Development of 225Ac- and 213Bi-labelled somatostatin analogues with improved tumour to kidney ratio for the treatment of neuroendocrine tumours

Ahenkorah Stephen


SCK•CEN Mentor

Ooms Maarten
+32 14 33 32 83

SCK•CEN Co-mentor

Derradji Hanane
+32 14 33 21 51

Expert group


PhD started


Short project description

Recently, the Committee for Medicinal Products for Human Use (CHMP) of the European Medicine Agency (EMA) recommended the marketing authorization of the radiopharmaceutical product Lutathera®. The active substance of Lutathera® is 177Lu-DOTATATE (also called 177Lu-DOTA-Tyr3-octreotate, 177Lu-octreotate or 177Lu-oxodotreotide), which is a radiolabelled peptide that targets subtype 2 somatostatin receptors, which are often overexpressed in neuroendocrine tumours. Lutathera® will be used for the improvement of progression-free survival (PFS) of patients with unresectable or metastatic, progressive, well-differentiated (grade 1 and grade 2), somatostatin receptor positive gastro-entero pancreatic neuroendocrine tumours (GEP-NETs). This recommendation is based on data of the randomized NETTER-1 trial (Strosberg et al. 2017), where patients treated with Lutathera® had a marked benefit in PFS compared to controls (PFS at 20 months: 65.2% for Lutathera vs. 10.8% in the control group) and preliminary evidence of overall survival benefit was observed (deaths in Lutathera® group: 14 vs. 26 in control group).

Uptake and retention in kidneys is often a problem for radiolabelled peptides and proteins with a molecular weight less than 60 kDa such as Lutathera® and other radiolabelled somatostatin analogues, which can pass the glomerular membrane. A substantial part of these radiolabelled peptides or proteins may be reabsorbed in the proximal tubule of the kidneys after glomerular filtration. This causes a high radiation burden for the kidneys, hinders diagnostic accuracy and limits therapeutic applications. An interesting approach to reduce radioactivity accumulation in the kidneys is based on the use of cleavable linkers. Suzuki et al. reported recently that the Gly-L-Phe-L-Lys (GFK) sequence is a substrate for renal brush border membrane enzymes that are present on the lumen of the renal tubule. Incorporation of the GFK linker (L-form) between a 55-kDa fragment antigen-binding (Fab) and technetium-99m chelate resulted in significantly lower renal radioactivity levels in comparison with the same construct using the control linker GFK (D-form) (Suzuki et al. 2016). The hypothesis is that the technetium-99m chelate is cleaved off specifically by renal brush border membrane enzymes and is excreted further to urinary bladder while the antibody fragments are taken up by the renal cells. Fujioka et al. (Fujioka et al. 2005) reported an in vitro system to estimate renal brush border enzyme-mediated cleavage of peptide linkages. This in vitro assay allows to evaluate in an efficient way possible cleavable linkers that are substrate for renal brush border membrane enzymes.

Radionuclide based targeted therapy is mainly focused on the use of β--emitters bound to targeting molecules such as small molecules, peptides or monoclonal antibodies. This is due to the availability and the favourable characteristics of many β--emitting nuclides. Other therapeutic particle emissions are Auger electrons, α-particles and conversion electrons. Despite the limited availability of α-particle emitters that are relevant for clinical use, targeted α-radionuclide therapy (TAT) is gaining more and more attention in preclinical and clinical studies (Dekempeneer et al. 2016). Due to the short path length and high linear energy transfer (LET) of α-particles, the DNA damage caused by α-particles is much more difficult to repair than the DNA damage caused by β-particles. Furthermore, because of the limited range of the α-particle, toxicity to the healthy surrounding cells is expected to be quite low. TAT is envisioned as being more potent than targeted radionuclide therapy with β--emitters for the treatment of metastases, in particular small ones, or residual tumours after surgery. On the other hand, β--emitters are considered more suitable for the eradication of larger solid tumours because radiopharmaceuticals are often not homogeneously distributed within the tumour. An additional advantage of TAT is that the cytocidal efficacy is generally considered to be independent of dose fractionation, dose rate, or hypoxia and it can reverse resistance to chemotherapy or conventional external beam radiotherapy (Yong and Brechbiel 2015). It is generally accepted that there is no effective resistance to α-particle lethality.

In this project, a somatostatin receptor agonist based upon an octreotate backbone will be derivatized with the chelator DOTA via a cleavable linker, and labelled with the α-emitters 225Ac (T1/2 = 10 days) and 213Bi (T1/2 = 46 minutes) and the β--emitter 177Lu (T1/2 = 7 days). Somatostatin analogues exhibit a fast tumour targeting and fast renal clearance (Mäcke et al. 1993), which should ideally match with radioisotopes with relatively short physical half-life such as 213Bi. The 177Lu-labelled somatostatin analogue 177Lu-DOTATATE will be used as a benchmark for comparison.

225Ac can be obtained from the decay of 229Th (T1/2 = 7880 years). SCK•CEN obtains a limited quantity of 229Th originating from the historical Actinium Programme. 213Bi can be obtained from an 225Ac/213Bi generator made with 225Ac originating from 229Th. 177Lu will be obtained from a commercial supplier.

This research project will be conducted by the Radiochemistry (RCA) and Radiobiology (RDB) groups of the Belgian Nuclear Research Centre (SCK•CEN) and the Laboratory of Radiopharmaceutical research (Prof. Guy Bormans; Dr. Frederik Cleeren) and the Nuclear Medicine & Molecular Imaging group (Prof. Christophe Deroose), both of the University of Leuven (KU Leuven).



Biber, Jurg, Bruno Stieger, Gerti Stange, and Heini Murer. 2007. 'Isolation of renal proximal tubular brush-border membranes', Nat. Protocols, 2: 1356-59.

Dekempeneer, Yana, Marleen Keyaerts, Ahmet Krasniqi, Janik Puttemans, Serge Muyldermans, Tony Lahoutte, Matthias D’huyvetter, and Nick Devoogdt. 2016. 'Targeted alpha therapy using short-lived alpha-particles and the promise of nanobodies as targeting vehicle', Expert Opinion on Biological Therapy, 16: 1035-47.

Fujioka, Y., S. Satake, T. Uehara, T. Mukai, H. Akizawa, K. Ogawa, H. Saji, K. Endo, and Y. Arano. 2005. 'In vitro system to estimate renal brush border enzyme-mediated cleavage of peptide linkages for designing radiolabelled antibody fragments of low renal radioactivity levels', Bioconjugate Chemistry, 16: 1610-16.

Mäcke, H. R., P. Smith-Jones, T. Maina, B. Stolz, R. Albert, C. Bruns, and H. Reist. 1993. 'New octreotide derivatives for in vivo targeting of somatostatin receptor-positive tumors for single photon emission computed tomography (SPECT) and positron emission tomography (PET)', Hormone and Metabolic Research. Supplement, 27: 12-17.

Strosberg, Jonathan, Ghassan El-Haddad, Edward Wolin, Andrew Hendifar, James Yao, Beth Chasen, Erik Mittra, Pamela L. Kunz, Matthew H. Kulke, Heather Jacene, David Bushnell, Thomas M. O’Dorisio, Richard P. Baum, Harshad R. Kulkarni, Martyn Caplin, Rachida Lebtahi, Timothy Hobday, Ebrahim Delpassand, Eric Van Cutsem, Al Benson, Rajaventhan Srirajaskanthan, Marianne Pavel, Jaime Mora, Jordan Berlin, Enrique Grande, Nicholas Reed, Ettore Seregni, Kjell Öberg, Maribel Lopera Sierra, Paola Santoro, Thomas Thevenet, Jack L. Erion, Philippe Ruszniewski, Dik Kwekkeboom, and Eric Krenning. 2017. 'Phase 3 Trial of 177Lu-Dotatate for Midgut Neuroendocrine Tumors', New England Journal of Medicine, 376: 125-35.

Suzuki, C., N. Kanazawa, S. Wada, H. Suzuki, T. Uehara, and Y. Arano. 2016. "A novel chelating agent with an enzyme-cleavable linkage for reducing renal radioactivity levels of Tc-labelled antibody fragments." In SNMMI, 188-89. San Diego.

Yong, Kwon, and Martin Brechbiel. 2015. 'Application of (212)Pb for Targeted α-particle Therapy (TAT): Pre-clinical and Mechanistic Understanding through to Clinical Translation', AIMS medical science, 2: 228-45.


This doctoral research project has the overall aim to improve radionuclide therapy for the treatment of neuroendocrine tumours by using 225Ac- or 213Bi-labelled somatostatin analogues with improved tumour to kidney ratio. At the end of this project, we intend to have preclinically validated 225Ac- or 213Bi-labelled somatostatin analogues for the treatment of neuroendocrine tumours. The 177Lu-labelled somatostatin analogue will be used as a benchmark for comparison.


Project work packages (WPs) include:


  1. Definition and optimization of radiochemical procedures for the radiolabelling of the somatostatin analogue by using a linker with sensitive chemical bonds between the DOTA chelator and the octreotide backbone.
  2. Assessment of the radiochemical stability of 177Lu-, 213Bi- and 225Ac-labelled somatostatin analogues in storage and physiological conditions, including stability in rodent and human plasma.
  3. In vitro studies on tumour cell lines, with a clear view on specificity of binding, affinity, and degree of internalisation.
  4. In vivo studies in normal mice and different human tumour xenografted mouse models for neuroendocrine tumours to assess biodistribution (especially the tumour to kidney ratio), tissue penetration and tumour targeting. In vivo toxicity studies (histopathology and analysis of functional parameters in serum or urine) based on the outcome of biodistribution studies with special emphasis on the renal toxicity.
  5. Comparison of the therapeutic efficacy of 177Lu-, 213Bi- and 225Ac-labelled somatostatin analogues in relevant preclinical cancer mouse models. 177Lu-DOTATATE serves as a benchmark.


1.Radiolabelling of the somatostatin analogue

The radiolabelling of the somatostatin analogue with 177Lu, 213Bi and 225Ac will be performed using the DOTA-chelator, which has a double functionality so that it can bind to the radionuclide on one side and to the somatostatin analogue on the other side. In order to allow a fast screening of new peptide-cleavable linker-DOTA constructs, these constructs will be first labelled with 68Ga. This allows the selection of the most promising peptide-cleavable linker-DOTA constructs using the validated in vitro assay reported by Fujioka et al. (Fujioka et al. 2005) – see below. Readouts of the radiochemical procedures will be yield and purity after radiolabelling.

Peptides based upon the somatostatin receptor agonist octreotide, containing potentially cleavable linkers (e.g. GFK) between the DOTA-chelator and the octreotide backbone, will be synthesized using standard solid phase peptide synthesis. As a control, the linker GFK (D-form) will be used. In addition, other potentially cleavable linkers (e.g. GYK) will be considered.

2. In vitro and in vivo evaluation of the radiolabelled somatostatin analogue

The characteristics of the labelled somatostatin analogues will be assessed in vitro and in vivo. The first in vitro tests will be performed with 68Ga-labelled peptide-cleavable linker-DOTA constructs in order to compare several constructs and select the most promising ones for further investigation. The in vitro assay reported by Fujioka et al. (Fujioka et al. 2005) using brush border membrane vesicles (BBMVs) isolated from rat kidney cortex will be implemented. However, because in vivo studies described in later WPs will be performed in mice, this assay will be adapted by using mouse kidney cortex. The renal proximal tubular brush-border membranes will be isolated according to the method reported by Biber J. et al. (Biber et al. 2007). The γ-glutamyl-transferase, aminopeptidase, and renal dipeptidase activities on BBMVs will be determined. After the in vitro assays, the kidney uptake of the 68Ga-labelled peptide-cleavable linker-DOTA constructs (D-aminoacid linker as control) will be investigated in healthy mice.

Based on the results of the in vitro assays and the kidney uptake tests performed in healthy mice the most promising 68Ga-labelled peptide-cleavable linker-DOTA constructs will be labelled with 177Lu, 213Bi and 225Ac. Cell binding and internalisation studies will be performed using a SSTR2 positive CA20948 cell line with acidic wash (20 mM NaOAc pH 5) protocols to evaluate cell binding affinity and internalisation of the new constructs. Biodistribution studies in pancreatic tumour AR42J (high SSTR2 expression) tumour-bearing athymic mice will be used for the comparison of retention of  177Lu-, 213Bi- and 225Ac-labelled constructs in tumour and kidneys. After titration of the peptide dose to select an optimal tumour/kidney uptake and retention ratio, the corresponding optimal mass dose of the new 177Lu-, 213Bi- and 225Ac-labelled constructs will be evaluated for therapeutic efficacy in the same mouse model at doses of 10-50 MBq.