Development of 161Tb-labelled nanobodies for the treatment of colorectal cancer

Cassells Irwin

Promoter

SCK•CEN Mentor

Ooms Maarten
maarten.ooms@sckcen.be
+32 14 33 32 83

SCK•CEN Co-mentor

Derradji Hanane
hanane.derradji@sckcen.be
+32 14 33 21 51

Expert group

Radiochemistry

PhD started

2018-10-29

Short project description

Colorectal cancer is one of the most frequently diagnosed cancers in the western world and third most common cause of cancer deaths. Patients with colon cancers that have not spread to distant sites usually have surgery as the main treatment, possibly followed by adjuvant chemotherapy. If the cancer has spread to distant organs, therapy consists out of a combination of surgery (resection of metastasis), chemotherapy, external X-ray beam therapy or selective targeted therapy with monoclonal antibodies (mAbs) such as bevacizumab (Avastin), cetuximab (Erbitux) and panitumumab (Vectibix) or small molecules such as regorafenib (Stivarga). To treat cancer efficiently, there is an urgent need for the development of new therapeutic molecules to enlarge and improve the treatment options offered to the patients nowadays. Apart from external high energy X-ray beam therapy, Targeted RadioNuclide Therapy (TRNT) is another approach to deliver radiation to cancer cells. TRNT is distributed within the body by the vascular system and allows targeted irradiation of a primary tumor and all its metastases, resulting in substantially less collateral damage to normal tissues as compared to external radiotherapy. It is a systemic cancer therapy, tackling systemic spread of the disease, which is the cause of death in the vast majority of cancer patients (Gudkov et al. 2015). TRNT with radionuclides with short-range emission (alpha, beta, Auger) is particularly of interest to target microscopic metastases in the adjuvant or pseudo-adjuvant setting.

The radiolanthanide terbium-161 (161Tb) has gathered increasing interest in recent years due to its favorable properties for medical application (Lehenberger et al. 2011). 161Tb decays, with a half-life of 6.9 days, by the emission of low-energy β- particles (Eβ−average = 154 keV) with a maximal tissue range of 0.29 mm and a linear energy transfer (LET) of around 0.32 keV/μm, which is suitable for the treatment of metastasized malignancies. Importantly, 161Tb exhibits a co-emission of γ-radiation, enabling imaging using single photon emission computed tomography (SPECT). 161Tb has similar chemical and physical properties to lutetium-177  (177Lu, T1/2 = 6.7 d, Eβ−average = 134 keV, LET of about 0.34 keV/μm), a feature which will allow us to exploit the already existing knowledge related to the radiolabelling chemistry techniques established for 177Lu. Therefore as both 161Tb and 177Lu are radiolanthanides, the chelator DOTA can be used.

The advantage of using 161Tb over 177Lu are numerous. Firstly, the decay process of 161Tb releases more Auger/conversion electrons (energy ≤ 50 keV) than 177Lu. These Auger/conversion electrons release much higher local dose density due to their shorter range in tissue (0.5 – 30 µm), thereby contributing to the therapeutic anti-tumor effects of 161Tb (Muller et al. 2014) without causing additional renal side effects (Haller et al. 2016). Secondly, 161Tb can be used as a theranostics agent as there are other Tb nuclides that emit diagnostic radiation only such as 155Tb (useful for SPECT imaging) and 152Tb (useful for PET imaging) (Muller et al. 2012; Muller et al. 2016).

Up to now, only few studies have used 161Tb for the evaluation of its therapeutic potential which makes it an innovative radionuclide. These studies have mostly compared the therapeutic efficacy of 161Tb to other similar β- emitting radionuclides. These preliminary therapy studies revealed that the therapeutic effect of 161Tb-labelled compounds was superior to the effect of their 177Lu-, 67Cu- or 47Sc -labelled counterparts when applied at the same dose (Muller et al. 2014; Grunberg et al. 2014; Champion et al, 2016)

CarcinoEmbryonic Antigen (CEA) is the preferred biomarker for in vivo colorectal cancer imaging and targeting. In terms of sensitivity and specificity, CEA is the most promising marker with a considerable margin, as compared with other reported colorectal cancer biomarkers such as tumor-associated glycoprotein-72 (TAG-72), folate receptor-α (FRα) and epithelial growth factor receptor (EGFR) (Tiernan et al, 2013). Both primary and metastatic colorectal cancer lesions show consistently high expression of CEA and expression in normal tissues is limited. Furthermore, adenocarcinomas of the lung, breast, other gastrointestinal organs and of the ovary show frequently elevated CEA levels.

This project focuses on the use of anti-carcinoembryonic antigen (anti-CEA) nanobodies as vector molecules. Nanobodies (Nb, VHH) are antigen-binding fragments derived from heavy-chain-only antibodies occurring naturally in Camelid species. Nbs bind their antigens very fast and specifically with high affinity in vivo, whereas unbound Nbs are rapidly cleared from the blood by the kidneys. Physiological uptake of Nbs in liver and in abdomen is limited, therefore, Nbs can be considered as ideal vectors for in vivo imaging and radionuclide therapy of colorectal cancer metastases.

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).

References

Gudkov, S. V., N. Y. Shilyagina, V. A. Vodeneev, and A. V. Zvyagin. 2015. 'Targeted Radionuclide Therapy of Human Tumors', Int J Mol Sci, 17.

Lehenberger, S., C. Barkhausen, S. Cohrs, E. Fischer, J. Grunberg, A. Hohn, U. Koster, R. Schibli, A. Turler, and K. Zhernosekov. 2011. 'The low-energy beta(-) and electron emitter (161)Tb as an alternative to (177)Lu for targeted radionuclide therapy', Nucl Med Biol, 38: 917-24.

Haller, S., G. Pellegrini, C. Vermeulen, N. P. van der Meulen, U. Koster, P. Bernhardt, R. Schibli, and C. Muller. 2016. 'Contribution of Auger/conversion electrons to renal side effects after radionuclide therapy: preclinical comparison of (161)Tb-folate and (177)Lu-folate', EJNMMI Res, 6: 13.

Muller, C., J. Reber, S. Haller, H. Dorrer, P. Bernhardt, K. Zhernosekov, A. Turler, and R. Schibli. 2014. 'Direct in vitro and in vivo comparison of (161)Tb and (177)Lu using a tumour-targeting folate conjugate', Eur J Nucl Med Mol Imaging, 41: 476-85.

Muller, C., C. Vermeulen, K. Johnston, U. Koster, R. Schmid, A. Turler, and N. P. van der Meulen. 2016. 'Preclinical in vivo application of (152)Tb-DOTANOC: a radiolanthanide for PET imaging', EJNMMI Res, 6: 35.

Muller, C., K. Zhernosekov, U. Koster, K. Johnston, H. Dorrer, A. Hohn, N. T. van der Walt, A. Turler, and R. Schibli. 2012. 'A unique matched quadruplet of terbium radioisotopes for PET and SPECT and for alpha- and beta- radionuclide therapy: an in vivo proof-of-concept study with a new receptor-targeted folate derivative', J Nucl Med, 53: 1951-9.

Grunberg, J., D. Lindenblatt, H. Dorrer, S. Cohrs, K. Zhernosekov, U. Koster, A. Turler, E. Fischer, and R. Schibli. 2014. 'Anti-L1CAM radioimmunotherapy is more effective with the radiolanthanide terbium-161 compared to lutetium-177 in an ovarian cancer model', Eur J Nucl Med Mol Imaging, 41: 1907-15.

Champion, C., M. A. Quinto, C. Morgat, P. Zanotti-Fregonara, and E. Hindie. 2016. 'Comparison between Three Promising ss-emitting Radionuclides, (67)Cu, (47)Sc and (161)Tb, with Emphasis on Doses Delivered to Minimal Residual Disease', Theranostics, 6: 1611-8.

Tiernan JP, Perry SL, Verghese ET, West NP, Yeluri S, Jayne DG, Hughes TA. Carcinoembryonic antigen is the preferred biomarker for in vivo colorectal cancer targeting. Br J Cancer. 2013 Feb 19;108(3):662-7.

 

Objective

The overall objective of this PhD proposal is to develop and to pre-clinically evaluate a 161Tb-based radiopharmaceutical for the treatment of minimal residual disease originating from colorectal cancer.

To achieve this overall objective, specific objectives are formulated:

  1. Optimization of site-specific derivatization of anti-CEA Nbs with the DOTA chelator using Sortase A, a transpeptidase derived from Staphylococcus aureus.

  2. Optimization of the radiochemical procedures for the radiolabelling with 161Tb. Readouts will be yield and purity after labelling.

  3. Investigation of the radiochemical stability of 161Tb-labelled anti-CEA nanobody in relevant storage and physiological conditions, including stability in rodent and human serum.

  4. Investigation of the specificity of 161Tb-anti-CEA Nb for CEA by in vitro binding assay to CHO (negative control) and CEA-transfected CHO cells. In addition, the internalization ratio will be determined.

  5. In vitro studies on tumor cell lines (LoVO, human colorectal adenocarcinoma cell line) to investigate the specificity of binding, the affinity, degree of internalization , and in vitro cytotoxicity.

  6. In vivo studies of the biodistribution and stability of 161Tb-anti-CEA Nbs in normal wild type mice.

  7. In vivo targeting of 161Tb-anti-CEA Nbs will be evaluated in a preclinical setting using LoVO (CEA+) tumor-bearing BALB/c nude mice to monitor the biodistribution, the stability, tissue penetration, tumor targeting and toxicity.

  8. Evaluation of the therapeutic potential of the 161Tb-labelled anti-CEA nanobody in a preclinical mouse model (orthotopic colorectal tumor mouse model) of minimal residual disease.

161Tb has similar chemical and physical properties as the clinically more established 177lutetium (177Lu) for RNT of metastatic tumors. Therefore, 177Lu will be used for comparison purposes.

 

Methodology

1. Radiolabeling of the radiopharmaceutical

161Tb will be produced in the Belgian Reactor 2 (BR2) at SCK•CEN by neutron irradiation of highly enriched gadolinium-160 (160Gd) and can thereafter be isolated from the target material chemically. The pure 161Tb will be provided by the radiochemistry group from SCK•CEN using the known separation method used by Lehenberger (Lehenberger et al, 2011).

In a first step, the sortase-mediated protein ligation method will be used for site-specific derivatization of the anti-CEA nanobody with the DOTA-chelator. Thereafter, the structural and functional integrity of the Nb will be monitored using an SDS-PAGE, western-blot and LC-HRMS methods. The binding properties of the derivatized NbCEA5 will be measured by surface plasmon resonance (SPR). The DOTA-derivatized anti-CEA Nb will be radiolabelled with 161Tb, labelling efficiency will be monitored by instant thin-layer chromatography (iTLC) and purification will be performed using a disposable size-exclusion column. The radiolabelled nanobody will be formulated in a formulation suitable for injection and quality control will be performed using radio-size exclusion chromatography. Stability will be evaluated in the formulation and in PBS and serum (37 °C).

  2- In vitro and in vivo evaluation of 161Tb-anti-CEA Nb   

The specificity of 161Tb-anti-CEA Nb for the CEA antigen will be monitored in vitro on CHO-CEA negative (Chinese hamster ovary cell line) and CEA-transfected CHO cell lines using binding assay. Degree of internalization will be estimated via the glycine wash method. Complementary in vitro studies will be performed on the human colorectal adenocarcinoma cell line (LoVO) to monitor the affinity, degree of internalization and cytotoxicity of 161Tb -anti-CEA Nb using the binding assay, glycine wash method and a colony forming assay, respectively.

In vivo evaluation of the biodistribution of 161Tb-anti-CEA Nb will be monitored first in normal wild type mice then in nude mice bearing implanted xenografts of CEA-positive and negative tumors by means of MicroSPECT-CT imaging technique. Stability analysis in blood and urine will be analyzed using a gamma counting system. At the end of the in vivo experiments, the animals will be euthanized and dosimetric analysis of the tumor and all major organs and tissues will be performed. Furthermore, dissected tumors will be sectioned using a cryostat after which autoradiography will be performed, allowing us to study the distribution of the radiopharmaceuticals within the tumor tissue and compare it with histological images. In addition, mouse tumor slices can be incubated in vitro directly with the tracer, thereafter, autoradiography will be performed to assess the distribution of receptors/tracer in tumor tissues. CEA negative tumor can be used as control tissue. Autoradiography on human tumor tissue (after approval by ethical committee and biobank from UZ Leuven) can be used to evaluate the specificity of 161Tb-anti-CEA Nb for human CEA by comparison of tracer distribution (autoradiography) and CEA expression (immunohistochemistry).

The therapeutic characteristics and the specificity of the 161Tb-anti-CEA  Nb will be investigated on nude mice bearing implanted xenografts of CEA-positive tumors and contralateral CEA-negative control tumors. These mice will be injected with 161Tb-anti-CEA Nb or PBS as a placebo at different stages during tumor development. Tumor growth will be analyzed at different time points. To this end, 2-[18F]fluoro-2-deoxy-D-glucose (FDG) micro-PET/MRI scans will be performed. In addition, tumor size will be physically measured using a vernier caliper. Finally, also weight, survival and toxicity to the liver and kidneys will be evaluated. If the results  on the xenografted mice are promising, a more relevant clinical mouse model bearing orthotopic colorectal tumor developing metastases will be used to monitor the therapeutic efficiency of the 161Tb-anti-CEA Nb.