title: Synthesis, preclinical validation, dosimetry, and toxicity of 68Ga-NOTA-anti-HER2 Nanobodies for iPET imaging of HER2 receptor expression in cancer.
abstract: Nanobodies are the smallest fully functional antigen-binding antibody fragments possessing ideal properties as probes for molecular imaging. In this study we labeled the anti-human epidermal growth factor receptor type 2 (HER2) Nanobody with (68)Ga via a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) derivative and assessed its use for HER2 iPET imaging.
  
  
  METHODS
  The 2Rs15dHis6 Nanobody and the lead optimized current-good-manufacturing-practice grade analog 2Rs15d were conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) to enable fast and efficient (68)Ga labeling. Biodistribution and PET/CT studies were performed on HER2-positive and -negative tumor xenografts. The effect of injected mass on biodistribution was evaluated. The biodistribution data were extrapolated to calculate radiation dose estimates for the adult female using OLINDA software. A single-dose extended-toxicity study for NOTA-2Rs15d was performed on healthy mice up to a dose of 10 mg/kg.
  
  
  RESULTS
  Radiolabeling was quantitative (>97%) after 5 min of incubation at room temperature; specific activity was 55-200 MBq/nmol. Biodistribution studies showed fast and specific uptake (percentage injected activity [%IA]) in HER2-positive tumors (3.13 ± 0.06 and 4.34 ± 0.90 %IA/g for (68)Ga-NOTA-2Rs15dHis6 and (68)Ga-NOTA-2Rs15d, respectively, at 1 h after injection) and high tumor-to-blood and tumor-to-muscle ratios at 1 h after injection, resulting in high-contrast PET/CT images with high specific tumor uptake. A remarkable finding of the biodistribution studies was that kidney uptake was reduced by 60% for the Nanobody lacking the C-terminal His6 tag. The injected mass showed an effect on the general biodistribution: a 100-fold increase in NOTA-2Rs15d mass decreased liver uptake from 7.43 ± 1.89 to 2.90 ± 0.26 %IA/g whereas tumor uptake increased from 2.49 ± 0.68 to 4.23 ± 0.99 %IA/g. The calculated effective dose, based on extrapolation of mouse data, was 0.0218 mSv/MBq, which would yield a radiation dose of 4 mSv to a patient after injection of 185 MBq of (68)Ga-NOTA-2Rs15d. In the toxicity study, no adverse effects were observed after injection of a 10 mg/kg dose of NOTA-2Rs15d.
  
  
  CONCLUSIONS
  A new anti-HER2 PET tracer, (68)Ga-NOTA-2Rs15d, was synthesized via a rapid procedure under mild conditions. Preclinical validation showed high-specific-contrast imaging of HER2-positive tumors with no observed toxicity. (68)Ga-NOTA-2Rs15d is ready for first-in-human clinical trials.
authors.affiliation: In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel, Brussels, Belgium. cxavier@vub.ac.be
authors.forename: Catarina
authors.initials: C
authors.lastname: Xavier
authors.forename: Ilse
authors.initials: I
authors.lastname: Vaneycken
authors.forename: Matthias
authors.initials: M
authors.lastname: D'huyvetter
authors.forename: Johannes
authors.initials: J
authors.lastname: Heemskerk
authors.forename: Marleen
authors.initials: M
authors.lastname: Keyaerts
authors.forename: Cécile
authors.initials: C
authors.lastname: Vincke
authors.forename: Nick
authors.initials: N
authors.lastname: Devoogdt
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Tony
authors.initials: T
authors.lastname: Lahoutte
authors.forename: Vicky
authors.initials: V
authors.lastname: Caveliers
mesh_terms: D000328:Adult; D000818:Animals; D045744:Cell Line, Tumor; D060326:Chemistry Techniques, Synthetic; D004355:Drug Stability; D005260:Female; D005710:Gallium Radioisotopes; D015972:Gene Expression Regulation, Neoplastic; D006571:Heterocyclic Compounds / Q000737:chemistry*; D006573:Heterocyclic Compounds, 1-Ring; D006801:Humans; D051379:Mice; D064847:Multimodal Imaging / Q000379:methods*; D009369:Neoplasms / Q000000981:diagnostic imaging / Q000473:pathology*; D049268:Positron-Emission Tomography; D011874:Radiometry; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism*; D015203:Reproducibility of Results; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000276:immunology / Q000633:toxicity; D014057:Tomography, X-Ray Computed
chemical_list: D005710:Gallium Radioisotopes; D006571:Heterocyclic Compounds; D006573:Heterocyclic Compounds, 1-Ring; D018719:Erb-b2 Receptor Tyrosine Kinases; D061905:Single-Domain Antibodies; C048993:1,4,7-triazacyclononane-N,N',N''-triacetic acid
keywords: 68Ga; HER2; NOTA; Nanobodies; iPET