The clinical success of radiolabeled somatostatin analogs in the diagnosis and therapy—“theranostics”—of tumors expressing the somatostatin subtype 2 receptor (SST₂R) has paved the way for the development of a broader panel of peptide radioligands targeting different human tumors. This approach relies on the overexpression of other receptor-targets in different cancer types. In recent years, a shift in paradigm from internalizing agonists to antagonists has occurred. Thus, SST₂R-antagonist radioligands were first shown to accumulate more efficiently in tumor lesions and clear faster from the background in animal models and patients. The switch to receptor antagonists was soon adopted in the field of radiolabeled bombesin (BBN). Unlike the stable cyclic octapeptides used in the case of somatostatin, BBN-like peptides are linear, fast to biodegradable and elicit adverse effects in the body. Thus, the advent of BBN-like antagonists provided an elegant way to obtain effective and safe radiotheranostics. Likewise, the pursuit of gastrin and exendin antagonist-based radioligands is advancing with exciting new outcomes on the horizon. In the present review, we discuss these developments with a focus on clinical results, commenting on challenges and opportunities for personalized treatment of cancer patients by means of state-of-the-art antagonist-based radiopharmaceuticals.

Diagnostic imaging and radionuclide therapy of prostate (PC) and breast cancer (BC) using radiolabeled gastrin-releasing peptide receptor (GRPR)-antagonists represents a promising approach. We herein propose the GRPR-antagonist based radiotracer [^99mTc]Tc-DB15 ([^99mTc]Tc-N₄-AMA-DGA-_DPhe⁶,Sar¹¹,LeuNHEt¹³]BBN(6-13); N₄: 6-carboxy-1,4,8,11-tetraazaundecane, AMA: aminomethyl-aniline, DGA: diglycolic acid) as a new diagnostic tool for GRPR-positive tumors applying SPECT/CT. The uptake of [^99mTc]Tc-DB15 was tested in vitro in mammary (T-47D) and prostate cancer (PC-3) cells and in vivo in T-47D or PC-3 xenograft-bearing mice as well as in BC patients. DB15 showed high GRPR-affinity (IC₅₀ = 0.37 ± 0.03 nM) and [^99mTc]Tc-DB15 strongly bound to the cell-membrane of T-47D and PC-3 cells, according to a radiolabeled antagonist profile. In mice, the radiotracer showed high and prolonged GRPR-specific uptake in PC-3 (e.g., 25.56 ± 2.78 %IA/g vs. 0.72 ± 0.12 %IA/g in block; 4 h pi) and T-47D (e.g., 15.82 ± 3.20 %IA/g vs. 3.82 ± 0.30 %IA/g in block; 4 h pi) tumors, while rapidly clearing from background. In patients with advanced BC, the tracer could reveal several bone and soft tissue metastases on SPECT/CT. The attractive pharmacokinetic profile of [^99mTc]DB15 in mice and its capability to target GRPR-positive BC lesions in patients highlight its prospects for a broader clinical use, an option currently being explored by ongoing clinical studies.

Background: Peptide radioligands may serve as radionuclide carriers to tumor sites overexpressing their cognate receptor for diagnostic or therapeutic purposes. Treatment of mice with the neprilysin (NEP)-inhibitor phosphoramidon was previously shown to improve the metabolic stability and tumor uptake of biodegradable radiopeptides. Aiming to clinical translation of this methodology, we herein investigated the impact of the approved pill Entresto, releasing the potent NEP-inhibitor LBQ657 in vivo, on the stability and tumor uptake of two radiopeptides. Methods: The metabolic stability of [^99mTc]Tc-DB4 (DB4, N₄-Pro-Gln-Arg-Tyr-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH₂) and [¹¹¹In]In-SG4 (SG4, DOTA-DGlu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH₂) was tested in LBQ657/Entresto-treated mice vs. untreated controls. The uptake in gastrin-releasing peptide receptor (GRPR)-, or cholecystokinin subtype 2 receptor (CCK₂R)-positive tumors respectively, was compared between LBQ657/Entresto-treated mice and untreated controls. Results: LBQ657/Entresto treatment induced marked stabilization of [^99mTc] Tc-DB4 and [¹¹¹In]In-SG4 in peripheral mice blood, resulting in equally enhanced tumor uptake at 4 h post-injection. Accordingly, the [^99mTc]Tc-DB4 uptake of 7.13 ± 1.76%IA/g in PC-3 tumors increased to 16.17 ± 0.71/17.50 ± 3.70%IA/g (LBQ657/Entresto) and the [¹¹¹In]In-SG4 uptake of 3.07 ± 0.87%IA/g in A431-CCK₂R(+) tumors to 8.11 ± 1.45/9.61 ± 1.70%IA/g. Findings were visualized by SPECT/CT. Conclusions: This study has shown the efficacy of Entresto to notably improve the profile of [^99mTc]Tc-DB4 and [¹¹¹In]In-SG4 in mice, paving the way for clinical translation of this approach.

Radiolabeled somatostatin subtype 2 receptor (SST2R)-antagonists have shown advantageous profiles for cancer theranostics compared with agonists. On the other hand, the newly introduced hybrid chelator (6-pentanoic acid)-6-(amino)methyl-1,4-diazepinetriacetate (DATA^5m) rapidly binds Ga-68 (t_1/2: 67.7 min) at much lower temperature, thus allowing for quick access to “ready-for-injection” [⁶⁸Ga]Ga-tracers in hospitals. We herein introduce [⁶⁸Ga]Ga-DATA^5m-LM4 for PET/CT imaging of SST₂R-positive human tumors. LM4 was obtained by 4Pal³/Tyr³-substitution in the known SST₂R antagonist LM3 (H-DPhe-c[DCys-Tyr-DAph(Cbm)-Lys-Thr-Cys]-DTyr-NH₂) and DATA^5m was coupled at the N-terminus for labeling with radiogallium (Ga-67/68). [⁶⁷Ga]Ga-DATA^5m-LM4 was evaluated in HEK293-SST₂R cells and mice models in a head-to-head comparison with [⁶⁷Ga]Ga-DOTA-LM3. Clinical grade [⁶⁸Ga]Ga-DATA^5m-LM4 was prepared and injected in a neuroendocrine tumor (NET) patient for PET/CT imaging. DATA^5m-LM4 displayed high SST₂R binding affinity. [⁶⁷Ga]Ga-DATA^5m-LM4 showed markedly higher uptake in HEK293-SST₂R cells versus [⁶⁷Ga]Ga-DOTA-LM3 and was stable in vivo. In HEK293-SST₂R xenograft-bearing mice, it achieved longer tumor retention and less kidney uptake than [⁶⁷Ga]Ga-DOTA-LM3. [⁶⁸Ga]Ga-DATA^5m-LM4 accurately visualized tumor lesions with high contrast on PET/CT. In short, [⁶⁸Ga]Ga-DATA^5m-LM4 has shown excellent prospects for the PET/CT diagnosis of SST₂R-positive tumors, further highlighting the benefits of Ga-68 labeling in a hospital environment via the DATA^5m-chelator route.

Radiolabeled gastrin analogues have been proposed for theranostics of cholecystokinin subtype 2 receptor (CCK₂R)-positive cancer. Peptide radioligands based on other receptor antagonists have displayed superior pharmacokinetics and higher biosafety than agonists. Here, we present DGA1, a derivative of the nonpeptidic CCK₂R antagonist Z-360 carrying an acyclic tetraamine, for [^99mTc]Tc labeling. Preclinical comparison of [^99mTc]Tc-DGA1 with [^99mTc]Tc-DG2 (CCK₂R-agonist reference) was conducted in HEK293-CCK₂R/CCK_2i4svR cells and mice models, qualifying [^99mTc]Tc-DGA1 for further study in patients with CCK₂R-positive tumors and single-photon emission computed tomography/CT.

Background: The GRPR-antagonist ⁶⁸Ga-SB3 visualized prostate cancer lesions in animal models and in patients. Switching radiometal from ⁶⁸Ga to ¹¹¹In impaired tumor targeting in mice, but coinjection of the neprilysin (NEP)-inhibitor phosphoramidon (PA) stabilized ¹¹¹In-SB3 in circulation and remarkably increased tumor uptake. We herein report on the biological profile of ¹¹¹In-SB4: ¹¹¹In-[dAla¹¹]SB3. Methods: The biological responses of ¹¹¹In-SB3/SB4 were compared in PC-3 cells and animal models. Results: Gly¹¹/dAla¹¹-replacement deteriorated GRPR-affinity (SB4 IC₅₀: 10.7 ± 0.9 nM vs. SB3 IC₅₀: 4.6 ± 0.3 nM) and uptake in PC-3 cells (¹¹¹In-SB4: 1.3 ± 0.4% vs. ¹¹¹In-SB3 16.2 ± 0.8% at 1 h). ¹¹¹In-SB4 was more stable than ¹¹¹In-SB3, but PA-coinjection stabilized both radiotracers in peripheral mice blood. Unmodified ¹¹¹In-SB3 showed higher uptake in PC-3 xenografts (8.8 ± 3.0%ID/g) vs. ¹¹¹In-SB4 (3.1 ± 1.1%ID/g) at 4 h pi. PA-coinjection improved tumor uptake, with ¹¹¹In-SB3 still showing superior tumor targeting (38.3 ± 7.9%ID/g vs. 7.4 ± 0.3%ID/g for ¹¹¹In-SB4). Conclusions: Replacement of Gly¹¹ by dAla¹¹ improved in vivo stability, however, at the cost of GRPR-affinity and cell uptake, eventually translating into inferior tumor uptake of ¹¹¹In-SB4 vs. unmodified ¹¹¹In-SB3. On the other hand, in-situ NEP-inhibition turned out to be a more efficient and direct strategy to optimize the in vivo profile of ¹¹¹In-SB3, and potentially other peptide radiotracers.