Targeted radionuclide therapy with A 177Lu-labeled anti-HER2 nanobody
Matthias D'Huyvetter, Cécile Vincke, Catarina Xavier, An Aerts, Nathalie Impens, Sarah Baatout, Hendrik De Raeve, Serge Muyldermans, Vicky Caveliers, Nick Devoogdt, Tony Lahoutte, Matthias D'Huyvetter, Cécile Vincke, Catarina Xavier, An Aerts, Nathalie Impens, Sarah Baatout, Hendrik De Raeve, Serge Muyldermans, Vicky Caveliers, Nick Devoogdt, Tony Lahoutte
Abstract
RIT has become an attractive strategy in cancer treatment, but still faces important drawbacks due to poor tumor penetration and undesirable pharmacokinetics of the targeting vehicles. Smaller radiolabeled antibody fragments and peptides feature highly specific target accumulation, resulting in low accumulation in healthy tissue, except for the kidneys. Nanobodies are the smallest (MW<15 kDa) functional antigen-binding fragments that are derived from heavy chain-only camelid antibodies. Here, we show that the extend of kidney retention of nanobodies is predominantly dictated by the number of polar residues in the C-terminal amino acid tag. Three nanobodies were produced with different C-terminal amino-acid tag sequences (Myc-His-tagged, His-tagged, and untagged). Dynamic planar imaging of Wistar rats with 111In-DTPA-nanobodies revealed that untagged nanobodies showed a 70% drop in kidney accumulation compared to Myc-His-tagged nanobodies at 50 min p.i.. In addition, coinfusion of untagged nanobodies with the plasma expander Gelofusin led to a final reduction of 90%. Similar findings were obtained with different 177Lu-DTPA-2Rs15d nanobody constructs in HER2pos tumor xenografted mice at 1 h p.i.. Kidney accumulation decreased 88% when comparing Myc-His-tagged to untagged 2Rs15d nanobody, and 95% with a coinfusion of Gelofusin, without affecting the tumor targeting capacity. Consequently, we identified a generic method to reduce kidney retention of radiolabeled nanobodies. Dosimetry calculations of Gelofusin-coinfused, untagged 177Lu-DTPA-2Rs15d revealed a dose of 0.90 Gy/MBq that was delivered to both tumor and kidneys and extremely low doses to healthy tissues. In a comparative study, 177Lu-DTPA-Trastuzumab supplied 6 times more radiation to the tumor than untagged 177Lu-DTPA-2Rs15d, but concomitantly also a 155, 34, 80, 26 and 4180 fold higher radioactivity burden to lung, liver, spleen, bone and blood. Most importantly, nanobody-based targeted radionuclide therapy in mice bearing small estiblashed HER2pos tumors led to an almost complete blockade of tumor growth and a significant difference in event-free survival between the treated and the control groups (P<0.0001). Based on histology analyses, no evidence of renal inflammation, apoptosis or necrosis was obtained. In conclusion, these data highlight the importance of the amino acid composition of the nanobody's C-terminus, as it has a predominant effect on kidney retention. Moreover, we show successful nanobody-based targeted radionuclide therapy in a xenograft model and highlight the potential of radiolabeled nanobodies as a valuable adjuvant therapy candidate for treatment of minimal residual and metastatic disease.
Keywords: 177-Lutetium.; HER2; nanobody; targeted radionuclide therapy.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interest exists.
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References
- Jain M, Gupta S, Kaur S, Ponnusamy MP, Batra SK. Emerging Trends for Radioimmunotherapy in Solid Tumors. Cancer Biother Radiopharm. 2013 [Epub ahead of print]
- Huhalov A, Chester KA. Engineerd single chain antibody fragments for radioimmunotherapy. Q J Nucl Mol Imaging. 2004;48:279–88.
- Christensen EL, Brin H. Megalin and cubulin: multifunctional endocytic receptors. Nat Rev Mol Cell Biol. 2002;3:256–266.
- De Jong M, Barone R, Krenning E, Bernard B, Melis M, Visser T, Gekle M, Willnow TE, Walrand S, Jamar F, Pauwels S. Megalin is essential for renal proximal tubule reabsorption of (111)In-DTPA-octreotide. J Nucl Med. 2005;46:1696–1700.
- Moll S, Nickeleit V, Mueller-Brand J, Brunner FP, Maecke HR, Mihatsch MJ. A new cause of renal thrombotic microangiopathy: yttrium 90-DOTATOC internal radiotherapy. Am J Kidney Dis. 2001;37:847–51.
- Stoffel MP, Pollok M, Fries J, Baldamus CA. Radiation nephropathy after radiotherapy in metastatic medullary thyroid carcinoma. Nephrol Dial Transplant. 2001;16:1082–3.
- Barone R, Borson-Chazot F, Valkema R, Walrand S, Chauvin F, Gogou L. et al. Patient-specific dosimetry in predicting renal toxicity with 90Y-DOTATOC: relevance of kidney volume and dose rate in finding a dose-effect relationship. J Nucl Med. 2005;46:99S–106S.
- Wållberg H, Orlova A, Altai M, Hosseinimehr SJ, Widström C, Malmberg J, Ståhl S, Tolmachev V. Molecular design and optimization of 99mTc-labeled recombinant affibody molecules improves their biodistribution and imaging properties. J Nucl Med. 2011;52(3):461–9.
- Rolleman EJ, Bernard BF, Breeman WA, Forrer F, de Blois E, Hoppin J. et al. Molecular imaging of reduced renal uptake of radiolabelled [DOTA0,Tyr3]octreotate by the combination of lysine and gelofusine in rats. Nuklearmedizin. 2008;47:110–5.
- Gainkam LO, Caveliers V, Devoogdt N, Vanhove C, Xavier C, Boerman O, Muyldermans S, Bossuyt A, Lahoutte T. Localization, mechanism and reduction of renal retention of technetium-99m labeled epidermal growth factor receptor-specific nanobody in mice. Contrast Media Mol Imaging. 2011;6(2):85–92.
- Hamers-Casterman C, Atarhouch T, Muyldermans S. et al. Naturally occurring antibodies devoid of light chains. Nature. 1993;363:446–448.
- Vaneycken I, D'huyvetter M, Hernot S, De Vos J, Xavier C, Devoogdt N, Caveliers V, Lahoutte T. Immuno-imaging using nanobodies. Curr Opin Biotechnol. 2011 Dec;22(6):877–81.
- Vaneycken I, Govaert J, Vincke C, Caveliers V, Lahoutte T, De Baetselier P, Raes G, Bossuyt A, Muyldermans S, Devoogdt N. In vitro analysis and in vivo tumor targeting of a humanized, grafted nanobody in mice using pinhole SPECT/micro-CT. J Nucl Med. 2010 Jul;51(7):1099–106.
- Gainkam LO, Huang L, Caveliers V, Keyaerts M, Hernot S, Vaneycken I, Vanhove C, Revets H, De Baetselier P, Lahoutte T. Comparison of the biodistribution and tumor targeting of two 99mTc-labeled anti-EGFR nanobodies in mice, using pinhole SPECT/micro-CT. J Nucl Med. 2008 May;49(5):788–95.
- Vaneycken I, Devoogdt N, Van Gassen N, Vincke C, Xavier C, Wernery U, Muyldermans S, Lahoutte T, Caveliers V. Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer. FASEB J. 2011 Jul;25(7):2433–46.
- Xavier C, Vaneycken I, D'huyvetter M, Heemskerk J, Keyaerts M, Vincke C, Devoogdt N, Muyldermans S, Lahoutte T, Caveliers V. Synthesis, Preclinical Validation, Dosimetry, and Toxicity of 68Ga-NOTA-Anti-HER2 Nanobodies for iPET Imaging of HER2 Receptor Expression in Cancer. J Nucl Med. 2013 [Epub ahead of print]
- Evazalipour M, D'Huyvetter M, Soltani Tehrani B, Abolhassani M, Omidfar K, Abdoli S, Arezumand R, Morovvati H, Lahoutte T, Muyldermans S, Devoogdt N. Generation and characterization of nanobodies targeting PSMA for molecular imaging of prostate cancer. Contrast Media and Molecular Imaging. 2013 Accepted.
- Hynes NE, Lane HA. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer. 2005;5:341–35.
- Yarden Y. Biology of HER2 and its importance in breast cancer. Oncology. 2001;61(2):1–13.
- D'Huyvetter M, Aerts A, Xavier C, Vaneycken I, Devoogdt N, Gijs M, Impens N, Baatout S, Ponsard B, Muyldermans S, Caveliers V, Lahoutte T. Development of 177Lu-nanobodies for radioimmunotherapy of HER2-positive breast cancer: evaluation of different bifunctional chelators. Contrast Media Mol Imaging. 2012;7(2):254–64.
- Keyaerts M, Verschueren J, Bos TJ, Tchouate-Gainkam LO, Peleman C, Breckpot K, Vanhove C, Caveliers V, Bossuyt A, Lahoutte T. Dynamic bioluminescence imaging for quantitative tumour burden assessment using IV or IP administration of D: -luciferin: effect on intensity, time kinetics and repeatability of photon emission. Eur J Nucl Med Mol Imaging. 2008;35(5):999–1007.
- Broisat A, Hernot S, Toczek J, De Vos J, Riou LM, Martin S, Ahmadi M, Thielens N, Wernery U, Caveliers V, Muyldermans S, Lahoutte T, Fagret D, Ghezzi C, Devoogdt N. Nanobodies Targeting Mouse/Human VCAM1 for the Nuclear Imaging of Atherosclerotic Lesions. Circulation Res. 2012;110:927–937.
- Saerens D, Pellis M, Loris R, Pardon E, Dumoulin M, Matagne A, Wyns L, Muyldermans S, Conrath K. Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol. 2005;352(3):597–607.
- Zimmerman JM, Eliezer N, Simha R. The characterization of amino acid sequences in proteins by statistical methods. J Theor Biol. 1968;21(2):170–201.
- Hmila I, Cosyns B, Tounsi H, Roosens B, Caveliers V, Abderrazek RB, Boubaker S, Muyldermans S, El Ayeb M, Bouhaouala-Zahar B, Lahoutte T. Pre-clinical studies of toxin-specific Nanobodies: Evidence of in vivo efficacy to prevent fatal disturbances provoked by scorpion envenoming. Toxicol Appl Pharmacol. 2012;264(2):222–31.
- Tolmachev V, Orlova A, Pehrson R, Galli J, Baastrup B, Andersson K, Sandström M, Rosik D, Carlsson J, Lundqvist H, Wennborg A, Nilsson FY. Radionuclide therapy of HER2-positive microxenografts using a 177Lu-labeled HER2-specific affibody molecule. Cancer Res. 2007;67(6):2773–2782.
- Ray GL, Baidoo KE, Keller LMM, Albert PS, Brechbiel MW, Milenic D. Pre-clinical assessment of 177Lu-labeled trastuzumab targeting HER2 for treatment and management of cancer patients with disseminated intraperitoneal disease. Pharmaceuticals. 2012;5:1–15.
- Persson M, Tolmachev V, Andersson K, Gedda L, Sandström M, Carlsson J. [(177)Lu]pertuzumab: experimental studies on targeting of HER-2 positive tumour cells. Eur J Nucl Med Mol Imaging. 2005;32(12):1457–62.
- van Eerd JE, Vegt E, Wetzels JF, Russel FG, Masereeuw R, Corstens FH. et al. Gelatin-based plasma expander effectively reduces renal uptake of 111In-octreotide in mice and rats. J Nucl Med. 2006;47:528–33.
- Vegt E, Wetzels JF, Russel FG, Masereeuw R, Boerman OC, van Eerd JE. et al. Renal uptake of radiolabeled octreotide in human subjects is efficiently inhibited by succinylated gelatin. J Nucl Med. 2006;47:432–36.
- Gotthardt M, van Eerd-Vismale J, Oyen WJ, de Jong M, Zhang H, Rolleman E. et al. Indication for different mechanisms of kidney uptake of radiolabeled peptides. J Nucl Med. 2007;48:596–601.
- Adams GP, Shaller CC, Dadachova E, Simmons HH, Horak EM, Tesfaye A, Klein-Szanto AJ, Marks JD, Brechbiel MW, Weiner LM. A single treatment of Yttrium-90-labeled CHX-A”-C6.5 diabody inhibits the growth of established human tumor xenografts in immunodeficient mice. Cancer Res. 2004;64:6200–6206.
- Rasaneh S, Rajabi H, Akhlaghpoor S, Sheybani S. Radioimmunotherapy of mice bearing breast tumors with 177Lu-labeled trastuzumab. Turk J Med Sci. 2012;42(1):1292–1298.
- Sampath L, Kwon S, Ke S, Wang W, Schiff R, Mawad ME, Sevick-Muraca EM. Dual-labeled Trastuzumab-based imaging agent for the detection of human epidermal growth factor receptor 2 overexpression in breast cancer. J Nucl Med. 2007;48:1501–1510.
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