labeled tracer ensures consistency in both imaging modalities. However, developing such molecule is challenging due to the need to preserve the biochemical properties of the tracer while introducing bulky labeling moieties. In our study, we designed a trifunctional chelate that facilitates the coupling of the targeting vector and fluorescent dye at opposite sites to avoid undesired steric hindrance effects. The synthesis of the trifunctional chelate N3-Py-DOTAGA-(tBu)3 (7) involved a five-step synthetic route, followed by conjugation to the linear peptidyl-resin 8 through solid-phase synthesis. After deprotection and cyclization, the near-infrared fluorescent dye sulfo-Cy.5 was introduced using copper free click chemistry, resulting in eTFC-01. Subsequently, eTFC-01 was labeled with [111In]InCl3. In vitro assessments of eTFC-01 binding, uptake, and internalization were conducted in SSTR2-transfected U2OS cells. Ex-vivo biodistribution and fluorescence imaging were performed in H69-tumor bearing mice. Results: eTFC-01 demonstrated a two-fold higher IC50 value for SSTR2 compared to the gold standard DOTA-TATE. Labeling of eTFC-01 with [111In]InCl3 gave a high radiochemical yield and purity. The uptake of [111In]In-eTFC-01 in U2OS.SSTR2 cells was two-fold lower than the uptake of [111In]In-DOTA-TATE, consistent with the binding affinity. Tumor uptake in H69-xenografted mice was lower for [111In]In-eTFC-01 at all-time points compared to [111In]In-DOTA-TATE. Prolonged blood circulation led to increased accumulation of [111In]In-eTFC-01 in highly vascularized tissues, such as lungs, skin, and heart. Fluorescence measurements in different organs correlated with the radioactive signal distributio

Multimodality reporter gene imaging combines the sensitivity, resolution and translational potential of two or more signals. The approach has not been widely adopted by the animal imaging community, mainly because its utility in this area is unproven. We developed a new complementation-based reporter gene system where the large component of split NanoLuc luciferase (LgBiT) presented on the surface of cells (TM-LgBiT) interacts with a radiotracer consisting of the high-affinity complementary HiBiT peptide labeled with a radionuclide. Radiotracer uptake could be imaged in mice using SPECT/CT and bioluminescence within two hours of implanting reporter-gene-expressing cells. Imaging data were validated by ex vivo biodistribution studies. Following the demonstration of complementation between the TM-LgBiT protein and HiBiT radiotracer, we validated the use of the technology in the highly specific in vivo multimodal imaging of cells. These findings highlight the potential of this new approach to facilitate the advancement of cell and gene therapies from bench to clinic.

Dual functionalization of targeting vectors, such as peptides and antibodies, is still synthetically challenging despite the increasing demand for such molecules serving multiple purposes (i.e., optical and nuclear imaging). Our strategy was to synthesize a versatile building block via the orthogonal incorporation of chemical entities (e.g., radionuclide chelator, fluorescent dye, cytotoxic drugs, click handle, and albumin binder) in order to prepare various dual functionalized biovectors. The functional groups were introduced on the building block using straightforward chemical reactions. Thus, an azidolysine and a biogenic lysine were installed into the building block to allow the coupling of the second functional group and the regioselective conjugation to the biovector via the strain-promoted azide-alkyne cycloaddition, while the first functional group was inserted during the solid-phase peptide synthesis. To extend the applicability of the building block to large biomolecules, such as antibodies, a DBCO-maleimide linker was clicked to the azidolysine to present a maleimide group that could react with the exposed sulfhydryl groups of the cysteine residues. To exemplify the possibilities offered by the building block, we synthesized two dual-functionalized compounds containing a 2,2′,2″′,2-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl) tetraacetic acid chelator and an albumin binder (4a) to extend the blood half-life of radiolabeled biovectors or a click handle (4b) to enable the late-stage click reaction; 4a and 4b were conjugated to a model cyclic peptide bearing a short thiolated linker at the N-terminal position, in a single step via the thiol-maleimide Michael addition. Both dual-functionalized peptides, 9a and 9b, were obtained rapidly in high chemical purity (>95%) and labeled with [111In]InCl3. Both radiopeptides showed good stability in mouse serum and PBS buffer.

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.

Bifunctional chelators (BFCs) are a key element in the design of radiopharmaceuticals. By selecting a BFC that efficiently complexes diagnostic and therapeutic radionuclides, a theranostic pair possessing almost similar biodistribution and pharmacokinetic properties can be developed. We have previously reported 3p-C-NETA as a promising theranostic BFC, and the encouraging preclinical outcomes obtained with [18F]AlF-3p-C-NETA-TATE led us to conjugate this chelator to a PSMA-targeting vector for imaging and treatment of prostate cancer. In this study, we synthesized 3p-C-NETA-ePSMA-16 and radiolabeled it with different diagnostic (111In, 18F) and therapeutic (177Lu, 213Bi) radionuclides. 3p-C-NETA-ePSMA-16 showed high affinity to PSMA (IC50 = 4.61 ± 1.33 nM), and [111In]In-3p-C-NETA-ePSMA-16 showed specific cell uptake (1.41 ± 0.20% ID/106 cells) in PSMA expressing LS174T cells. Specific tumor uptake of [111In]In-3p-C-NETA-ePSMA-16 was observed up to 4 h p.i. (1.62 ± 0.55% ID/g at 1 h p.i.; 0.89 ± 0.58% ID/g at 4 h p.i.) in LS174T tumor-bearing mice. Only a faint signal could be seen at 1 h p.i. in the SPECT/CT scans, whereas dynamic PET/CT scans performed after administration of [18F]AlF-3p-C-NETA-ePSMA-16 in PC3-Pip tumor xenografted mice resulted in a better tumor visualization and imaging contrast. Therapy studies with short-lived radionuclides such as 213Bi could further elucidate the therapeutic potential of 3p-C-NETA-ePSMA-16 as a radiotheranostic.

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.

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.).

Image-guided surgery using a gastrin-releasing peptide receptor (GRPR)-targeting dual-modality probe could improve the accuracy of the resection of various solid tumors. The aim of this study was to further characterize our four previously developed GRPR-targeting dual-modality probes that vary in linker structures and were labeled with indium-111 and sulfo-cyanine 5. Cell uptake studies with GRPR-positive PC-3 cells and GRPR-negative NCI-H69 cells confirmed receptor specificity. Imaging and biodistribution studies at 4 and 24 h with 20 MBq/1 nmol [ 111In]In- 12- 15 were performed in nude mice bearing a PC-3 and NCI-H69 xenograft, and showed that the probe with only a pADA linker in the backbone had the highest tumor-to-organ ratios (T/O) at 24 h after injection (T/O > 5 for, e.g., prostate, muscle and blood). For this probe, a dose optimization study with three doses (0.75, 1.25 and 1.75 nmol; 20 MBq) revealed that the maximum image contrast was achieved with the lowest dose. Subsequently, the probe was successfully used for tumor excision in a simulated image-guided surgery setting. Moreover, it demonstrated binding to tissue sections of human prostate, breast and gastro-intestinal stromal tumors. In summary, our findings demonstrate that the developed dual-modality probe has the potential to aid in the complete surgical removal of GRPR-positive tumors.

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. [Lu-177]Lu-8a and [Lu-177]Lu-8b were obtained with high radiochemical purity (>97%) and demonstrated excellent stability in PBS and mouse serum (>95%). [Lu-177]Lu-8a showed better affinity towards human albumin compared to [Lu-177]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 [Lu-177]Lu-8a and [Lu-177]Lu-JR11 at 4, 24, 48, and 72 h post-injection, but no tumor uptake was observed for [Lu-177]Lu-8b. Ex vivo biodistribution studies revealed high and increasing tumor uptake for [Lu-177]Lu-8a. However, its extended blood circulation led to an unfavorable biodistribution profile for radionuclide therapy.

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 [111 In]In-12 and [111 In]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.