Radiolabeled Somatostatin Analogs—A Continuously Evolving Class of Radiopharmaceuticals

Simple Summary Somatostatin receptors (SSTs) are of particular interest in oncology because these proteins are overexpressed on the cell membranes of different human malignancies, especially neuroendocrine tumors (NETs) and neuroendocrine neoplasms (NENs). Radiolabeled short peptide analogs of the natural hormone somatostatin have been developed over the years to target SST-expressing tumors and are used for both imaging (diagnosis) and therapy. Today, this type of radiopharmaceutical plays a pivotal role in the management of NET and NEN patients. Despite their clinical success, new developments in recent years, in terms of peptide analogs and radionuclides, have shown certain advantages and hold promise for further improvement in both the diagnosis and therapy of SST-expressing tumors, even beyond NETs and NENs. Abstract 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.


Introduction
The somatostatin family consists of two cyclic disulfide-bond-containing peptide hormones, one with 14 amino acids (SS-14, primary form in the brain) and one with 28 amino acids (SS-28, primary form in the gut). The biologic actions of somatostatin are mediated by five somatostatin receptor subtypes (SST [1][2][3][4][5], which belong to a distinct group within the G-protein-coupled receptor superfamily, also known as 7-transmembrane In the amino acid sequence of the endogenous hormone somatostatin, the small tetrapeptide Phe-Trp-Lys-Thr (corresponding to the amino acid residues 7-10 in the natural hormone somatostatin-14) was identified as essential for receptor recognition and biological activity [9,10]. The introduction of D-amino acids for improved in vivo stability and stepwise optimization, based on the minimal amino acid chain length in somatostatin, resulted in an octapeptide with a type II β-turn, formed by the active core Phe-D-Trp-Lys-Thr in a six-member ring via a disulfide bridge, known as octreotide (OC, Table 1) [11]. Octreotide (Sandostatin ® ) is used for the management of growth-hormone-producing tumors (e.g., acromegaly), and tumor and symptom control of neuroendocrine tumors [12], and it has been the starting point for the development of radiolabeled somatostatin analogs ( Figure 1). It is worth mentioning that while the natural hormones somatostatin-14 and somatostatin-28 bind to all subtypes with high (though not the same) affinity, short somatostatin analogs, such as octreotide, only bind to the first subgroup of receptor subtypes (Table 1). More precisely, octreotide has high affinity to SST 2 and SST 5 and moderate affinity to SST 3 . The most interesting structural features on octreotide-based analogs are position 3 (Phe), which is involved in the critical β-turn, and position 8 (Thr(ol)), modifications of which have led to analogs with different receptor subtype selectivities and affinities. Briefly, the well-known Tyr 3  shows high affinity to SST 2 and moderate affinity to SST 5 , while 1-Nal 3 -octreotide (NOC) and BzThi 3 -octreotide (BOC) show additional affinity to SST 3 . The analog with substitution in both positions, [Tyr 3 , Thr 8 ]-octreotide ([Tyr 3 ]-octreotate or TATE), binds almost selectively to SST 2 , while the corresponding [1-Nal 3 , Thr 8 ]-octreotide (NOC-ATE) and [BzThi 3 , Thr 8 ]-octreotide (BOC-ATE) show additional affinity to SST 5 and SST 3 [13,14]. See Table 1 for affinity data.
Cancers 2022, 13, x FOR PEER REVIEW 3 of 14 position 3 (Phe), which is involved in the critical β-turn, and position 8 (Thr(ol)), modifications of which have led to analogs with different receptor subtype selectivities and affinities. Briefly, the well-known Tyr 3 -octreotide (TOC), where Phe is substituted by Tyr, shows high affinity to SST2 and moderate affinity to SST5, while 1-Nal 3 -octreotide (NOC) and BzThi 3 [13,14]. See Table 1 for affinity data. Color code: orange for L-amino acids (also showing the essential amino acids (tetrapeptide) in the somatostatin sequence for receptor recognition), green for D-amino acids, blue for chelators, and yellow for radionuclides.
After the pioneering work of Lamberts et al. in 1989, where endocrine-related tumors could be visualized using 123 I-labeled Tyr 3 -octreotide (TOC), the conjugation of chelators for labeling with radiometals revolutionized the field (Figure 1) [15]. More specifically, the following advances can be noted: (a) the clinical success of 111 In-DTPA-octreotide (Oc-treoScan ® , where DTPA: diethylenetriaminepentaacetic acid); (b) the introduction of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), which is able to form thermodynamically and kinetically stable complexes with a series of 3+ radiometals, like the β --emitter 90 Y; (c) the introduction of peptide receptor radionuclide therapy (PRRT) with 90 Y-or 177 Lu-labeled SST agonists, such as 90 Y-or 177 Lu-DOTA-TOC and 177 Lu-DOTA-TATE [16]; and (d) the accelerated development of 68 Ga radiochemistry/radiopharmacy, establishing SST PET/CT with somatostatin analogs, such as 68 Ga-DOTA-TOC, 68 Ga-DOTA-TATE, and 68 Ga-DOTA-NOC, allowing the most sensitive staging and restaging of NETs, as well as the identification of patients who would benefit from PRRT (theranostic approach), which made radiolabeled somatostatin analogs the archetype of peptide-based radiopharmaceuticals. Nowadays, a plethora of radiolabeled somatostatin analogs have been developed in order to optimize affinity, specificity, and/or pharmacokinetics (many reviews are available, see, for example, Eychenne R et al. [17]). Among them, DOTA-TOC and DOTA-TATE remain the most widely used analogs, with DOTA-TATE (NETSPOT ® ) and DOTA-TOC (SomaKit TOC ® ) kits having approval by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) for 68 Galabeling, and 177 Lu-DOTA-TATE ( 177 Lu-oxodotreotide or Lutathera ® ) being the only agent approved for therapy to date. It is expected that the approval of 177 Lu-DOTA-TOC ( 177 Luedotreotide) will follow the completion of the COMPETE (NCT03049189) phase III trial. After the pioneering work of Lamberts et al. in 1989, where endocrine-related tumors could be visualized using 123 I-labeled Tyr 3 -octreotide (TOC), the conjugation of chelators for labeling with radiometals revolutionized the field (Figure 1) [15]. More specifically, the following advances can be noted: (a) the clinical success of 111 In-DTPA-octreotide (OctreoScan ® , where DTPA: diethylenetriaminepentaacetic acid); (b) the introduction of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), which is able to form thermodynamically and kinetically stable complexes with a series of 3+ radiometals, like the β --emitter 90 Y; (c) the introduction of peptide receptor radionuclide therapy (PRRT) with 90 Y-or 177 Lu-labeled SST agonists, such as 90 Y-or 177 Lu-DOTA-TOC and 177 Lu-DOTA-TATE [16]; and (d) the accelerated development of 68 Ga radiochemistry/radiopharmacy, establishing SST PET/CT with somatostatin analogs, such as 68 Ga-DOTA-TOC, 68 Ga-DOTA-TATE, and 68 Ga-DOTA-NOC, allowing the most sensitive staging and restaging of NETs, as well as the identification of patients who would benefit from PRRT (theranostic approach), which made radiolabeled somatostatin analogs the archetype of peptide-based radiopharmaceuticals. Nowadays, a plethora of radiolabeled somatostatin analogs have been developed in order to optimize affinity, specificity, and/or pharmacokinetics (many reviews are available, see, for example, Eychenne R et al. [17]). Among them, DOTA-TOC and DOTA-TATE remain the most widely used analogs, with DOTA-TATE (NETSPOT ® ) and DOTA-TOC (SomaKit TOC ® ) kits having approval by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) for 68 Ga-labeling, and 177 Lu-DOTA-TATE ( 177 Lu-oxodotreotide or Lutathera ® ) being the only agent approved for therapy to date. It is expected that the approval of 177 Lu-DOTA-TOC ( 177 Lu-edotreotide) will follow the completion of the COMPETE (NCT03049189) phase III trial.

Clinical Studies and Approvals
Today, PRRT with radiolabeled SST agonists (e.g., DOTA-TOC or DOTA-TATE, Table 1) is part of the standard of care of NENs. NETTER-1 (NCT01578239; EudraCT number 2011-005049-11) was the first prospective, open-label, randomized, phase III trial to compare four cycles of 177 Lu-DOTA-TATE (4 × 7.4 GBq) plus 30 mg long-acting release octreotide (PRRT group, n = 117) with high-dose (60 mg double dose) long-acting release octreotide (control group, n = 114) in advanced, progressive midgut NET patients. There was a significantly longer progression-free survival for the PRRT arm (p < 0.001) [22] and a significant improvement in quality of life [23]. Consequently, 177 Lu-DOTA-TATE ( 177 Lu-oxodotreotide) received marketing authorization for the treatment of adult patients with SST-positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs). At the final analysis of overall survival (OS), the median OS was improved by 11.7 months for the 177 Lu-DOTA-TATE arm versus the control arm (48.0 (95% CI, 37.4-55.2) vs. 36.3 (95% CI, 25.9-51.7) months, respectively), which, however, did not reach statistical significance in the long-term follow-up with a median of 6.3 years [24]. Regarding safety, the NETTER-1 data show a low incidence of long-term side-effects regarding hematotoxicity and nephrotoxicity.
Currently, a second prospective, randomized, controlled, open-label, multi-center, phase III trial, COMPETE (NCT03049189), is ongoing, in which PRRT using 177 Lu-DOTA-TOC ( 177 Lu-edotreotide, four cycles with 7.5 GBq/cycle) is being compared with the mTOR inhibitor everolimus (10 mg daily) in patients with progressive, SST-positive GEP-NETs. Upon completion of the study, the approval of 177 Lu-DOTA-TOC is expected. These trials and other trials (e.g., OCCLURANDOM, NCT02230176) should further precisely determine the position of PRRT in the current clinical algorithm with regard to other systemic therapies, such as everolimus and sunitinib.
Using routes other than intravenous administration may be an interesting approach to enhance the therapeutic and safety window of PRRT. NENs and their liver metastases are often highly perfused, and the intra-arterial route can exploit the first-pass effect to treat liver-dominant disease more efficiently. Such an approach can also be used for inoperable primary tumors to downstage the disease in the neoadjuvant setting [25,26]. However, large comparative prospective trials supporting its wider use are missing.

Combination with Alpha-Emitters
Alpha particles have a very short range in tissues (20-100 µm), irradiating volumes with cellular dimensions and therefore sparing normal surrounding tissues from cytotoxic radiation. At the same time, their linear energy transfer (LET) is much higher compared to that of βparticles (50-230 vs. 0.2 keV/µm), which makes alpha radiation far more cytotoxic. Among the α-emitters, 213 Bi was initially used in combination with DOTA-TOC. Kratochwil et al. performed the first clinical study (retrospective) with an α-emitter in combination with DOTA-TOC ( 213 Bi-DOTA-TOC) in seven patients with metastatic NETs (activities ranging from 3.3 to 21 GBq in one-five cycles) after progressing under 90 Y-/ 177 Lu-DOTA-TOC therapy, and it is already available [27]. The report showed moderate renal and hematological toxicity but possible long-term bone marrow toxicity, with the diagnosis of multiple dysplastic syndrome/acute myeloid lymphoma MDS/AML in one heavily pretreated patient. However, the current supply limitations of high-activity 225 Ac/ 213 Bi generators have prevented larger confirmatory prospective studies and have instead motivated the use of the α-emitters 225 Ac or 212 Pb. 212 Pb-DOTAMTATE (AlphaMedix™) is in a phase I, non-randomized, open-label, dose-escalation, single-center study in 20 PRRT naïve NET patients (NCT03466216), with the highest dose level being four cycles of 2.50 MBq/kg/cycle. Previously, at the highest dose level in a small cohort of 10 NET patients, the objective radiological response (ORR) was 80%, and it had mild adverse effects and a tolerable safety profile [28]. 225 Ac-DOTA-TOC was administered in 40 patients with progressive NENs, where the maximum tolerated dose was established at 40 MBq as a single fraction and at 25 MBq in two fractions at a 4-month interval [29]. In another study, 225 Ac-DOTA-TATE was reported in 32 patients with metastatic GEP-NETs, who were stable or had progressive disease and were on 177 Lu-DOTA-TATE therapy. After the administration of 7.8-44.4 MBq 225 Ac-DOTA-TATE in one-five portions, partial remission was achieved in 15 patients and stable disease in 9 of them. At a median of 8-month follow-up, no disease progression or deaths were documented [30]. Recently, a retrospective analysis was performed in 39 patients who received 225 Ac-DOTA-TOC in an attempt to define the safety levels of 225 Ac-DOTA-TOC [31]. The analysis was mainly conclusive regarding acute hematological toxicity but not regarding chronic nephrotoxicity due to pre-existing risk factors. Overall, it was found that a single dose of up to 29 MBq, repeated doses of~20 MBq in 4-month intervals, and a cumulative dose of 60-80 MBq were hematologically tolerable and avoided high-grade (3/4) hematotoxicity.
Although α-emitters offer potential advantages over β --emitters therapeutically, longterm toxicity data are still lacking to properly assess the therapeutic benefit. Importantly, the translocation of radioactive daughter nuclides from the chelator should also be considered as a potential safety hazard for α-emitters with multiple α-emitting daughters, such as 225 Ac.

Conjugates with Prolonged Circulation
Despite the successful outcome of the NETTER-1 study, the objective response rate of patients treated with 177 Lu-DOTA-TATE was, at most, 18% [22], probably due to the rapid blood clearance of 177 Lu-DOTA-TATE, leading to a suboptimal tumor residence time. An attempt to overcome this drawback was the incorporation of Evans Blue (EB) motifs, which prolongs the half-life of the conjugate in the blood by having low micromolar affinity to albumin. This concept was applied to DOTA-TATE, for which it was shown that treatment with 177 Lu-DOTA-EB-TATE was more effective in SST 2 -expressing xenografts than 177 Lu-DOTA-TATE [32,33]. The first dosimetry data of the long-circulating SST 2 agonist 177 Lu-DOTA-EB-TATE versus 177 Lu-DOTA-TATE showed a 7.9-fold increase in tumor dose, which was counterbalanced with an even greater increase in renal and bone marrow absorbed doses [34]. A better response rate (assessed by 68 Ga-DOTA-TATE PET/CT) after one cycle of treatment was reported for the EB conjugate [35], but matching doses in the kidneys and bone marrow were not provided. The superiority of 177 Lu-DOTA-EB-TATE was not confirmed in an intraindividual comparison versus 177 Lu-DOTA-TOC in a limited number (n = 5) of patients [36], where the tumor-to-critical organs' absorbed dose ratios (defined as therapeutic index) were mainly higher for 177 Lu-DOTA-TOC and not for 177 Lu-DOTA-EB-TATE. Whether 177 Lu-DOTA-EB-TATE has any benefit over the established radiopharmaceuticals is still debatable.

Preclinical Development
The observation that GPCR antagonists may bind to more binding sites than agonists, since their binding is independent of the fraction of receptors coupled to the GTP-binding proteins [37], was the primary reason for the development of radiolabeled SST antagonists. Structurally, the main feature to convert an agonist to an antagonist was shown to be the inversion of chirality at positions 1 and 2 of the octreotide family [38]. From the very first preclinical evaluation, the superiority of radiolabeled SST antagonists over agonists was illustrated in terms of targeting SST-expressing tumors [20]. For example, the first SST 2 antagonist 111 In-DOTA-BASS (Table 1) showed almost twice higher tumor uptake compared to the agonist 111 In-DTPA-TATE, despite its lower affinity (IC 50 = 9.4 ± 0.4 nM vs. 1.3 ± 0.2 nM [18,20]), and it also showed binding to a higher number of sites on the cell membrane (B max ) [20]. This was, further on, confirmed on human tumor tissues by autoradiography when comparing 177 Lu-DOTA-BASS with 177 Lu-DOTA-TATE [39], and the long-lasting tumor uptake of 177 Lu-DOTA-BASS in xenografts in vivo holds promise for therapeutic applications of the antagonists [40]. A series of analogs were developed by systematic substitutions of different amino acids with the aim of identifying the structural features that lead to SST 2 -selective antagonists with high affinity [41]. The analogs JR11 and LM3 (Table 1) were selected among the ones with the best affinity and highest hydrophilicity, and they were studied in combination with different chelators and various radiometals [21,42].
Several reports in the past had shown that adding a radiometal to a chelator-SST agonist conjugate could alter its affinity, with 68 Ga systematically improving the SST 2 affinity of DOTA-conjugated agonists, as well as their pharmacokinetics, compared to 111 In, 90 Y, and 177 Lu [13,18]. The effect of the radiometal, but also of the chelator, was far more impressive, and even unexpected, for the SST 2 antagonists [21,42]. Comprehensive studies with JR11 and LM3 in combination with different chelators, such as DOTA and NODAGA, and various (radio)metals, including Ga, Cu, In, Y, and Lu, have illustrated a very high sensitivity of the SST 2 antagonists to the N-terminal modification needed for radiolabeling, and they have shed light on the most promising metal-chelator-antagonist combinations for further development, having the following major impacts: (1) All Ga-DOTA conjugates lost affinity for SST 2 , contrary to the (radio)metalated In-, Y-, and Lu-DOTA conjugates. The affinity of the Ga-complexes was recovered by replacing DOTA with NODAGA. For instance, 68 Ga-NODAGA-LM3 has a 10-fold higher SST 2 affinity than 68 Ga-DOTA-LM3, and 68 Ga-NODAGA-JR11 has an almost 25-fold higher affinity than 68 Ga-DOTA-JR11 (Table 1). Therefore, 68 Ga-NODAGA conjugates of SST 2 antagonists were selected for clinical development. (2) The great potential of using SST 2 antagonists became obvious when the low-affinity 68 Ga-DOTA-JR11 was compared to 68 Ga-DOTA-TATE, which had approx. a 150-fold higher affinity (Table 1). It was found in vivo that 68 Ga-DOTA-JR11 outweighed the affinity differences, being even slightly better than the high-affinity 68 Ga-DOTA-TATE. Not to mention that the high affinity 68 Ga-NODAGA-JR11 was better distinguished than 68 Ga-DOTA-TATE in terms of tumor uptake [21].
Similarly, the therapeutic counterpart 177 Lu-DOTA-JR11 compared to 177 Lu-DOTA-TATE showed a higher tumor uptake and, more importantly, a longer tumor residence time, leading to a higher radiation tumor dose [43] and, consequently, delayed tumor growth and longer median survival [44]. The reasons for these observed in vivo differences can be found, at least partially, in the differences between the two radiopharmaceuticals on the cellular level, which were recently investigated [45]. 177 Lu-DOTA-JR11 showed faster association, slower dissociation, and longer cellular retention than 177 Lu-DOTA-TATE. Despite a comparable high affinity, 177 Lu-DOTA-JR11 recognized four times more receptor binding sites than 177 Lu-DOTA-TATE. However, more interestingly, while a high excess of antagonist was able to entirely displace the agonist bound on the cell membrane, the agonist could not completely displace the antagonist. Taken together, the antagonist binds not only to additional binding sites but also to different binding sites that are not recognized by the agonist (e.g., uncoupled G proteins) [45]. This observation is clinically relevant, as it indicates that the interruption of somatostatin agonists before treatment with radiolabeled analogs may not be necessary if SST 2 antagonists are used.
Last but not least, SPECT tracers based on antagonists are missing, but they are also important considering that more than 70% of nuclear medicine procedures still use 99m Tc. The first attempts to label SST 2 antagonists with 99m Tc via the monodentate ligand hydrazinonicotinamide (HYNIC) using ethylenediamine N,N diacetic acid (EDDA) as a co-ligand (similarly to the clinically used agonist [ 99m Tc]Tc-HYNIC/EDDA-TOC) failed because the antagonist entirely lost its affinity for SST 2 [46], once more depicting the extreme sensitivity of the antagonists to N-terminal modifications. Further studies illustrated that the loss of affinity can be circumvented, to a certain extent, when a spacer of appropriate length and nature (e.g., aminohexanoic acid) is introduced between the antagonist and HYNIC [47]. Nevertheless, the alternative chelating system 6-carboxy-1,4,8,11-tetraazaundecane (N4) seems to be better suited to 99m Tc-based SST 2 antagonists. In fact, 99m Tc-labeled LM3 via N4 ([ 99m Tc]Tc-TECANT-1) has been selected as the first 99m Tc-based antagonist for clinical translation [48] under the ERAPerMED project "TECANT" (Ref No. ERAPERMED2018-125). The clinical trial is expected to start soon.

Clinical Translation
The first clinical evidence indicating that imaging with SST 2 antagonists may be superior to that with agonists was provided by a prospective study, which included five patients with NETs or thyroid cancer after total-body scintigraphy and a SPECT/CT scan with 111 In-DOTA-BASS versus OctreoScan [49]. 111 In-DOTA-BASS had a higher tumor detection rate (25/28 lesions) than 111 In-DTPA-octreotide (17/28 lesions) in a lesion-based analysis. Meanwhile, based on affinity studies and preclinical results, 68 Ga-NODAGA-JR11 (= 68 Ga-OPS202) was selected for PET/CT imaging studies. Nicolas et al. performed a single-center, prospective, phase I/II study with 12 GEP-NET patients, comparing PET/CT with two micro doses of 68 Ga-NODAGA-JR11 (15 and 50 µg/150 MBq) and one micro dose of the potent SST 2 agonist 68 Ga-DOTA-TOC (NCT02162446). 68 Ga-NODAGA-JR11 showed favorable dosimetry results and imaging properties, with the best tumor contrast between 1 and 2 h after injection [50]. 68 Ga-NODAGA-JR11 PET/CT showed a significantly higher sensitivity in a lesion-based comparison with 68 Ga-DOTA-TOC PET/CT: 93.7% (95% CI: 85.3-97.6%) vs. 59.2% (95% CI: 36.3-79.1%) [51]. In this study, diagnostic efficacy measures were compared against contrast-enhanced CT or MRI. 68 Ga-DOTA-JR11 was also assessed clinically, despite its >20 times lower affinity compared to 68 Ga-NODAGA-JR11 (Table 1) [21,[52][53][54]. Zhu et al. prospectively compared 68 Ga-DOTA-JR11 and 68 Ga-DOTA-TATE PET/CT in the same patients with NETs [54]. As in the study of Nicolas et al., they detected significantly more liver lesions with the SST 2 antagonist (552 vs. 365) but, at the same time, significantly less bone lesions (158 vs. 388) compared to 68 Ga-DOTA-TATE. Importantly, 68 Ga-DOTA-JR11 showed a lower tumor uptake than 68 Ga-DOTA-TATE, which is in contrast to the study of Nicolas et al., who prospectively compared 68 Ga-NODAGA-JR11 and 68 Ga-DOTA-TOC PET/CT in the same patients [51]. This finding can be explained by the much lower SST 2 affinity of 68 Ga-DOTA-JR11 in comparison to 68 Ga-NODAGA-JR11 (Table 1) and/or by the study design, which may have caused a bias, as 68 Ga-DOTA-TATE PET/CT was always performed 24 h ahead of 68 Ga-DOTA-JR11 PET/CT, creating the risk of receptor occupation and/or internalization [55].
The therapeutic companion 177 Lu-DOTA-JR11 (= 177 Lu-OPS201), which was initially assessed in a single-center, prospective, proof-of-principle study (phase 0 study), was com-pared with 177 Lu-DOTA-TATE in the same four patients with advanced, metastatic neuroendocrine neoplasia (NEN) (grades 1-3) [56]. The median tumor dose was 3.5-fold higher for the antagonist. At the same time, tumor-to-kidney dose ratios were >2-fold higher with 177 Lu-DOTA-JR11 compared to 177 Lu-DOTA-TATE. Overall, tumor doses with 177 Lu-DOTA-JR11 were up to 487 Gy, with moderate adverse events with grade 3 thrombocytopenia after treatment with three cycles (total 15.2 GBq) in one patient. Figure 2 illustrates a direct comparison of the antagonist 177 Lu-DOTA-JR11 versus the agonist 177 Lu-DOTA-TOC in the same patient with lung NETs (G2).
trast to the study of Nicolas et al., who prospectively compared Ga-NODAGA-JR11 and 68 Ga-DOTA-TOC PET/CT in the same patients [51]. This finding can be explained by the much lower SST2 affinity of 68 Ga-DOTA-JR11 in comparison to 68 Ga-NODAGA-JR11 (Table 1) and/or by the study design, which may have caused a bias, as 68 Ga-DOTA-TATE PET/CT was always performed 24 h ahead of 68 Ga-DOTA-JR11 PET/CT, creating the risk of receptor occupation and/or internalization [55].
The therapeutic companion 177 Lu-DOTA-JR11 (= 177 Lu-OPS201), which was initially assessed in a single-center, prospective, proof-of-principle study (phase 0 study), was compared with 177 Lu-DOTA-TATE in the same four patients with advanced, metastatic neuroendocrine neoplasia (NEN) (grades 1-3) [56]. The median tumor dose was 3.5-fold higher for the antagonist. At the same time, tumor-to-kidney dose ratios were >2-fold higher with 177 Lu-DOTA-JR11 compared to 177 Lu-DOTA-TATE. Overall, tumor doses with 177 Lu-DOTA-JR11 were up to 487 Gy, with moderate adverse events with grade 3 thrombocytopenia after treatment with three cycles (total 15.2 GBq) in one patient. Figure 2 illustrates a direct comparison of the antagonist 177 Lu-DOTA-JR11 versus the agonist 177 Lu-DOTA-TOC in the same patient with lung NETs (G2). Later on, a single-center phase I study with 20 NET patients (grades 1-3) reported a best overall response (RECIST 1.1 criteria) of 45%, and the median progression-free survival (PFS) was 21 months (95% CI, 13.6-NR), accompanied, however, with grade 4 hematotoxicity (leukopenia, neutropenia, and thrombocytopenia) in four out of seven patients treated with two cycles of 177 Lu-DOTA-JR11 (cumulative activity between 10.5 and 14.7 GBq) [57]. Hence, the study was suspended, and the protocol was modified to limit the cumulative absorbed bone marrow dose. Later on, a single-center phase I study with 20 NET patients (grades 1-3) reported a best overall response (RECIST 1.1 criteria) of 45%, and the median progression-free survival (PFS) was 21 months (95% CI, 13.6-NR), accompanied, however, with grade 4 hematotoxicity (leukopenia, neutropenia, and thrombocytopenia) in four out of seven patients treated with two cycles of 177 Lu-DOTA-JR11 (cumulative activity between 10.5 and 14.7 GBq) [57]. Hence, the study was suspended, and the protocol was modified to limit the cumulative absorbed bone marrow dose. 177 Lu-DOTA-JR11 ( 177 Lu-OPS201) is currently being evaluated in a phase I/II, multi-center, open-label study (NCT02592707-active, not recruiting). To date, there is only an abstract available with a brief summary of the results of 20 NET patients with an adequate follow-up [58]. The disease control rate (DCR) at 12 months was 90% (95% CI: 68.3-98.8) for these 20 patients.
The therapeutic companion, 177 Lu-DOTA-LM3, was evaluated in a single-center, compassionate-use study, which included 51 patients with metastatic NENs of grades 1-3, who were selected after 68 Ga-NODAGA-LM3 PET/CT imaging [62]. There were few adverse events (maximal grade 3 thrombocytopenia in 5.9% of patients) after treatment with one-four cycles of 177 Lu-DOTA-LM3, with mean cumulative activity between 6.1 and 26.1 GBq. The partial response and DCR (RECIST 1.1 criteria in 47 patients) were 36% and 85% at 3-6 months, respectively.

Novel Indications for Radiolabeled Somatostatin Analogs
The binding capacities of radiolabeled SST antagonists and agonists were compared in human tissue samples from nine different tumors using in vitro autoradiography with 177 Lu-DOTA-BASS vs. 177 Lu-DOTA-TATE [39], as mentioned above, and with 125 I-JR11 vs. 125 I-TOC [63]. A summary of the outcome is provided in Figure 3.
The therapeutic companion, 177 Lu-DOTA-LM3, was evaluated in a single-center, compassionate-use study, which included 51 patients with metastatic NENs of grades 1-3, who were selected after 68 Ga-NODAGA-LM3 PET/CT imaging [62]. There were few adverse events (maximal grade 3 thrombocytopenia in 5.9% of patients) after treatment with one-four cycles of 177 Lu-DOTA-LM3, with mean cumulative activity between 6.1 and 26.1 GBq. The partial response and DCR (RECIST 1.1 criteria in 47 patients) were 36% and 85% at 3-6 months, respectively.

Novel Indications for Radiolabeled Somatostatin Analogs
The binding capacities of radiolabeled SST antagonists and agonists were compared in human tissue samples from nine different tumors using in vitro autoradiography with 177 Lu-DOTA-BASS vs. 177 Lu-DOTA-TATE [39], as mentioned above, and with 125 I-JR11 vs. 125 I-TOC [63]. A summary of the outcome is provided in Figure 3.  In all tested cases, the radiolabeled SST 2 antagonist bound to more SST 2 sites in all tumors, with an uptake that was 3.8-21.8 times higher than that with the agonist. Interestingly, in some non-neuroendocrine neoplasias, the level of binding of the antagonists reached the same level as that of the agonists (e.g., 177 Lu-DOTA-TATE) in well-differentiated NENs. Of particular interest is the fact that tumors other than GEP-NETs and lung NETs have the potential to become targets for radiolabeled SST 2 antagonists, despite the relatively low SST 2 expression, for example, non-Hodgkin lymphomas, renal cell carcinoma, breast cancer, pheochromocytoma, paraganglioma, medullary thyroid cancer, small-cell lung cancer, and paraganglioma.
SSTs are also expressed in peritumoral vessel endothelial cells; in inflammatory cells; and in immune system cells, such as activated lymphocytes, monocytes, and epithelioid cells. This suggests that clinical indications can be found in benign and chronic inflammatory diseases, besides oncology [64]. PET imaging with 68 Ga-labeled SST agonists (DOTA-TOC, DOTA-TATE, and DOTA-NOC) have shown relevance in detecting vulnerable, atherosclerotic plaques and have been correlated to other risk factors in patients (summarized in [65]). The use of antagonists in this context has, to date, only been explored preclinically [66]. In terms of PRRT, a retrospective analysis of a limited number of oncological patients indicated that 177 Lu-DOTA-TATE results in a reduction in atherosclerotic plaque activity [67], while 177 Lu-DOTA-TOC showed treatment effects in a feasibility study involving two patients with refractory multi-organ involvement of sarcoidosis [68]. Nevertheless, radiolabeled somatostatin analogs have not yet found clinical relevance in these indications, and their impact on clinical outcome needs to be assessed in large-scale clinical trials.

Perspectives
The use of alternative theranostic pairs of radionuclides, such as radioisotopes of scandium ( 43/44/47 Sc) and terbium ( 149/152/155/161 Tb), might open novel theranostic applications. Recently, a preclinical study demonstrated clear therapeutic benefit when using 161 Tb instead of 177 Lu in combination with SST analogs; 161 Tb has similar decay properties to 177 Lu but, additionally, emits a substantial number of conversion and Auger electrons [69]. The most important finding of the study was the identification of the cellular localization of the 161 Tb-labeled SST analog, which leads to the best therapeutic outcome. It was shown that the combination of 161 Tb with the SST 2 antagonist DOTA-LM3, which is not internalizing but remains on the cell membrane, was a better combination than the internalized cytoplasm agonist DOTA-TOC and the internalized and partially nucleus-localized DOTA-TOC-NLS bearing a nucleus-targeting unit (nuclear localization signal (NLS)) [69]. Overall, the preclinical data suggest a benefit of treating NENs with 161 Tb-DOTA-LM3 (or 161 Tb-labeled SST antagonists) vs. 177 Lu-DOTA-TOC ( 161 Tb-labeled SST agonists).
To date, radiolabeled somatostatin analogs used for treatment bind with high affinity to the most predominantly expressed SST 2 . However, various expression and co-expression patterns have been described for the five somatostatin receptor subtypes (SST 1-5 ), depending on the tumor type and origin [3,70,71]. Interestingly, tumor areas lacking expression of a given subtype may be populated by another one [70,71]. In addition, the downregulation or loss of SST 2 in advanced disease stages is associated with an inherently worse disease prognosis, a lower sensitivity in imaging, and ineffective therapy with SST 2 -specific analogs due to inadequate tumor targeting. Hence, somatostatin analogs with affinity to more than one receptor subtype are of great interest, as they address receptor subtype co-expression and heterogeneous expression patterns [72]. Analogs targeting more subtypes than SST 2 potentially target a broader spectrum of tumors and/or increase the uptake of a given tumor and are, therefore, a field to explore.