# Combination of terbium-161 with somatostatin receptor antagonists-a potential paradigm shift for the treatment of neuroendocrine neoplasms

Francesca Borgna, Stephanie Haller, Josep M. Monné Rodriguez, Mihaela

Ginj, Pascal V. Grundler, Jan Rijn Zeevaart, Ulli Köster, Roger Schibli,

Nicholas P. van der Meulen, Cristina Müller

► To cite this version:

Francesca Borgna, Stephanie Haller, Josep M. Monné Rodriguez, Mihaela Ginj, Pascal V. Grundler, et al.. Combination of terbium-161 with somatostatin receptor antagonists-a potential paradigm shift for the treatment of neuroendocrine neoplasms. European Journal of Nuclear Medicine and Molecular Imaging, 2021, 49 (4), pp.1113-1126. 10.1007/s00259-021-05564-0 . hal-05330191

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# Combination of terbium-161 with somatostatin receptor antagonists—a potential paradigm shift for the treatment of neuroendocrine neoplasms

Francesca Borgna<fnref n="1" /> · Stephanie Haller<fnref n="1" /> · Josep M. Monné Rodriguez<fnref n="2" /> · Mihaela Ginj<fnref n="3" /> · Pascal V. Grundler<fnref n="1" /> ·

Jan Rijn Zeevaart<fnref n="4" /> · Ulli Köster<fnref n="5" /> · Roger Schibli<fnref n="1" />,<fnref n="6" /> · Nicholas P. van der Meulen<fnref n="1" />,<fnref n="7" /> · Cristina Müller<fnref n="1" />,<fnref n="6" />

Received: 21 July 2021 / Accepted: 9 September 2021 / Published online: 8 October 2021

© The Author(s) 2021

## Abstract

**Purpose** The β⁻-emitting terbium-161 also emits conversion and Auger electrons, which are believed to be effective in killing single cancer cells. Terbium-161 was applied with somatostatin receptor (SSTR) agonists that localize in the cytoplasm (DOTATOC) and cellular nucleus (DOTATOC-NLS) or with a SSTR antagonist that localizes at the cell membrane (DOTA-LM3). The aim was to identify the most favorable peptide/terbium-161 combination for the treatment of neuroendocrine neoplasms (NENs).

**Methods** The capability of the ¹⁶¹Tb- and ¹⁷⁷Lu-labeled somatostatin (SST) analogues to reduce viability and survival of SSTR-positive AR42J tumor cells was investigated in vitro. The radiopeptides' tissue distribution profiles were assessed in tumor-bearing mice. The efficacy of terbium-161 compared to lutetium-177 was investigated in therapy studies in mice using DOTATOC or DOTA-LM3, respectively.

**Results** In vitro, [¹⁶¹Tb]Tb-DOTA-LM3 was 102-fold more potent than [¹⁷⁷Lu]Lu-DOTA-LM3; however, ¹⁶¹Tb-labeled DOTATOC and DOTATOC-NLS were only 4- to fivefold more effective inhibiting tumor cell viability than their ¹⁷⁷Lu-labeled counterparts. This result was confirmed in vivo and demonstrated that [¹⁶¹Tb]Tb-DOTA-LM3 was significantly more effective in delaying tumor growth than [¹⁷⁷Lu]Lu-DOTA-LM3, thereby, prolonging survival of the mice. A therapeutic advantage of terbium-161 over lutetium-177 was also manifest when applied with DOTATOC. Since the nuclear localizing sequence (NLS) compromised the in vivo tissue distribution of DOTATOC-NLS, it was not used for therapy.

**Conclusion** The use of membrane-localizing DOTA-LM3 was beneficial and profited from the short-ranged electrons emitted by terbium-161. Based on these preclinical data, [¹⁶¹Tb]Tb-DOTA-LM3 may outperform the clinically employed [¹⁷⁷Lu]Lu-DOTATOC for the treatment of patients with NENs.

**Keywords** Terbium-161 · Auger electrons · NEN · SSTR antagonists · PRRT · Radionuclide therapy

This article is part of the Topical Collection on Radiopharmacy

<figure><img src="image_2.png" /></figure>

<footnote>Cristina Müller
cristina.mueller@psi.ch</footnote>

<footnote n="1">¹ Center for Radiopharmaceutical Sciences, ETH-PSI-USZ, Paul Scherrer Institute, 5232 Villigen-PSI, Switzerland</footnote>

<footnote n="2">² Laboratory for Animal Model Pathology, Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, 8057 Zurich, Switzerland</footnote>

<footnote n="3">³ The Joint Department of Medical Imaging, University Health Network, 200 Elizabeth St, Toronto, ON M5G 2C4, Canada</footnote>

<footnote n="4">⁴ South African Nuclear Energy Corporation (Necsa), Pelindaba, Brits 0240, South Africa</footnote>

<footnote n="5">⁵ Institut Laue-Langevin, 38042 Grenoble, France</footnote>

<footnote n="6">⁶ Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland</footnote>

<footnote n="7">⁷ Laboratory of Radiochemistry, Paul Scherrer Institute, 5232 Villigen-PSI, Switzerland</footnote>
Introduction

Neuroendocrine neoplasms (NENs) are clinically hetero-
geneous malignancies, which originate in the neuroen-
docrine system mostly in the gastro-pancreatic or bron-
chopulmonary tract [1]. Peptide receptor radionuclide
therapy (PRRT) using radiolabeled somatostatin (SST)
analogues has been employed since the early 1990s to
treat somatostatin receptor (SSTR)-positive NENs [1,
2]. The initially used [¹¹¹In]In-octreotide, a cell-inter-
nalizing SSTR agonist, was effective for symptom pal-
liation, but the short tissue penetration of the emitted
Auger electrons ( < 10 µm) was not sufficient for an effec-
tive cancer therapy [3–5]. The application of yttrium-90
was introduced in the late 1990s using the next-genera-
tion SSTR agonists, DOTATOC and DOTATATE [6, 7].
Yttrium-90 was more successfully used for PRRT; how-
ever, the high energy β⁻-particles (Eβ<sub>average</sub> = 932 keV;
max. tissue range: ~ 10 mm) were unfavorable due to the
risk of renal damage [8]. The β⁻-particle-emitting lute-
tium-177 (Eβ<sub>average</sub> = 134 keV, T<sub>1/2</sub> = 6.65 days; max. tis-
sue range: ~ 2 mm [9]) has a more favorable safety profile
[8] and is currently the most often employed radiometal
for PRRT using DOTATATE (Lutathera™) or DOTA-
TOC [10, 11]. Additionally, the emission of γ-rays makes
lutetium-177 useful for γ-scintigraphy and single photon
emission computed tomography (SPECT) enabling therapy
monitoring and dosimetry [12].

More recently, the challenge of tumor radioresistance
triggered the concept of targeted α-therapy, which is par-
ticularly effective due to the high linear energy transfer
(LET) of α-particles (50–230 keV/µm) [13–15]. PRRT
using [²¹³Bi]Bi-DOTATOC or [²²⁵Ac]Ac-DOTATATE
in patients, refractory to ⁹⁰Y- and/or ¹⁷⁷Lu-based PRRT,
resulted in partial remission or stable disease [16, 17].
There are, however, a number of uncertainties regarding
the application of α-emitters, among those, the unfavora-
bly short half-life of bismuth-213 and the complicated
decay scheme of actinium-225, which comprises a con-
siderable risk of undesired side effects. The inability of
imaging these α-emitters and, finally, the difficult produc-
tion scenarios make their application challenging [18–20].

In 2006, Ginj et al. introduced the concept of SSTR
antagonists for targeting NENs [21]. Preclinical studies
demonstrated much higher tumor accumulation of these
non-internalizing SST analogues than for SSTR agonists.
In a first-in-human clinical application with four patients
with metastatic NENs, the antagonist [¹⁷⁷Lu]Lu-DOTA-
JR11 performed better than [¹⁷⁷Lu]Lu-DOTATATE
[22], which led to a Phase 1/2 multicenter clinical study
(NCT02592707). More recently, [¹⁷⁷Lu]Lu-DOTA-LM3,
another SSTR antagonist [23], was tested in 51 patients

with metastatic NENs [24]. It resulted in higher tumor
doses compared to [¹⁷⁷Lu]Lu-DOTATOC and was well
tolerated in patients [24]. Unfortunately, this promis-
ing new class of SSTR antagonists cannot be combined
with α-particle emitters with complex decay chain, such
as actinium-225, for which effective internalization is an
essential prerequisite to avoid toxicity to healthy tissues as
a result of released daughter nuclides [18].

Over the last decade, terbium-161 has gained increas-
ing attention as a potential therapy radionuclide [25]. Simi-
lar to lutetium-177, it decays by the emission of medium-
energy β⁻-particles (Eβ<sub>average</sub> = 154 keV; T<sub>1/2</sub> = 6.95 days
[26]) and emits γ-radiation suitable for imaging purposes
(Eγ = 48.9 keV, I = 17% and 74.6 keV, I = 10%) [27, 28].
Most importantly, terbium-161 co-emits a substantial num-
ber of short-ranged electrons (conversion and Auger elec-
trons) [25, 29], thought to be effective for the treatment of
single cancer cells due to their high LET (4–26 keV/µm)
[30–32]. Several preclinical studies using folate conjugates
and ligands targeting the prostate-specific membrane anti-
gen (PSMA) demonstrated the superior therapeutic effect of
terbium-161 over lutetium-177 and the absence of additional
side effects [33–35].

A central question in the context of using terbium-161
refers to the type of targeting agent which would ensure the
greatest benefit from the effect of the co-emitted conversion
and Auger electrons. It is commonly believed that nuclear
localization of a peptide would be essential to induce DNA
double-strand breaks (DSBs) by short-ranged electrons [36,
37]. It was, however, also shown that the cell membrane
may be a suitable target for Auger electron emitters [38].
We believe that the non-internalizing SSTR antagonists
may, thus, benefit from the co-emitted Auger electrons of
terbium-161, which could eventually lead to a paradigm shift
in PRRT.

The aim of this study was, therefore, to investigate the
impact of the localization of ¹⁶¹Tb-based SST analogues to
benefit from the short-ranged electrons in PRRT of NENs.
The therapeutic efficacy of three ¹⁶¹Tb-labeled SST ana-
logues supposed to localize (i) in the cytoplasm (DOTA-
TOC, SSTR agonist [39]), (ii) in the cytoplasm and cell
nucleus (DOTATOC-NLS [40, 41]) or at the cell membrane
(DOTA-LM3, SSTR antagonist [23]) was compared with
those of their ¹⁷⁷Lu-labeled counterparts.

**Materials and methods**

Radiopeptide preparation

No-carrier-added (n.c.a.) terbium-161 was produced at Paul
Scherrer Institute, Switzerland, as previously reported [42]
and n.c.a. lutetium-177 was obtained from ITM Medical

<figure><img src="image_3.png" /></figure>
Isotopes GmbH, Germany. Radiolabeling and quality con-
trol of the SST analogues were performed as previously
reported [43]. [¹⁶¹Tb]Tb-DOTATOC and [¹⁷⁷Lu]Lu-DOTA-
TOC, [¹⁶¹Tb]Tb-DOTATOC-NLS, and [¹⁷⁷Lu]Lu-DOTA-
TOC-NLS as well as [¹⁶¹Tb]Tb-DOTA-LM3 and [¹⁷⁷Lu]
Lu-DOTA-LM3 were obtained at radiochemical purity
of ≥ 98% (up to 100 MBq/nmol) (Supplementary Material,
Fig. S1/S2).

Study design

Initially, the tumor uptake, internalization, and subcellular localization of the three SST analogues were assessed in SSTR-positive cancer cells. Afterwards, the therapeutic effect of the ¹⁷⁷Lu- and ¹⁶¹Tb-labeled somatostatin analogues was investigated in vitro using cell viability and survival assays. In vivo, the biodistribution of the radiopeptides was determined under variable conditions, after which the most promising setting was used for an in vivo comparison of the therapeutic effects of the ¹⁷⁷Lu- and ¹⁶¹Tb-labeled SST analogues.

Since it was previously confirmed that the SST analogues
have equal in vitro and in vivo behavior regarding (sub)cel-
lular uptake and tissue distribution, irrespective of whether
they were labeled with lutetium-177 or terbium-161 [43],
those experiments were performed with only one version of
the radiolabeled peptides.

AR42J tumor cell uptake and internalization

AR42J tumor cells, a SSTR-positive exocrine rat pancre-
atic cancer cell line [44] (ECACC 93,100,618, Health Pro-
tection Agency Culture Collections, Salisbury, UK), were
cultured as previously reported (Supplementary Material)
[43]. Cell uptake and internalization studies were performed
to determine the fractions of radiopeptides localized at the
membrane or in the cytoplasm, respectively. AR42J tumor
cells were grown as monolayers in 12-well plates overnight,
followed by incubation of the cells with each of the ¹⁷⁷Lu-
labeled radiopeptides (~ 15 kBq, ~ 0.375 pmol per well) for
2 h. The total uptake and internalization were determined
after rinsing the cells with phosphate-buffered saline (PBS)
and acidic glycine buffer, respectively. The uptake after
SSTR saturation was evaluated after incubating the cells
with increasing molar amounts of the SST analogues (Sup-
plementary Material).

Determination of the cellular localization
of the radiopeptides

The nuclear accumulation of activity was determined after
a 2 h-incubation period of the AR42J tumor cells with the
respective radiopeptide using a nuclei isolation kit (Nuclei

EZ Prep Kit, Sigma-Aldrich, St. Louis, U.S.) (Supplemen-
tary Material). The nuclei isolation assay was performed in
triplicate with each ¹⁷⁷Lu-labeled radiopeptide. The nuclear
fraction of the respective radiopeptide was expressed as per-
centage of total cell uptake determined as described above.

Cell viability and survival after treatment

The viability of AR42J tumor cells after incubation with ¹⁶¹Tb- or ¹⁷⁷Lu-labeled DOTATOC, DOTATOC-NLS or DOTA-LM3 (0.001–40 MBq/mL, 0.01–400 pmol/mL) was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [33, 45]. The cell viability of treated cells was presented as average ± standard deviation (SD) of 4–5 independent experiments and expressed as percentage of sham-treated cells (set as 100%). The data were plotted against the applied activity concentration in logarithmic scale and fitted with a dose-response curve to determine the activity concentration necessary to reduce AR42J tumor cell viability to 50% of untreated control cells (EC₅₀). The survival of the treated AR42J tumor cells was investigated using a clonogenic assay [46]. AR42J tumor cells were seeded as single cells in 6-well plates (2000 cells per well) and exposed to variable activity concentrations (0.01–5 MBq/mL, 0.33–170 pmol/mL) of ¹⁶¹Tb- and ¹⁷⁷Lu-labeled SST analogues. Two hours later, the activity was removed and the cells were rinsed and let to grow into colonies over 2 weeks. The colonies were colored with crystal violet and counted as previously described (Supplementary Material) [46]. The survival of treated cells was expressed as average ± SD of 3 independent experiments performed in triplicates and expressed as percentage of sham-treated cells (set as 100%). The survival data were analyzed with a two-way ANOVA with Sidak's multiple comparisons post-test.

DNA damage evaluation

The number of DNA DSBs induced in AR42J tumor cells
after exposure to the radiopeptides was assessed by immu-
nostaining of γH2AX. The cells were treated in Petri dishes
for 2 h using 2.5 MBq/mL or 10 MBq/mL of each radio-
peptide followed by incubation with fresh culture medium
for additional 24 h. Cell pellets obtained after centrifuga-
tion were fixed and embedded in paraffin and cut into sec-
tions. The immunostaining was performed using a phospho-
histone H2A.X (Ser139) rabbit monoclonal antibody (Cell
Signaling Techonology, Danvers, Massachusetts, USA) and
an anti-rabbit, horseradish peroxidase-derivatized second-
ary antibody with DAB substrate buffer (Agilent Tech-
nologies, Santa Clara, California, USA) (Supplementary
Material). The sections were scanned using a digital slide
scanner (NanoZoomer-XR C12000; Hamamatsu, Japan)
and the positively and negatively stained AR42J tumor cells were quantified with the pathology image analysis software VIS (Visiopharm Integrator System, Version 208 2019.02.2.6239, Visiopharm, Hoersholm, Denmark) (Supplementary Material). The percentage of γH2AX-positive cells in sham-treated samples was in average 1%. Data were analyzed with a one-way ANOVA with Dunnet's multiple comparisons post-test.

## In vivo studies

The animal experiments were carried out according to the guidelines of Swiss Regulations for Animal Welfare, ethically approved by the Cantonal Committee of Animal Experimentation and permitted by the responsible cantonal authorities (license N° 75721 and 79692). Five-week-old female, athymic nude mice (CD-1 Foxn-1/nu) were obtained from Charles River Laboratories (Sulzfeld, Germany). Mice were subcutaneously inoculated with AR42J tumor cells ($5 \times 10^6$ cells in 100 µL PBS) for SPECT/CT imaging, biodistribution, and therapy studies.

## SPECT/CT imaging and biodistribution studies

The studies were performed 10–14 days after tumor cell inoculation when the tumor size reached a volume of ~250 mm³. $[^{161}Tb]Tb-DOTATOC$, $[^{161}Tb]Tb-DOTATOC-NLS$, or $[^{161}Tb]Tb-DOTA-LM3$ (15 MBq, 1 nmol) was injected to acquire scans under isoflurane/oxygen anesthesia using a dedicated small-animal SPECT/CT scanner (NanoSPECT/CT, Mediso Medical Imaging Systems, Budapest, Hungary) as previously reported (Supplementary Material) [43]. In a first series of biodistribution studies, it was subsequently assessed that a molar peptide amount of 0.2 nmol per mouse was the optimum to achieve high tumor-to-background ratios (Supplementary Material). The time-dependent biodistribution studies were, therefore, performed with mice ($n=3$ per group) intravenously injected with 0.2 nmol of the

radiolabeled DOTATOC or DOTA-LM3 (5 MBq, in 100 µL PBS containing 0.05% bovine serum albumin (BSA)), respectively. Selected tissues and organs were collected, weighed, and counted for activity using a γ-counter (Perkin Elmer). The decay-corrected data were listed as a percentage of the injected activity per gram of tissue mass (% IA/g). The data were analyzed for significance using a two-way ANOVA with Tukey's multiple comparisons post-test.

## Therapy study

The therapy study was initiated with mice randomly assigned to five groups ($n=6$) when the AR42J tumors reached an average volume of $99 \pm 16$ mm³. At day 0 and day 7 of the study, the mice were intravenously injected with vehicle only (Group A: PBS with 0.05% BSA; sham-treatment), $[^{161}Tb]Tb-DOTATOC$ (Group B: 10 MBq, 0.2 nmol), $[^{177}Lu]Lu-DOTATOC$ (Group C: 10 MBq, 0.2 nmol), $[^{161}Tb]Tb-DOTA-LM3$ (Group D: 10 MBq, 0.2 nmol), and $[^{177}Lu]Lu-DOTA-LM3$ (Group E: 10 MBq, 0.2 nmol) (Table 1). The relative body weight (RBW) and the relative tumor volume (RTV) were defined based on the values at therapy start as previously described (Supplementary Material) [47]. The endpoint criteria were defined according to a scoring system which required euthanasia of mice with a score $\ge 3$ (Supplementary Material).

## Assessment of the therapeutic efficacy

The efficacy of the treatment was assessed by comparison of the RTVs, measured every second day, of mice in each group using a two-way ANOVA with Sidak's multiple comparisons post-test. The average tumor growth delay, herein defined as the time during which the tumors did not grow or even decreased in size, was determined for mice of each group. For the subsequent phase, in which the tumors started to regrow, the doubling time of the tumor volume was calculated based on the fitted exponential tumor growth curve.

<table><caption>**Table 1** Design of the therapy study including the average tumor volumes and body weights of mice at therapy start. The mice were injected at day 0 and day 7 with the radiopeptide at 0.2 nmol per mouse ($n=6$)</caption><thead><tr><th rowspan="2">Group</th><th rowspan="2">Treatment</th><th>Injected activity</th><th>Tumor volume<sup>b</sup></th><th>Body weight<sup>c</sup></th></tr><tr><th>day 0 and day 7</th><th>(mm<sup>3</sup>)<br>(average ± SD)</th><th>(g)<br>(average ± SD)</th></tr><tr><th></th><th></th><th>0.2 nmol/mouse</th><th>Day 0</th><th>Day 0</th></tr></thead><tbody><tr><td>A</td><td>Vehicle<sup>a</sup></td><td>-</td><td>118 ± 90</td><td>23 ± 2</td></tr><tr><td>B</td><td><sup>[161]Tb]Tb-DOTATOC</sup></td><td>2 × 10 MBq</td><td>92 ± 48</td><td>24 ± 2</td></tr><tr><td>C</td><td><sup>[177]Lu]Lu-DOTATOC</sup></td><td>2 × 10 MBq</td><td>102 ± 56</td><td>24 ± 2</td></tr><tr><td>D</td><td><sup>[161]Tb]Tb-DOTA-LM3</sup></td><td>2 × 10 MBq</td><td>76 ± 32</td><td>23 ± 1</td></tr><tr><td>E</td><td><sup>[177]Lu]Lu-DOTA-LM3</sup></td><td>2 × 10 MBq</td><td>109 ± 72</td><td>24 ± 2</td></tr></tbody></table>

<footnote><sup>a</sup>Vehicle: 0.05% BSA in PBS; <sup>b</sup>no significant differences determined between the tumor volumes measured for each group ($p>0.05$); <sup>c</sup>no significant differences determined between the body weights measured for each group ($p>0.05$)</footnote>
The average ± SD of tumor growth delay and of the doubling time of the tumor volume in single mice, respectively, were compared among groups with a one-way ANOVA with Tukey's multiple comparisons post-test. The survival times of mice were presented by Kaplan–Meier curves and analyzed using a log-rank test (Mantel-Cox).

## Assessment of early side effects

Early side effects were assessed based on the RBW of the mice. When an endpoint was reached or at the end of the study (at day 49), blood plasma parameters (albumin (ALB), creatinine (CRE), blood urea nitrogen (BUN), alkaline phosphatase (ALP), and total bilirubin (TBIL)) were determined (Supplementary Material). At the same time, relevant organs and tissues were collected, and the masses were put into relation to the brain mass of the respective mouse in order to allow the comparison of organ-to-brain and organ-to-body weight ratios among the single treatment groups (Supplementary Material). Data were analyzed for significance using a one-way ANOVA test with a Tukey's multiple comparisons post-test.

## Statistical analysis and graphs preparation

The GraphPad Prism software (version 8) was used for preparation of the graphs, for the analysis of the data and to

perform statistical analysis. A *p* value < 0.05 was considered statistically significant. SPECT/CT images were prepared using the software CorelDRAW (version X7).

## Results

### Tumor cell uptake and localization

DOTA-LM3, irrespective of whether it was labeled with terbium-161 or lutetium-177 [43], showed the highest AR42J tumor cell uptake in vitro with ~70% of total added activity after a 2-h incubation period. This was 4- to sixfold higher than the uptake of radiolabeled DOTATOC (~10%) and DOTATOC-NLS (~15%), respectively (Fig. 1a–c); (Supplementary Material, Fig. S3).

About 9% of the cellular uptake of radiolabeled DOTA-LM3 was internalized whereas in the case of DOTATOC and DOTATOC-NLS, the internalized fraction was much higher (~81% and ~84%, respectively) (Fig. 1e–g). The fraction of accumulated radiopeptide in the cellular nucleus was ~6% of the total uptake in the case of radiolabeled DOTATOC-NLS but <2% for radiolabeled DOTATOC and DOTA-LM3 (Fig. 1e–g).

<figure><img src="image_4.png" /><figcaption>**Fig. 1** Graphs representing cell uptake and localization of *a/e* radiolabeled DOTATOC; *b/f* radiolabeled DOTATOC-NLS, and *c/g* radiolabeled DOTA-LM3</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2** Graphs representing AR42J tumor cell viability and survival after treatment. **a/b/c** Results of the cell viability assessment (MTT assay); **d/e/f** results of the cell survival assessment (clonogenic assay)</figcaption></figure>

**In vitro tumor cell viability**

It was observed that, in all cases, the ¹⁶¹Tb-labeled SST analogue was more potent in reducing AR42J tumor cell viability than the ¹⁷⁷Lu-labeled analogue (Fig. 2a–c, Table S1, Supplementary Material). [¹⁶¹Tb]Tb-DOTATOC and [¹⁶¹Tb]Tb-DOTATOC-NLS were ~5- and ~fourfold more potent than [¹⁷⁷Lu]Lu-DOTATOC and [¹⁷⁷Lu]Lu-DOTATOC-NLS, respectively, but [¹⁶¹Tb]Tb-DOTA-LM3 was 102-fold more potent than [¹⁷⁷Lu]Lu-DOTA-LM3.

Importantly, [¹⁶¹Tb]Tb-DOTA-LM3, the most potent radiopeptide, revealed an EC₅₀ of 0.010 MBq/mL (CI 0.008–0.014), which was 820-fold more potent than the clinically used [¹⁷⁷Lu]Lu-DOTATOC (EC₅₀ of 8.2 MBq/mL, CI 6.4–10).

**In vitro tumor cell survival**

Colony-forming assays confirmed that the ¹⁶¹Tb-labeled peptides reduced AR42J tumor cell survival more effectively than the respective ¹⁷⁷Lu-labeled analogues (Fig. 2d/e/f). Compared to untreated control cells, less than 3% of cells treated with 0.50 MBq/mL [¹⁶¹Tb]Tb-DOTATOC survived. To reach a similar effect with [¹⁷⁷Lu]Lu-DOTATOC, a tenfold higher activity concentration had to be applied (data not shown). [¹⁶¹Tb]Tb-DOTATOC-NLS and [¹⁶¹Tb]Tb-DOTA-LM3 reduced the cell survival to <5% at an activity concentration of 0.1 MBq/mL, while [¹⁷⁷Lu]Lu-DOTATOC-NLS and [¹⁷⁷Lu]Lu-DOTA-LM3 applied at a tenfold higher concentration reduced the survival to only ~13% and ~15%, respectively.
<figure><img src="image_6.png" /><figcaption>**Fig. 3** DSBs quantification (γH2AX) in AR42J cells. **a** Representative γH2AX-staining in sham-treated cells or cells treated with the respective radiopeptide (scale bar: 50 µm). Quantification of γH2AX staining in **b** sham-treated cells; or cells treated with **c** [¹⁶¹Tb]Tb-/</figcaption></figure>

[¹⁷⁷Lu]Lu-DOTATOC; **d** [¹⁶¹Tb]Tb-/[¹⁷⁷Lu]Lu-DOTATOC-NLS or **e** [¹⁶¹Tb]Tb-/[¹⁷⁷Lu]Lu-DOTA-LM3. The number of positive cells is expressed in percent relative to the 1% positive cases detected in average in sham-treated cells

## DNA damage determined by the number of induced γH2AX foci

[¹⁶¹Tb]Tb-DOTATOC showed a tendency of inducing a higher number of DNA DSBs compared to [¹⁷⁷Lu]Lu-DOTATOC (*p* > 0.05) (Fig. 3). Exposure of tumor cells to [¹⁶¹Tb]Tb-DOTATOC-NLS increased the number of γH2AX foci more than [¹⁷⁷Lu]Lu-DOTATOC-NLS (> 11% γH2AX-positive cells vs ~5% at an activity concentration of 10 MBq/mL, *p* > 0.05). When applied at 2.5 MBq/mL, [¹⁶¹Tb]Tb-DOTA-LM3 had a similar effect as [¹⁷⁷Lu]Lu-DOTA-LM3 (*p* > 0.05), but at 10 MBq/mL, it induced more DNA DSBs than [¹⁷⁷Lu]Lu-DOTA-LM3 (~8% γH2AX-positive cells vs ~3%, *p* > 0.05).

## SPECT/CT imaging studies

As previously reported [43], [¹⁶¹Tb]Tb-DOTATOC accumulated in the tumors, but retention in the kidneys was also visible on the 2 h p.i.-SPECT/CT scan (Fig. 4a). At

the same timepoint after injection of [¹⁶¹Tb]Tb-DOTATOC-NLS, the tumor uptake was slightly increased, but retention in the liver and kidneys was exceedingly high. As a result, this radiopeptide would not be applicable for therapeutic purposes (Fig. 4b) (Supplementary Material, Fig. S4). [¹⁶¹Tb]Tb-DOTA-LM3 showed the most favorable tissue distribution, with an increased tumor uptake, resulting in the highest tumor-to-kidney ratios (Fig. 4c) [43]. The tumor uptake was SSTR-specific for all radiopeptides, as previously reported (Supplementary Material) [43].

## Biodistribution studies

Time-dependent biodistribution studies were performed with radiolabeled DOTATOC and DOTA-LM3 using the peptide amount (0.2 nmol per mouse) that was evaluated in this work as most favorable to achieve high tumor-to-background ratios (Supplementary Material, Fig. S5, Tables S2/S3). Due to the unfavorable in vivo distribution of [¹⁶¹Tb]Tb-DOTATOC-NLS with high uptake in
**Fig. 4** SPECT/CT images of AR42J tumor-bearing mice shown as maximum intensity projections 2 h after injection of the radiopeptides (15 MBq, 1.0 nmol per mouse). Mice injected with a [¹⁶¹Tb]Tb-DOTATOC [43]; b [¹⁶¹Tb]Tb-DOTATOC-NLS (b), and c [¹⁶¹Tb]Tb-DOTA-LM3 [43]. AR42J = SSTR-positive xeno-graft; Ki = kidneys; Li = liver; Bl = urinary bladder

<figure><img src="image_7.png" /></figure>

liver and kidneys, as demonstrated by SPECT/CT imaging, DOTATOC-NLS was excluded from further in vivo studies. [¹⁶¹Tb]Tb-DOTATOC reached the highest tumor uptake early after injection (15 ± 1% IA/g; 0.5 h p.i.), which was retained over the following 4 h, but dropped afterward to 6.3 ± 0.6% IA/g (24 h p.i.) and 3.7 ± 0.7% IA/g (48 h p.i.). Significant activity accumulation was also observed in the lungs, stomach, and pancreas shortly after injection; however, it was effectively cleared over time (≤ 1%IA/g; 4 h p.i.). Renal clearance was slower (~ 10% IA/g; 4 h p.i. and ~ 4–5% IA/g at 24 h p.i.) (Fig. 5a; Supplementary Material Table S4).

The tumor uptake and retention of [¹⁶¹Tb]Tb-DOTA-LM3 was significantly higher (35 ± 7% IA/g; 4 h p.i.; 21 ± 4%IA/g at 48 h p.i.) than for [¹⁶¹Tb]Tb-DOTATOC at all investigated timepoints (*p* < 0.05). In the lungs, stomach, and pancreas, activity was detected during the first 4 h but effectively cleared over the hours that followed. The accumulated activity of [¹⁶¹Tb]Tb-DOTA-LM3 in the pancreas and stomach was significantly higher than in the case of [¹⁶¹Tb]Tb-DOTATOC (*p* < 0.05) at all investigated timepoints, whereas renal uptake was similar as observed for [¹⁶¹Tb]Tb-DOTATOC (*p* > 0.05) (Fig. 5b and Supplementary Material Table S5). As previously demonstrated [43], the in vivo distribution of ¹⁶¹Tb- and ¹⁷⁷Lu-labeled SST-analogues is equal; hence, the results hold true irrespective of which radionuclide was employed.

## Therapy study in AR42J tumor-bearing mice

Sham-treated mice of Group A showed an exponential tumor growth so that the endpoint was reached within the first 14 days in all cases. The tumor growth was delayed in treated mice of Groups B–E, resulting in significantly prolonged median survival times compared to the 9 days in the control group (Fig. 6, Table 2). Mice treated with [¹⁶¹Tb]Tb-DOTATOC (Group B) showed a slightly slower tumor growth compared to mice treated with [¹⁷⁷Lu]Lu-DOTATOC (Group C). After 12 days from the therapy start, the RTV of mice of these two groups were significantly different (2.0 ± 0.7 vs 4.0 ± 3.3, *p* < 0.05). The tumor growth delay and doubling time were higher in Group B (9.0 ± 5.5 days and 3.4 ± 3.6 days, respectively) compared to those of Group C (6.0 ± 4.4 days and 3.4 ± 0.8 days, respectively, *p* > 0.05). Mice of Group B were, thus, euthanized at a later stage (day 20–26) compared to Group C (day 12–22). The median survival (21 vs 19.5 days) was, however, comparable between the groups (*p* > 0.05).

The tumor growth delay for mice treated with [¹⁶¹Tb]Tb-DOTA-LM3 was 44 ± 5 days, but only 35 ± 7 days in mice treated with [¹⁷⁷Lu]Lu-DOTA-LM3 (*p* < 0.05). Afterwards, the tumors started to regrow exponentially in 5 out of 6 mice of both groups; however, the doubling time was considerably longer for mice treated with
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 5** Biodistribution data obtained in AR42J tumor-bearing mice. Results after injection of **a** [¹⁶¹Tb]Tb-DOTATOC (0.2 nmol/mouse) and **b** [¹⁶¹Tb]Tb-DOTA-LM3 (0.2 nmol/mouse) shown in percentage of injected activity per tissue mass (% IA/g) and presented as the average ± SD of n = 3</figcaption></figure>

[¹⁶¹Tb]Tb-DOTA-LM3 compared to the tumor growth in mice treated with [¹⁷⁷Lu]Lu-DOTA-LM3 (7.4 ± 4.6 days vs 3.8 ± 1.1 days, *p* > 0.05). All mice treated with [¹⁶¹Tb] Tb-DOTA-LM3 survived until the end of the study, while this was the case only for three out of six mice treated with [¹⁷⁷Lu]Lu-DOTA-LM3.

## Assessment of potential early side effects

No signs of early side effects were detected in any of the treated groups (Fig. 7, Supplementary Material, Tables S6–S9). The mice gained weight over the course of the study and the body weights did not differ between the groups at any timepoint (*p* > 0.05) (Fig. 7a/b). ALB plasma levels, an indicator of general health status of mice, were comparable among the groups (Fig. 7c). No signs of kidney

toxicity were observed at the time of euthanasia, as kidney-to-brain mass ratios and plasma CRE levels showed no significant difference among the groups with the exception of one outlier (Fig. 7d/e). The BUN levels were elevated in mice treated with [¹⁶¹Tb]Tb-DOTA-LM3 (9.9 ± 1.5 mmol/L) and [¹⁷⁷Lu]Lu-DOTA-LM3 (8.2 ± 1.9 mmol/L) compared to control mice (6.2 ± 0.7 mmol/L) and mice treated with [¹⁶¹Tb] Tb- or [¹⁷⁷Lu]Lu-DOTATOC, but in the physiological range reported for this particular mouse strain by the breeding company (Charles River, Germany) (Fig. 7f). No liver toxicity was observed based on liver-to-brain mass ratios, plasma ALP, and TBIL which were comparable among all groups (*p* > 0.05) (Fig. 7g/h/i).

## Discussion

Terbium-161 and lutetium-177 share similar physical decay properties in terms of half-life and β⁻-energy; however, the co-emission of short-ranged electrons is a specific characteristic of terbium-161. A direct comparison of the therapeutic effect of terbium-161 and lutetium-177 is feasible due to the similar chemical properties of these radiolanthanides and, thus, equal tumor cell uptake and biodistribution profiles of the investigated pairs of radiopeptides herein [43]. In this study, we investigated the impact of the subcellular localization of SST analogues on the therapeutic efficacy of terbium-161 and lutetium-177, respectively. Both DOTATOC and DOTATOC-NLS internalized to a large degree into the cytoplasm (80% and 84%) whereof, in the case of DOTATOC-NLS, ~15% of the internalized fraction were shuttled to the nucleus. In contrast, the largest fraction of the SSTR antagonist DOTA-LM3 remained at the cell membrane (> 90%).

Indeed, terbium-161 was more effective in reducing AR42J tumor cell viability and survival than lutetium-177, irrespective of the SST analogue employed. This indicated clearly that the conversion and Auger electrons emitted by terbium-161 contributed positively to its therapeutic efficacy. The difference in therapeutic efficacy between ¹⁶¹Tb- and ¹⁷⁷Lu-labeled peptides was, however, not the same among the three types of SST analogues.

In line with the common belief that Auger electron emitters should be shuttled in close proximity to the cell DNA [36, 37], [¹⁶¹Tb]Tb-DOTATOC-NLS, which accumulated to about 6% in the cell nucleus, induced the highest number of DNA DSBs after 24 h incubation. [¹⁶¹Tb] Tb-DOTATOC and [¹⁶¹Tb]Tb-DOTA-LM3 induced the formation of far fewer DSBs at this timepoint. Despite the positive effect of the NLS to induce DSBs, the overall advantage of using [¹⁶¹Tb]Tb-DOTATOC-NLS instead of [¹⁷⁷Lu]Lu-DOTATOC-NLS was similar to the advantage of [¹⁶¹Tb]Tb-DOTATOC over [¹⁷⁷Lu]Lu-DOTATOC in
<caption>**Fig. 6** Results of the therapy study performed with <sup>161</sup>Tb- and <sup>177</sup>Lu-SSTR agonist and antagonist (2×10 MBq; 0.2 nmol) in AR42J tumor-bearing mice.</caption>

a The RTV at day 0 (set as 1) for mice of Group A (sham), B ([<sup>161</sup>Tb]Tb-DOTATOC), C ([<sup>177</sup>Lu]Lu-DOTATOC), D ([<sup>161</sup>Tb]Tb-DOTA-LM3), and E ([<sup>177</sup>Lu]Lu-DOTA-LM3). Data are shown until the first mouse of the respective group reached an endpoint. **b/c/d/e/f** Absolute TV of single mice (grey lines) and average (colored line) of Groups A–E; **g** tumor growth delay of Group A–E; **h** tumor doubling time of Groups A–E; **i** Kaplan–Meier plot of Groups A–E

<figure><img src="image_10.png" /></figure>

both viability and survival assays. These findings, together with the unfavorably high uptake of [<sup>161</sup>Tb]Tb/[<sup>177</sup>Lu]Lu-DOTATOC-NLS in kidneys and liver, clearly indicated

that the functionalization of a SST analogue with a NLS is not a feasible strategy to ideally exploit the therapeutic potential of terbium-161.
<table><caption>**Table 2** Data regarding euthanasia period and median survival</caption><thead><tr><th>Group</th><th>Treatment</th><th>Time frame of euthanasia [day]</th><th>Median survival [day]</th></tr></thead><tbody><tr><td>A</td><td>Vehicle</td><td>6–15</td><td>9</td></tr><tr><td>B</td><td>[<sup>161</sup>Tb]Tb-DOTATOC<sup>a</sup></td><td>20–26</td><td>21</td></tr><tr><td>C</td><td>[<sup>177</sup>Lu]Lu-DOTATOC<sup>a</sup></td><td>12–22</td><td>19.5</td></tr><tr><td>D</td><td>[<sup>161</sup>Tb]Tb-DOTA-LM3<sup>a</sup></td><td>end of study (n = 6)</td><td>&gt; &gt; 49<sup>b</sup></td></tr><tr><td>E</td><td>[<sup>177</sup>Lu]Lu-DOTA-LM3<sup>a</sup></td><td>42–48 (n = 3)<br>end of study (n = 3)</td><td>48.5</td></tr></tbody></table>

<figure><img src="image_11.png" /><figcaption>**Fig. 7** Parameters of potential side effects in therapy mice. **a/b/c** Indicators for the general health status of the mice: RBW during the therapy (a) shown until the first mouse of the group reached an endpoint, RBW at the endpoint (b), and plasma ALB levels (c). **d/e/f** Indicators for kidney toxicity: kidney-to-brain mass ratios (d), plasma CRE</figcaption></figure>

(e), plasma BUN levels (f). **g/h/i** Indicators for liver toxicity: liver-to-brain mass ratios (g), plasma ALP levels (h), and plasma TBIL levels (i). Organ-to-brain mass ratios and plasma parameters refer to mice at the endpoint

<footnote><sup>a</sup>1st injection: 10 MBq, 0.2 nmol at day 0; 2nd injection: 10 MBq, 0.2 nmol at day 7.</footnote>

<footnote><sup>b</sup>End of study at day 49.</footnote>
Interestingly, the non-internalizing [¹⁶¹Tb]Tb-DOTA-LM3 revealed a 102-fold increased efficacy to reduce cell viability in vitro compared to [¹⁷⁷Lu]Lu-DOTA-LM3 (Fig. 2). These findings were in line with a previous observation made with internalizing and non-internalizing antibodies, wherof the latter were more effective in killing tumor cells when labeled with the Auger electron-emitting iodine-125 [38, 48, 49]. Obviously, the cell membrane is a better target to exploit the effect of short-ranged electrons than the cytoplasm. Current endeavors in our laboratories focus, thus, on a further radio-biological investigation of the effect of [¹⁶¹Tb]Tb-DOTA-LM3 to better understand the “membrane effect” of short-ranged electrons.

Preclinical therapy studies with AR42J tumor-bearing mice confirmed the superior efficacy of ¹⁶¹Tb-labeled SST analogues over those labeled with lutetium-177. The obvious positive effect of Auger electrons emitted by terbium-161 was particularly pronounced when using DOTA-LM3. The overall efficacy of DOTA-LM3-based PRRT was much better than the results obtained with DOTATOC, irrespective of which radionuclide was applied. It can be ascribed to the higher tumor accumulation of SSTR antagonists compared to agonists. [¹⁶¹Tb]Tb-DOTA-LM3 induced a tumor growth delay of over 44 days and was, thus, the most powerful candidate, with a significant advantage over the clinically applied [¹⁷⁷Lu] Lu-DOTATOC, which induced a tumor growth delay of only 6±4 days. No significant early side effects nor signs of liver or kidney toxicity were observed in any of the treated mice, yet further investigations are necessary in order to assess potentially negative effects to SSTR-expressing normal tissues or bone marrow toxicity.

Even though AR42J tumor cells show neuroendocrine properties [50] and have, thus, been extensively employed for the evaluation of SST analogues, it can be considered a limitation of this study to have only used rat tumor cells. It will, thus, be crucial to confirm the reported findings using human endocrine pancreatic tumor cells. The investigation of further “agonist/antagonist” pairs that target SSTR or other receptors will provide clarification about the general validity of the proposed advantage of terbium-161 when applied in combination with non-internalizing tumor-targeting agents. Both topics are in the focus of our currently-ongoing research activities.

Our previous work already showed that terbium-161 provides an advantage over the use of lutetium-177 [33–35]. If the concept presented herein will be confirmed in follow-up studies, terbium-161 may be used preferentially with non-internalizing tumor-targeting agents, thereby, complementing the use of α-particle emitters, which should be combined with fast-internalizing molecules.

Finally, our results, together with the recently reported data of the successful application of [¹⁷⁷Lu]Lu-DOTA-LM3 in patients [24, 51], encourage a clinical translation of

[¹⁶¹Tb]Tb-DOTA-LM3 for the treatment of NEN’s. Current efforts at PSI are, therefore, dedicated to the scale-up of the terbium-161 production, which is performed in analogy to the production of n.c.a. lutetium-177 (Supplementary Material, Fig. S6) [25, 42] to enable the preparation of therapeutic quantities of [¹⁶¹Tb]Tb-DOTA-LM3 under good manufacturing practice (GMP) for clinical application.

## Conclusion

This study showed that the cellular localization of ¹⁶¹Tb-labeled SST analogues is relevant to exploit the therapeutic potential of short-ranged electrons. [¹⁶¹Tb]Tb-DOTA-LM3 revealed to be powerful for the treatment of NENs, as it combines the favorable properties of SSTR antagonists with the advantages of terbium-161. Should our results be confirmed with other tumor models and targeting concepts, these results may initiate a paradigm shift towards the application of Auger electron emitters with membrane-targeting biomolecules to enable more effective PRRT in NENs.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s00259-021-05564-0.

**Acknowledgements** The authors thank Dr. Christoph Umbricht, Susan Cohrs, Fan Sozzi-Guo, Raffaella Schmid, and Patrick Barrit for assistance of the preclinical experiments and Dr. Zeynep Talip, Chiara Favaretto, and Colin Hillhouse for assisting the production of terbium-161 at PSI, Switzerland; they also thank Lebogang Sepini for assistance with the target irradiations at Necsa, South Africa.

**Funding** Open Access funding provided by Lib4RI – Library for the Research Institutes within the ETH Domain: Eawag, Empa, PSI & WSL. The project was supported by the NET Research Foundation Petersen Investigator Award 2018 U.S.A. (PI: Roger Schibli). Dr. Francesca Borgna received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 701647 and from the Swiss Cancer Research Foundation, Grant No. KFS-4678-02-2019-R (PI: Cristina Müller). ITM Medical Isotopes GmbH, Germany, provided n.c.a. lutetium-177 for this study.

**Data availability** The raw data presented in this study are available on request from the corresponding author.

**Code availability** Not applicable.

## Declarations

**Ethics approval** In vivo experiments were approved by the local veterinary department and ethics committee and conducted in accordance with the Swiss law of animal protection.

**Conflict of interest** Francesca Borgna, Roger Schibli, Nicholas P. van der Meulen, and Cristina Müller are co-inventors on a patent application filed by the Paul Scherrer Institute and the University Hospital Basel, Switzerland. All other authors declare no competing interests.
**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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