Simultaneous targeting of the prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) could improve the diagnostic accuracy in prostate cancer (PCa). The aim of this study was to develop a PSMA/GRPR-targeting bispecific heterodimer for SPECT and positron emission tomography (PET) diagnostic imaging of PCa. The heterodimer NOTA-DUPA-RM26 was produced by manual solid-phase peptide synthesis. NOTA-DUPA-RM26 was labeled with ¹¹¹In and ⁶⁸Ga, with yields >98%, and demonstrated a high stability and binding specificity to PSMA and GRPR. IC₅₀ values for ^natIn-NOTA-DUPA-RM26 were 4 ± 1 nM towards GRPR and 824 ± 230 nM towards PSMA. An in vivo binding specificity 1 h pi of ¹¹¹In-NOTA-DUPA-RM26 in PC3-PIP-xenografted mice demonstrated partially blockable tumor uptake when co-injected with an excess of PSMA- or GRPR-targeting agents. Simultaneous co-injection of both agents induced pronounced blocking. The biodistribution of ¹¹¹In-NOTA-DUPA-RM26 and ⁶⁸Ga-NOTA-DUPA-RM26 revealed fast activity clearance from the blood and normal organs via the kidneys. Tumor uptake exceeded normal organ uptake for both analogs 1 h pi. ⁶⁸Ga-NOTA-DUPA-RM26 had a significantly lower tumor uptake (8 ± 2%ID/g) compared to ¹¹¹In-NOTA-DUPA-RM26 (12 ± 2%ID/g) 1 h pi. Tumor-to-organ ratios increased 3 h pi, but decreased 24 h pi, for ¹¹¹In-NOTA-DUPA-RM26. MicroPET/CT and microSPECT/CT scans confirmed biodistribution data, suggesting that ⁶⁸Ga-NOTA-DUPA-RM26 and ¹¹¹In-NOTA-DUPA-RM26 are suitable candidates for the imaging of GRPR and PSMA expression in PCa shortly after administration.

Gastrin‐releasing peptide receptors (GRPRs) are overexpressed in prostate cancer and are suitable for targeted radionuclide therapy (TRT). We optimized the bombesin‐derived GRPR‐antagonist PEG₂‐RM26 for labeling with ¹⁷⁷Lu and further determined the effect of treatment with ¹⁷⁷Lu‐labeled peptide alone or in combination with the anti‐HER2 antibody trastuzumab in a murine model. The PEG₂‐RM26 analog was coupled to NOTA, NODAGA, DOTA and DOTAGA chelators. The peptide‐chelator conjugates were labeled with ¹⁷⁷Lu and characterized in vitro and in vivo. A preclinical therapeutic study was performed in PC‐3 xenografted mice. Mice were treated with intravenous injections (6 cycles) of (A) PBS, (B) DOTAGA‐PEG₂‐RM26, (C) ¹⁷⁷Lu‐DOTAGA‐PEG₂‐RM26, (D) trastuzumab or (E) ¹⁷⁷Lu‐DOTAGA‐PEG₂‐RM26 in combination with trastuzumab. ¹⁷⁷Lu‐DOTAGA‐PEG₂‐RM26 demonstrated quantitative labeling yield at high molar activity (450 GBq/μmol), high in vivo stability (5 min pi >98% of radioligand remained when coinjected with phosphoramidon), high affinity to GRPR (K_D = 0.4 ± 0.2 nM), and favorable biodistribution (1 hr pi tumor uptake was higher than in healthy tissues, including the kidneys). Therapy with ¹⁷⁷Lu‐DOTAGA‐PEG₂‐RM26 induced a significant inhibition of tumor growth. The median survival for control groups was significantly shorter than for treated groups (Group C 66 days, Group E 74 days). Trastuzumab together with radionuclide therapy significantly improved survival. No treatment‐related toxicity was observed. In conclusion, based on in vitro and in vivo characterization of the four ¹⁷⁷Lu‐labeled PEG₂‐RM26 analogs, we concluded that ¹⁷⁷Lu‐DOTAGA‐PEG₂‐RM26 was the most promising analog for TRT. Radiotherapy using ¹⁷⁷Lu‐DOTAGA‐PEG₂‐RM26 effectively inhibited tumor growth in vivo in a murine prostate cancer model. Anti‐HER2 therapy additionally improved survival.

Prostate-specific membrane antigen (PSMA) is overexpressed in the majority of prostate cancer cells and is considered to be an important target for the molecular imaging and therapy of prostate cancer. Herein, we present the design, synthesis, and evaluation of 11 PSMA-binding radioligands with modified linker structures, focusing on the relationship between molecular structure and targeting properties. The linker design was based on 2-naphthyl-L-alanine-tranexamic acid, the linker structure of PSMA-617. X-ray crystal-structure analysis of PSMA and structure-based design were used to generate the linker modifications, suggesting that substitution of tranexamic acid could lead to interactions with Phe546, Trp541, and Arg43 within the binding cavity. After synthesis through SPPS, analogues were labelled with indium-111 and evaluated in vitro for their specific binding, affinity, and cellular retention. Selected compounds were further evaluated in vivo in PSMA-expressing tumour-bearing mice. Based on the results, 2-naphthyl-L-alanine appears to be crucial for good targeting properties, whereas tranexamic acid could be replaced by other substituents. [¹¹¹In]In-BQ7859, consisting of a 2-naphthyl-L-alanine-L-tyrosine linker, demonstrated favourable targeting properties. The substitution of tranexamic acid for L-tyrosine in the linker led to an improved tumour-to-blood ratio, highlighting [¹¹¹In]In-BQ7859 as a promising PSMA-targeting radioligand.

Prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) are promising targets for molecular imaging of prostate cancer (PCa) lesions. Due to the heterogenic overexpression of PSMA and GRPR in PCa, a heterodimeric radiotracer with the ability to bind to both targets could be beneficial. Recently, our group reported the novel heterodimer BQ7800 consisting of a urea-based PSMA inhibitor, the peptide-based GRPR antagonist RM26 and NOTA chelator. The study reported herein, aimed to improve the affinity of BQ7800 towards PSMA by changing the composition of the two linkers connecting the PSMA- and GRPR-targeting motifs. Three novel heterodimeric analogues were synthesized by incorporation of phenylalanine in the functional linker of the PSMA-binding motif and/or shortening the PEG-linker coupled to RM26. The heterodimers were labeled with indium-111 and evaluated in vitro. In the competitive binding assay, BQ7812, featuring phenylalanine and shorter PEG-linker, demonstrated a nine-fold improved affinity towards PSMA. In the in vivo biodistribution study of [¹¹¹In]In-BQ7812 in PC3-pip tumor-bearing mice (PSMA and GRPR positive), the activity uptake was two-fold higher in the tumor and three-fold higher in kidneys than for [¹¹¹In]In-BQ7800. Herein, we showed that the affinity of a bispecific PSMA/GRPR heterodimer towards PSMA could be improved by linker modification.

Gastrin-releasing peptide receptor (GRPR) and prostate-specific membrane antigen (PSMA) are overexpressed in most prostate cancers. GRPR expression is higher in early stages while PSMA expression increases with progression. The possibility of targeting both markers with a single theranostics radiotracer could improve patient management. Three GRPR/PSMA-targeting bispecific heterodimers (urea derivative PSMA-617 and bombesin-based antagonist RM26 linked via X-triazolyl-Tyr-PEG2, X = PEG2 (BO530), (CH₂)₈ (BO535), none (BO536)) were synthesized by solid-phase peptide synthesis. Peptides were radio-iodinated and evaluated in vitro for binding specificity, cellular retention, and affinity. In vivo specificity for all heterodimers was studied in PC-3 (GRPR-positive) and LNCaP (PSMA-positive) xenografts. [¹²⁵I]I-BO530 was evaluated in PC-3pip (GRPR/PSMA-positive) xenografts. Micro single-photon emission computed tomography/computed tomography (microSPECT/CT) scans were acquired. The heterodimers were radiolabeled with high radiochemical yields, bound specifically to both targets, and demonstrated high degree of activity retention in PC-3pip cells. Only [¹²⁵I]I-BO530 demonstrated in vivo specificity to both targets. A biodistribution study of [¹²⁵I]I-BO530 in PC-3pip xenografted mice showed high tumor activity uptake (30%–35%ID/g at 3 h post injection (pi)). Activity uptake in tumors was stable and exceeded all other organs 24 h pi. Activity uptake decreased only two-fold 72 h pi. The GRPR/PSMA-targeting heterodimer [¹²⁵I]I-BO530 is a promising agent for theranostics application in prostate cancer.

Upregulation of the human epidermal growth factor receptor type 3 (HER3) is a common mechanism to bypass HER-targeted cancer therapy. Affibody-based molecular imaging has the potential for detecting and monitoring HER3 expression during treatment. In this study, we compared the imaging properties of newly generated ⁶⁸Ga-labeled anti-HER3 affibody molecules (HE)₃-Z_HER3-DOTA and (HE)₃-Z_HER3-DOTAGA with previously reported [⁶⁸Ga]Ga-(HE)₃-Z_HER3-NODAGA. We hypothesized that increasing the negative charge of the gallium-68/chelator complex would reduce hepatic uptake, which could lead to improved contrast of anti-HER3 affibody-based PET-imaging of HER3 expression. (HE)₃-Z_HER3-X (X = DOTA, DOTAGA) were produced and labeled with gallium-68. Binding of the new conjugates was specific in HER3 expressing BxPC-3 and DU145 human cancer cells. Biodistribution and in vivo specificity was studied in BxPC-3 xenograft bearing Balb/c nu/nu mice 3 h pi. DOTA- and DOTAGA-containing conjugates had significantly higher concentration in blood than [⁶⁸Ga]Ga-(HE)₃-Z_HER3-NODAGA. Presence of the negatively charged ⁶⁸Ga-DOTAGA complex reduced the unspecific hepatic uptake, but did not improve overall biodistribution of the conjugate. [⁶⁸Ga]Ga-(HE)₃-Z_HER3-DOTAGA and [⁶⁸Ga]Ga-(HE)₃-Z_HER3-NODAGA had similar tumor-to-liver ratios, but [⁶⁸Ga]Ga-(HE)₃-Z_HER3-NODAGA had the highest tumor uptake and tumor-to-blood ratio among the tested conjugates. In conclusion, [⁶⁸Ga]Ga-(HE)₃-Z_HER3-NODAGA remains the favorable variant for PET imaging of HER3 expression.