title: Improved tumor targeting of anti-HER2 nanobody through N-succinimidyl 4-guanidinomethyl-3-iodobenzoate radiolabeling.
abstract: Nanobodies are approximately 15-kDa proteins based on the smallest functional fragments of naturally occurring heavy chain-only antibodies and represent an attractive platform for the development of molecularly targeted agents for cancer diagnosis and therapy. Because the human epidermal growth factor receptor type 2 (HER2) is overexpressed in breast and ovarian carcinoma, as well as in other malignancies, HER2-specific Nanobodies may be valuable radiodiagnostics and therapeutics for these diseases. The aim of the present study was to evaluate the tumor-targeting potential of anti-HER2 5F7GGC Nanobody after radioiodination with the residualizing agent N-succinimidyl 4-guanidinomethyl 3-(125/131)I-iodobenzoate (*I-SGMIB).
  
  
  METHODS
  The 5F7GGC Nanobody was radiolabeled using *I-SGMIB and, for comparison, with N(ε)-(3-*I-iodobenzoyl)-Lys(5)-N(α)-maleimido-Gly(1)-GEEEK (*I-IB-Mal-d-GEEEK), another residualizing agent, and by direct radioiodination using IODO-GEN ((125)I-Nanobody). The 3 labeled Nanobodies were evaluated in affinity measurements, and paired-label internalization assays were performed on HER2-expressing BT474M1 breast carcinoma cells and in paired-label tissue distribution measurements in mice bearing subcutaneous BT474M1 xenografts.
  
  
  RESULTS
  *I-SGMIB-Nanobody was produced in 50.4% ± 3.6% radiochemical yield and exhibited a dissociation constant of 1.5 ± 0.5 nM. Internalization assays demonstrated that intracellular retention of radioactivity was up to 1.5-fold higher for *I-SGMIB-Nanobody than for coincubated (125)I-Nanobody or *I-IB-Mal-d-GEEEK-Nanobody. Peak tumor uptake for *I-SGMIB-Nanobody was 24.50% ± 9.89% injected dose/g at 2 h, 2- to 4-fold higher than observed with other labeling methods, and was reduced by 90% with trastuzumab blocking, confirming the HER2 specificity of localization. Moreover, normal-organ clearance was fastest for *I-SGMIB-Nanobody, such that tumor-to-normal-organ ratios greater than 50:1 were reached by 24 h in all tissues except lungs and kidneys, for which the values were 10.4 ± 4.5 and 5.2 ± 1.5, respectively.
  
  
  CONCLUSIONS
  Labeling anti-HER2 Nanobody 5F7GGC with *I-SGMIB yields a promising new conjugate for targeting HER2-expressing malignancies. Further research is needed to determine the potential utility of *I-SGMIB-5F7GGC labeled with (124)I, (123)I, and (131)I for PET and SPECT imaging and for targeted radiotherapy, respectively.
authors.affiliation: Department of Radiology, Duke University Medical Center, Durham, North Carolina.
authors.forename: Marek
authors.initials: M
authors.lastname: Pruszynski
authors.forename: Eftychia
authors.initials: E
authors.lastname: Koumarianou
authors.forename: Ganesan
authors.initials: G
authors.lastname: Vaidyanathan
authors.forename: Hilde
authors.initials: H
authors.lastname: Revets
authors.forename: Nick
authors.initials: N
authors.lastname: Devoogdt
authors.forename: Tony
authors.initials: T
authors.lastname: Lahoutte
authors.forename: H Kim
authors.initials: HK
authors.lastname: Lyerly
authors.forename: Michael R
authors.initials: MR
authors.lastname: Zalutsky
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D001565:Benzoates / Q000138:chemical synthesis; D001943:Breast Neoplasms / Q000000981:diagnostic imaging / Q000378:metabolism; D005260:Female; D019791:Guanidine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D006801:Humans; D007553:Isotope Labeling; D051379:Mice; D008819:Mice, Nude; D049268:Positron-Emission Tomography; D019275:Radiopharmaceuticals / Q000138:chemical synthesis; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D061905:Single-Domain Antibodies / Q000378:metabolism*; D014018:Tissue Distribution; D015899:Tomography, Emission-Computed, Single-Photon; D000068878:Trastuzumab; D023041:Xenograft Model Antitumor Assays
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D001565:Benzoates; D019791:Guanidine; D019275:Radiopharmaceuticals; D018719:Erb-b2 Receptor Tyrosine Kinases; D061905:Single-Domain Antibodies; D000068878:Trastuzumab; C438937:N-succinimidyl 4-guanidinomethyl-3-iodobenzoate; C508053:ERBB2 protein, human
keywords: HER2; Nanobody; SGMIB; breast cancer; radioiodination