[68Ga]Ga-DATA5m-LM4, a PET Radiotracer in the Diagnosis of SST2R-Positive Tumors: Preclinical and First Clinical Results

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 (DATA5m) rapidly binds Ga-68 (t1/2: 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-DATA5m-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 DATA5m was coupled at the N-terminus for labeling with radiogallium (Ga-67/68). [67Ga]Ga-DATA5m-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-DATA5m-LM4 was prepared and injected in a neuroendocrine tumor (NET) patient for PET/CT imaging. DATA5m-LM4 displayed high SST2R binding affinity. [67Ga]Ga-DATA5m-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-DATA5m-LM4 accurately visualized tumor lesions with high contrast on PET/CT. In short, [68Ga]Ga-DATA5m-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 DATA5m-chelator route.

The above class of octreotide-derived radioligands has shown agonistic properties at the SST2R, associated with fast internalization in target cells after binding to the cell membrane receptor. In fact, high radioligand uptake and retention in tumor lesions have been directly linked to fast internalization in cancer cells [7,8]. In the course of these developments, though radiolabeled octapeptides with antagonistic properties at the SST2R, it turned out to perform better than agonists [14][15][16][17][18][19]. Despite their lack of internalization, radiolabeled SST2R-antagonists displayed higher and more persistent uptake in tumor-bearing mice combined with faster background clearance. Such properties were next translated in NET patients, challenging the initial reading of SST2R-agonism as a major element of the success for somatostatin radioligands. Studies at the molecular and cellular level revealed that radiolabeled SST2R-antagonists bind to both active and inactive conformations of the receptor. Hence, antagonists have a far higher number of binding sites at their disposal than agonists, binding to the sub-population of active SST2Rs only [20]. A fair number of SST2Rantagonists have been developed in recent years carrying suitable chelators for labeling with radiometals of interest [21,22]. Such an example is DOTA-LM3 (DOTA-DPhe-c[DCys-Tyr-DAph(Cbm)-Lys-Thr-Cys]-DTyr-NH2; DAph(Cbm), D-4-(carbamoyl)amino-phenylalanine) (Figure 1). DOTA-LM3 labeled with Ga-68 and other radiometals has shown promising qualities during preclinical and clinical studies for use in NET theranostics [21,22]. It should be noted however that incorporation of Ga-68 to DOTA requires heating, complicating hospital logistics in view of the short half-life of Ga-68 (t1/2: 67.7 min) [23,24]. An elegant way to tackle this problem is provided by the hybrid chelator (6-pentanoic acid)-6-(amino)methy-1,4-diazepinetriacetate (DATA 5m ). DATA 5m rapidly coordinates Ga-68 at much lower temperature, thus allowing for quick and convenient access to "ready-for-injection" [ 68 Ga]Ga-tracers in hospitals [25][26][27][28][29][30]. We herein present [ 68 Ga]Ga-DATA 5m -LM4, as a new candidate for PET/CT imaging of SST2R-positive tumors. LM4 is a 4Pal 3 /Tyr 3 -analog of LM3 [21,22,31,32] (4Pal 3 , 3-(4-pyridyl)alanine), which was coupled to DATA 5m (Figure 1). In the present work, DOTA-LM3 and DATA 5m -LM4 were labeled with the longer-lived Ga-67 (t1/2: 78.3 h) surrogate for convenient and direct comparison of their preclinical profiles in HEK293-SST2R cells and HEK293-SST2R tumor-bearing mice. Moreover, clinical grade [ 68 Ga]Ga-DATA 5m -LM4 was easily produced in a hospital setting and evaluated in a NET patient applying PET/CT in a "proof-of-principle" approach.  It should be noted however that incorporation of Ga-68 to DOTA requires heating, complicating hospital logistics in view of the short half-life of Ga-68 (t 1/2 : 67.7 min) [23,24]. An elegant way to tackle this problem is provided by the hybrid chelator (6-pentanoic acid)-6-(amino)methy-1,4-diazepinetriacetate (DATA 5m ). DATA 5m rapidly coordinates Ga-68 at much lower temperature, thus allowing for quick and convenient access to "ready-forinjection" [ 68 Ga]Ga-tracers in hospitals [25][26][27][28][29][30]. We herein present [ 68 Ga]Ga-DATA 5m -LM4, as a new candidate for PET/CT imaging of SST 2 R-positive tumors. LM4 is a 4Pal 3 /Tyr 3analog of LM3 [21,22,31,32] (4Pal 3 , 3-(4-pyridyl)alanine), which was coupled to DATA 5m ( Figure 1). In the present work, DOTA-LM3 and DATA 5m -LM4 were labeled with the longerlived Ga-67 (t 1/2 : 78.3 h) surrogate for convenient and direct comparison of their preclinical profiles in HEK293-SST 2 R cells and HEK293-SST 2 R tumor-bearing mice. Moreover, clinical grade [ 68 Ga]Ga-DATA 5m -LM4 was easily produced in a hospital setting and evaluated in a NET patient applying PET/CT in a "proof-of-principle" approach.

Ligands and Radioligands
Labeling of the new DATA 5m -LM4 analog with Ga-67 was accomplished within 15 min at room temperature affording the [ 67 Ga]Ga-DATA 5m -LM4 in high radiochemical purity (RCP > 96%) at a molar activity of 3.7 MBq/nmol, as verified by radioanalytical HPLC methods (Supplementary Materials Figure S1). Similar results could be obtained with the DOTA-derivatized analogs DOTA-TOC and DOTA-LM3 only by heating of the labeling reaction mixture for 30 min at 90 • C. Radioligands were used as such in all further experiments without purification; the respective solutions were tested before and after all in vitro assays and animal studies (analytical data for DATA 5m -LM4 and DOTA-LM3 in Table S1, Supplementary Materials).

Ligands and Radioligands
Labeling of the new DATA 5m -LM4 analog with Ga-67 was accomplished within 15 min at room temperature affording the [ 67 Ga]Ga-DATA 5m -LM4 in high radiochemical purity (RCP > 96%) at a molar activity of 3.7 MBq/nmol, as verified by radioanalytical HPLC methods (Supplementary Materials Figure S1). Similar results could be obtained with the DOTAderivatized analogs DOTA-TOC and DOTA-LM3 only by heating of the labeling reaction mixture for 30 min at 90 °C. Radioligands were used as such in all further experiments without purification; the respective solutions were tested before and after all in vitro assays and animal studies (analytical data for DATA 5m -LM4 and DOTA-LM3 in Table S1, Supplementary Materials).
Labeling of DATA 5m -LM4 with Ga-68 for clinical use was conducted by an automatic system. Reaction at 50 °C provided RCP ≥ 98%. The automated synthesis led to activities between 413 MBq and 596 MBq for 50 µg of DATA 5m -LM4 (corresponding to 12.5-18 MBq/nmol molar activity; Supplementary Materials Figure S3).

Uptake/Internalization in HEK293-SST 2 R Cells
Comparative uptake and internalization of [ 67 Figure 3. We observe that [ 67 Ga]Ga-DATA 5m -LM4 is taken up by the cells most efficiently (28.86 ± 1.69%), followed by [ 67 Ga]Ga-DOTA-TOC (20.98 ± 0.71%; p < 0.0001) and [ 67 Ga]Ga-DOTA-LM3, which achieved very low cell uptake (2.51 ± 0.36%; p < 0.0001) within this group of radioligands. Notably, the bulk of radioactivity was found in the cell membrane in the case of [ 67 [14,16]. In all cases, cell uptake was banned in the presence of excess TATE, revealing an SST 2 R-mediated process. and [ 67 Ga]Ga-DOTA-TOC is consistent with an SST2R antagonist and agonist behavior, respectively [14,16]. In all cases, cell uptake was banned in the presence of excess TATE, revealing an SST2R-mediated process.

Animal Studies
The in vivo formation of radio metabolites following injection of [ 67 Ga]Ga-DATA 5m -LM4 in healthy mice could be ruled out by radio-HPLC analysis of blood samples collected 5 min post-injection (pi) [34], revealing the high metabolic stability of the octapeptide radiotracer (Supplementary Materials Figure S2).  Table S2, Supplementary Materials).
The radioligands displayed fast background clearance predominantly via the kidneys and the urinary tract. Interestingly, [ 67 Ga]Ga-DATA 5m -LM4 showed higher uptake in a number of SST2R-positive mice tissues at 1 h pi compared with [ 67 Ga]Ga-DOTA-LM3, such as the pancreas (22.96 ± 4.06%IA/g vs. 1.49 ± 0.71%IA/g for [ 67 Ga]Ga-DOTA-LM3; p < 0.0001) and the stomach (6.16 ± 3.00%IA/g vs. 0.80 ± 0.17%IA/g for [ 67 Ga]Ga-DOTA-LM3; p < 0.001). These discrepancies however drastically diminished at 4 h pi. A further striking difference in the biodistribution of the two analogs was observed in their kidney uptake and retention.

In Vivo Metabolic Stability of [ 67 Ga]Ga-DATA 5m -LM4
The in vivo formation of radio metabolites following injection of [ 67 Ga]Ga-DATA 5m -LM4 in healthy mice could be ruled out by radio-HPLC analysis of blood samples collected 5 min post-injection (pi) [34], revealing the high metabolic stability of the octapeptide radiotracer (Supplementary Materials Figure S2).

Discussion
We herein introduce [ 68 Ga]Ga-DATA 5m -LM4, as an SST 2 R antagonist in the diagnosis of NETs with PET/CT. The new tracer is based on the known SST 2 R antagonist LM3 [21,22] following 4Pal 3 /Tyr 3 -substitution [32] in the cyclic octapeptide chain. Notably, DATA 5m -LM4 can be labeled with Ga-67/68 at much lower temperatures than other DOTAderivatized peptides (including DOTA-LM3) by virtue of the hybrid DATA 5m chelator attached on its N-terminus (Figure 1) [25][26][27][28][29][30]. For convenient completion of the preclinical study, DATA 5m -LM4, DOTA-LM3 and the SST 2 R-agonist reference DOTA-TOC were labeled with the longer-lived Ga-67 (t 1/2 : 78.3 h) as a Ga-68 surrogate (t 1/2 : 67.7 min). Unlike the above two DOTA-conjugates requiring heating at 90 • C for 30 min during labeling, DATA 5m -LM4 was successfully labeled by a mere 15 min incubation at room temperature. The quality control of radiolabeled products adopting radioanalytical HPLC methods confirmed in all cases a >98% RCP at a molar activity of 3.7 MBq/nmol. Therefore, all radioligands were further used without purification in all preclinical experiments that followed.
During competition binding assays against [ 125 I-Tyr 25 ]LTT-SS28 on HEK293-SST 2 R cellmembranes [30], DATA 5m -LM4 and its Ga-tagged version [ nat Ga]Ga-DATA 5m -LM4 showed high receptor affinity, reflected in the single-digit nanomolar IC 50 values (1.24 ± 0.20 nM and 1.61 ± 0.32 nM, respectively; Figure 2). It is interesting to note that the IC 50 values reported for DOTA-LM3 and [ nat Ga]Ga-DOTA-LM3 during SST 2 R autoradiography against the same radioligand were 1.4 ± 0.5 nM and 12.5 ± 4.3 nM, respectively [15,22]. On the other hand, by changing the metal to Lu the IC 50 determined for [ nat Lu]Lu-DOTA-LM3 was 1.61 ± 0.32 nM and by replacing DOTA by NODAGA, the IC 50 value for [ nat Ga]Ga-NODAGA-LM3 reached 1.3 ± 0.3 nM [21,22]. These results reveal the strong impact of the metal chelate on SST 2 R affinity. Thus, Ga-labeling of DOTA-LM3 leads to a loss of receptor binding affinity compared with unlabeled DOTA-LM3 by one order of magnitude. In turn, these data reveal that the binding affinity of Ga-labeled DATA 5m -LM4 is 10-fold better than that of Ga-labeled DOTA-LM3, while both unlabeled compounds show similar binding affinity. Specific uptake of the [ 67 Ga]Ga-radioligands in HEK293-SST 2 R cells followed this affinity pattern (Figure 3), with [ 67 Ga]Ga-DATA 5m -LM4 taken up much more successfully by the cells at 1 h incubation compared with [ 67 Ga]Ga-DOTA-LM3 (28.86 ± 1.69 % vs. 2.51 ± 0.36%, respectively). It is also interesting to compare the cell distribution pattern of radioactivity between [ 67 Ga]Ga-DATA 5m -LM4 with the radioactivity predominantly found on the cell membrane and [ 67 Ga]Ga-DOTA-TOC with the bulk of radioactivity internalized. These two distinct patterns correspond to a non-internalizing receptor antagonist and an internalizing agonist profile.
The biodistribution of [ 67 Ga]Ga-DATA 5m -LM4 and [ 67 Ga]Ga-DOTA-LM3 were directly compared in male SCID mice bearing twin HEK293-SST 2 R and wtHEK293 xenografts in their flanks, with the latter serving as negative controls being devoid of SST 2 R expression. The new tracer [ 67 Ga]Ga-DATA 5m -LM4 showed a favorably constant uptake in the implanted HEK293-SST 2 R tumors in the period between 1 and 4 h pi, as opposed to [ 67 Ga]Ga-DOTA-LM3 declining from the tumor within this interval. Another interesting advantage of [ 67 Ga]Ga-DATA 5m -LM4 is the significantly lower kidney uptake at 1 h pi, which further decreased at 4 h pi. In contrast, [ 67 Ga]Ga-DOTA-LM3 retained a much higher kidney uptake within this time frame (Figure 4; Table S2, Supplementary Materials). It is interesting to note that biodistribution results on [ 67 Ga]Ga-DOTA-LM3 at 1 h pi are well comparable with those previously reported for [ 68 Ga]Ga-DOTA-LM3 on a similar HEK293-SST 2 R tumor model in female nude mice; the same total peptide amount (10 pmol) was injected in both cases [22]. Summarizing the above observations, [ 67  The above discussed promising preclinical results prompted us to test clinical grade [ 68 Ga]Ga-DATA 5m -LM4 in a NET patient in a preliminary "proof-of-principal" experiment. It should be noted that routine labeling with Ga-68 was successful and reproducible via an automated module in the hospital and mild heating at 50 • C in an overall production time of 15 min. Notably, tumor lesions were accurately visualized in the patient with a high contrast on PET/CT ( Figure 5).
The present study has revealed attractive features for [ 67 [28].
The [ 125 I][I-Tyr 25 ]LTT-SS28 was obtained from LTT-SS28 according to the chloramine-T method using [ 125 I]NaI in a 0.1 M NaOH solution for the radioiodination [30]. The radioligand was isolated in a pure form by HPLC and aliquots thereof in 0.1% BSA-PBS buffer were kept at −20 • C; these were used for competition binding experiments (molar activity of 74 GBq/µmol). For the preparation of [ nat Ga]Ga-DATA 5m -LM4, stock solution of DATA 5m -LM4 (60 µL, 2 mM, 120 nmol) was placed in an Eppendorf Protein LoBind ® centrifuge tube and a [ nat Ga]Ga(NO 3 ) 3 solution in 1 M sodium acetate buffer (pH 4.0) was added. The mixture was heated at 75 • C for 1 h and analysis was conducted applying reversed-phase high performance liquid chromatography (RP-HPLC).

Radiochemical Analysis
Analyses were performed on a Waters Chromatograph based on a 600E multi-solvent delivery system applying twin detection modes: (a) for photometric detection, a Waters 2998 photodiode array detector (Waters, Vienna, Austria) was used (DATA 5m -LM4 and [ nat Ga]Ga-DATA 5m -LM4), (b) for radiometric detection, a Gabi gamma-detector (Raytest, RSM Analytische Instrumente GmbH, Straubenhardt, Germany) was applied (detection of radioligands). The system was monitored and data was processed by the Empower Software (Waters, Milford, MA, USA). For radiochemical analyses, samples were eluted through a Xterra RP18 cartridge column (5 µm, 3.9 mm × 20 mm, Waters, Eschborn, Germany), applying the following linear gradient: 100%A/0% B to 60%A/40% B in 40 min, whereby A = 0.1% TFA in H 2 O (v/v) and B = MeCN (system 1). Radioligands were used without further purification in all subsequent experiments and suitable samples were tested before and after the end of all biological experiments. Retention times (t R ) were as follows: [ 67  The handling of solutions containing beta-/gamma-emitting radionuclides was conducted by authorized personnel in compliance with European radiation safety guidelines. For quality control of clinical grade [ 68 Ga]Ga-DATA 5m -LM4, radio-TLC was applied (TLC silica gel 60 F254 Merck) whereby the plates were developed with (1) 0.1 M citrate buffer pH 4 and (2) a 1:1 mixture of 1 M ammonium acetate and MeOH (v:v) as the mobile phase. TLC plates were measured on a miniGita TLC scanner from Elysia-Raytest (Angleur, Belgium) applying the GINA analysis software (Elysia-Raytest; Angleur, Belgium). Radio-HPLC was conducted on an Infinity 1200 analytical HPLC system from Agilent Technologies (Waldbronn, Germany), coupled to a Ramona * radiodetector from Elysia-Raytest (Angleur, Belgium). The VDSpher PUR 150 C18-E column (5 µm, 100 × 40 m) was eluted applying a linear gradient of 5-45% MeCN (+0.1% TFA)/95-55% H 2 O (+0.1% TFA) in 10 min (system 2). No indices for tracer instability were detected in the period between synthesis and image acquisition.

Cell Culture
The HEK293 cell line transfected to stably express the human SST 2 R, tagged with the T7-epitope (HEK293-SST 2 R), was offered by S. Schultz (Jena, Germany) [33]. Wild type HEK293 cells, devoid of SST 2 R expression, served as negative controls (wtHEK293). Cells were cultured in controlled humidified air with 5% CO 2 at 37 • C in Dulbecco's Modified Eagle Medium (DMEM), containing Glutamax-I and supplemented with 10% (v/v) heatinactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin and 400 µg/mL G418; the latter was not added during culturing of wtHEK293 cells. All culture media were provided by Gibco BRL, Life Technologies (Grand Island, NY, USA) and R.P.B. and T.M.; funding acquisition, F.R. and T.M. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded via a grant by International Centers for Precision Oncology (ICPO) Foundation, Wiesbaden, Germany, grant: "New SST 2 R-antagonists in the diagnosis of neuroendocrine tumors with PET/CT".
Institutional Review Board Statement: The animal study protocols were approved by the Department of Agriculture and Veterinary Service of the Prefecture of Athens (#1609, 24-04-2019: for stability studies, #1610, 24-04-2019: for biodistribution and imaging studies). The patient study did not require ethical approval due to its retrospective nature. Additionally, all imaging procedures were part of routine follow-up examinations. This study was conducted in accordance with the Declaration of Helsinki. [ 68 Ga]Ga-DATA 5m -LM4 was administered in compliance with The German Medicinal Products Act (AMG § 13 2b) and in accordance with the responsible regulatory body.
Informed Consent Statement: Informed consent was obtained from the patient in the study.
Data Availability Statement: Not applicable.