title: Targeted radionuclide therapy with A 177Lu-labeled anti-HER2 nanobody.
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.
authors.affiliation: 1. Radiobiology Unit, Molecular and Cellular Biology Expert Group, Belgian Nuclear Research Center (SCK•CEN), Mol, Belgium. ; 2. In vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel (VUB), Brussels, Belgium.
authors.forename: Matthias
authors.initials: M
authors.lastname: D'Huyvetter
authors.affiliation: 3. Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium. ; 4. Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
authors.forename: Cécile
authors.initials: C
authors.lastname: Vincke
authors.affiliation: 2. In vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel (VUB), Brussels, Belgium.
authors.forename: Catarina
authors.initials: C
authors.lastname: Xavier
authors.affiliation: 1. Radiobiology Unit, Molecular and Cellular Biology Expert Group, Belgian Nuclear Research Center (SCK•CEN), Mol, Belgium.
authors.forename: An
authors.initials: A
authors.lastname: Aerts
authors.affiliation: 1. Radiobiology Unit, Molecular and Cellular Biology Expert Group, Belgian Nuclear Research Center (SCK•CEN), Mol, Belgium.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Impens
authors.affiliation: 1. Radiobiology Unit, Molecular and Cellular Biology Expert Group, Belgian Nuclear Research Center (SCK•CEN), Mol, Belgium.
authors.forename: Sarah
authors.initials: S
authors.lastname: Baatout
authors.affiliation: 5. Department of Pathology, OLV Hospital Aalst, Aalst, Belgium. ; 6. Department of Pathology, UZ Brussel, Brussels, Belgium.
authors.forename: Hendrik
authors.initials: H
authors.lastname: De Raeve
authors.affiliation: 3. Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB), Brussels, Belgium. ; 4. Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.affiliation: 2. In vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel (VUB), Brussels, Belgium. ; 7. Department of Nuclear Medicine, UZ Brussel, Brussels, Belgium.
authors.forename: Vicky
authors.initials: V
authors.lastname: Caveliers
authors.affiliation: 2. In vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel (VUB), Brussels, Belgium. ; 4. Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
authors.forename: Nick
authors.initials: N
authors.lastname: Devoogdt
authors.affiliation: 2. In vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel (VUB), Brussels, Belgium. ; 7. Department of Nuclear Medicine, UZ Brussel, Brussels, Belgium.
authors.forename: Tony
authors.initials: T
authors.lastname: Lahoutte
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000009:adverse effects / Q000493:pharmacokinetics / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D008187:Lutetium / Q000009:adverse effects / Q000493:pharmacokinetics / Q000627:therapeutic use*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D010051:Ovarian Neoplasms / Q000532:radiotherapy*; D016499:Radioimmunotherapy / Q000379:methods*; D011868:Radioisotopes / Q000009:adverse effects / Q000493:pharmacokinetics / Q000627:therapeutic use*; D019275:Radiopharmaceuticals / Q000009:adverse effects / Q000493:pharmacokinetics / Q000627:therapeutic use*; D051381:Rats; D017208:Rats, Wistar; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000009:adverse effects / Q000493:pharmacokinetics / Q000627:therapeutic use; D000068878:Trastuzumab
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D008187:Lutetium; D011868:Radioisotopes; D019275:Radiopharmaceuticals; D018719:Erb-b2 Receptor Tyrosine Kinases; D057127:Single-Chain Antibodies; D000068878:Trastuzumab; C508053:ERBB2 protein, human
keywords: 177-Lutetium.; HER2; nanobody; targeted radionuclide therapy