The gastrin-releasing peptide receptor (GRPR) is expressed in high numbers in a variety of human tumors, including the frequently occurring prostate and breast cancers, and therefore provides the rationale for directing diagnostic or therapeutic radionuclides on cancer lesions after administration of anti-GRPR peptide analogs. This concept has been initially explored with analogs of the frog 14-peptide bombesin, suitably modified at the N-terminus with a number of radiometal chelates. Radiotracers that were selected for clinical testing revealed inherent problems associated with these GRPR agonists, related to low metabolic stability, unfavorable abdominal accumulation, and adverse effects. A shift toward GRPR antagonists soon followed, with safer analogs becoming available, whereby, metabolic stability and background clearance issues were gradually improved. Clinical testing of three main major antagonist types led to promising outcomes, but at the same time brought to light several limitations of this concept, partly related to the variation of GRPR expression levels across cancer types, stages, previous treatments, and other factors. Currently, these parameters are being rigorously addressed by cell biologists, chemists, nuclear medicine physicians, and other discipline practitioners in a common effort to make available more effective and safe state-of-the-art molecular tools to combat GRPR-positive tumors. In the present review, we present the background, current status, and future perspectives of this endeavor.

Patients with metastatic medullary thyroid cancer (MTC) have limited systemic treatment options. The use of radiolabeled gastrin analogs targeting the cholecystokinin-2 receptor (CCK2R) is an attractive approach. However, their therapeutic efficacy is presumably decreased by their enzymatic degradation in vivo. We aimed to investigate whether the chemically stabilized analog ¹⁷⁷Lu-DOTA-PP-F11N (¹⁷⁷Lu-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2) performs better than reference analogs with varying in vivo stability, namely ¹⁷⁷Lu-DOTA-MG11 (¹⁷⁷Lu-DOTA-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) and ¹⁷⁷Lu-DOTA-PP-F11 (¹⁷⁷Lu-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2), and if the use of protease inhibitors further improves CCKR2-targeting. First human data on ¹⁷⁷Lu-DOTA-PP-F11N are also reported. Methods: In vitro stability of all analogs was assessed against a panel of extra- and intra-cellular endoproteases, while their in vitro evaluation was performed using the human MTC MZ-CRC-1 and the transfected A431-CCK2R(+) cell lines. Biodistribution without and with the protease inhibitors phosphoramidon (PA) and thiorphan (TO) was assessed 4h post-injection in MZ-CRC-1 and A431-CCK2R(+) dual xenografts. Autoradiography of ¹⁷⁷Lu-DOTA-PP-F11N (without and with PA) and nanoSPECT/CT images were performed. SPECT/CT images of ¹⁷⁷Lu-DOTA-PP-F11N in a metastatic MTC patient were also acquired. Results: natLu-DOTA-PP-F11N is less of a substrate for neprilysines than the other analogs, while intracellular cysteine proteases, like cathepsins-L, might be involved in the degradation of gastrin analogs. The uptake of all radiotracers was higher in MZ-CRC-1 tumors, compared to A431-CCK2R(+), apparently due to the higher number of binding sites on MZ-CRC-1 cells. ¹⁷⁷Lu-DOTA-PP-F11N has the same biodistribution as ¹⁷⁷Lu-DOTA-PP-F11, however, the uptake in the MZ-CRC-1 tumors is almost double (20.7±1.71 vs 11.2±2.94 %IA/g, P = 0.0002). Co-administration of PA or TO increases significantly the ¹⁷⁷Lu-DOTA-MG11 uptake in the CCK2R(+) tumors and stomach. Less profound is the effect on ¹⁷⁷Lu-DOTA-PP-F11, while no influence or even reduction is observed for ¹⁷⁷Lu-DOTA-PP-F11N (20.7±1.71 vs 15.6±3.80 (with PA) %IA/g, p<0.05 in MZ-CRC-1 tumors). First clinical data show high ¹⁷⁷Lu-DOTA-PP-F11N accumulation in the tumors, stomach, kidneys and colon. Conclusion: The performance of ¹⁷⁷Lu-DOTA-PP-F11N without protease inhibitors is as good as the performance of ¹⁷⁷Lu-DOTA-MG11 in the presence of inhibitors. The human application of single compounds without unessential additives is preferable. Preliminary clinical data spotlight stomach as a potential dose-limiting organ beside the kidneys.

Somatostatin receptors (SSTs) are recognized as favorable molecular targets in neuroendocrine tumors (NETs) and neuroendocrine neoplasms (NENs), with subtype 2 (SST₂) being the predominantly and most frequently expressed. PET/CT imaging with ⁶⁸Ga-labeled SST agonists, e.g., ⁶⁸Ga-DOTA-TOC (SomaKit TOC^®) or ⁶⁸Ga-DOTA-TATE (NETSPOT^®), plays an important role in staging and restaging these tumors and can identify patients who qualify and would potentially benefit from peptide receptor radionuclide therapy (PRRT) with the therapeutic counterparts ¹⁷⁷Lu-DOTA-TOC or ¹⁷⁷Lu-DOTA-TATE (Lutathera^®). This is an important feature of SST targeting, as it allows a personalized treatment approach (theranostic approach). Today, new developments hold promise for enhancing diagnostic accuracy and therapeutic efficacy. Among them, the use of SST₂ antagonists, such as JR11 and LM3, has shown certain advantages in improving image sensitivity and tumor radiation dose, and there is evidence that they may find application in other oncological indications beyond NETs and NENs. In addition, PRRT performed with more cytotoxic α-emitters, such as ²²⁵Ac, or β^- and Auger electrons, such as ¹⁶¹Tb, presents higher efficacy. It remains to be seen if any of these new developments will overpower the established radiolabeled SST analogs and PRRT with β^--emitters.

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.

Treatment of neuroendocrine tumours with the radiolabelled somatostatin receptor subtype 2 (SST₂) peptide agonist [¹⁷⁷Lu]Lu-DOTA-TATE is effective and well-established. Recent studies suggest improved therapeutic efficacy using the SST₂ peptide antagonist [¹⁷⁷Lu]Lu-OPS201. However, little is known about the cellular mechanisms that lead to the observed differences. In the present in vitro study, we compared kinetic binding, saturation binding, competition binding, cellular uptake and release of [¹⁷⁷Lu]Lu-OPS201 versus [¹⁷⁷Lu]Lu-DOTA-TATE using HEK cells stably transfected with the human SST₂. While [¹⁷⁷Lu]Lu-OPS201 and [¹⁷⁷Lu]Lu-DOTA-TATE exhibited comparable affinity (K_D, 0.15 ± 0.003 and 0.08 ± 0.02 nM, respectively), [¹⁷⁷Lu]Lu-OPS201 recognized four times more binding sites than [¹⁷⁷Lu]Lu-DOTA-TATE. Competition assays demonstrated that a high concentration of the agonist displaced only 30% of [¹⁷⁷Lu]Lu-OPS201 bound to HEK-SST₂ cell membranes; an indication that the antagonist binds to additional sites that are not recognized by the agonist. [¹⁷⁷Lu]Lu-OPS201 showed faster association and slower dissociation than [¹⁷⁷Lu]Lu-DOTA-TATE. Whereas most of [¹⁷⁷Lu]Lu-OPS201 remained at the cell surface, [¹⁷⁷Lu]Lu-DOTA-TATE was almost completely internalised inside the cell. The present data identified distinct differences between [¹⁷⁷Lu]Lu-OPS201 and [¹⁷⁷Lu]Lu-DOTA-TATE regarding the recognition of receptor binding sites (higher for [¹⁷⁷Lu]Lu-OPS201) and their kinetics (faster association and slower dissociation of [¹⁷⁷Lu]Lu-OPS201) that explain, to a great extent, the improved therapeutic efficacy of [¹⁷⁷Lu]Lu-OPS201 compared to [¹⁷⁷Lu]Lu-DOTA-TATE.