title: N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: influence of isomeric substitution on radiolabeling and target cell residualization.
abstract: BACKGROUND
  N-succinimidyl 4-guanidinomethyl-3-[(*)I]iodobenzoate ([(*)I]SGMIB) has shown promise for the radioiodination of monoclonal antibodies (mAbs) and other proteins that undergo extensive internalization after receptor binding, enhancing tumor targeting compared to direct electrophilic radioiodination. However, radiochemical yields for [(131)I]SGMIB synthesis are low, which we hypothesize is due to steric hindrance from the Boc-protected guanidinomethyl group ortho to the tin moiety. To overcome this, we developed the isomeric compound, N-succinimidyl 3-guanidinomethyl-5-[(131)I]iodobenzoate (iso-[(131)I]SGMIB) wherein this bulky group was moved from ortho to meta position.
  
  
  METHODS
  Boc2-iso-SGMIB standard and its tin precursor, N-succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-(trimethylstannyl)benzoate (Boc2-iso-SGMTB), were synthesized using two disparate routes, and iso-[*I]SGMIB synthesized from the tin precursor. Two HER2-targeted vectors - trastuzumab (Tras) and a nanobody 5F7 (Nb) - were labeled using iso-[(*)I]SGMIB and [(*)I]SGMIB. Paired-label internalization assays in vitro with both proteins, and biodistribution in vivo with trastuzumab, labeled using the two isomeric prosthetic agents were performed.
  
  
  RESULTS
  When the reactions were performed under identical conditions, radioiodination yields for the synthesis of Boc2-iso-[(131)I]SGMIB were significantly higher than those for Boc2-[(131)I]SGMIB (70.7±2.0% vs 56.5±5.5%). With both Nb and trastuzumab, conjugation efficiency also was higher with iso-[(131)I]SGMIB than with [(131)I]SGMIB (Nb, 33.1±7.1% vs 28.9±13.0%; Tras, 45.1±4.5% vs 34.8±10.3%); however, the differences were not statistically significant. Internalization assays performed on BT474 cells with 5F7 Nb indicated similar residualizing capacity over 6h; however, at 24h, radioactivity retained intracellularly for iso-[(131)I]SGMIB-Nb was lower than for [(125)I]SGMIB-Nb (46.4±1.3% vs 56.5±2.5%); similar results were obtained using Tras. Likewise, a paired-label biodistribution of Tras labeled using iso-[(125)I]SGMIB and [(131)I]SGMIB indicated an up to 22% tumor uptake advantage at later time points for [(131)I]SGMIB-Tras.
  
  
  CONCLUSIONS
  Given the higher labeling efficiency obtained with iso-SGMIB, this residualizing agent might be of value for use with shorter half-life radiohalogens.
authors.affiliation: Department of Radiology, Duke University Medical Center, Durham, NC, USA.
authors.forename: Jaeyeon
authors.initials: J
authors.lastname: Choi
authors.affiliation: Department of Radiology, Duke University Medical Center, Durham, NC, USA. Electronic address: ganesan.v@duke.edu.
authors.forename: Ganesan
authors.initials: G
authors.lastname: Vaidyanathan
authors.affiliation: Department of Radiology, Duke University Medical Center, Durham, NC, USA.
authors.forename: Eftychia
authors.initials: E
authors.lastname: Koumarianou
authors.affiliation: Department of Radiology, Duke University Medical Center, Durham, NC, USA.
authors.forename: Darryl
authors.initials: D
authors.lastname: McDougald
authors.affiliation: Department of Radiology, Duke University Medical Center, Durham, NC, USA.
authors.forename: Marek
authors.initials: M
authors.lastname: Pruszynski
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, USA.
authors.forename: Takuya
authors.initials: T
authors.lastname: Osada
authors.affiliation: Free University of Brussels, Brussels, Belgium.
authors.forename: Tony
authors.initials: T
authors.lastname: Lahoutte
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, NC, USA.
authors.forename: H Kim
authors.initials: HK
authors.lastname: Lyerly
authors.affiliation: Department of Radiology, Duke University Medical Center, Durham, NC, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Zalutsky
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology / Q000378:metabolism*; D001565:Benzoates / Q000493:pharmacokinetics; D001943:Breast Neoplasms / Q000000981:diagnostic imaging* / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D019791:Guanidine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D007457:Iodine Radioisotopes / Q000493:pharmacokinetics; D007553:Isotope Labeling; D051379:Mice; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000493:pharmacokinetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D013237:Stereoisomerism; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D001565:Benzoates; D019791:Guanidine; D007457:Iodine Radioisotopes; D019275:Radiopharmaceuticals; D018719:Erb-b2 Receptor Tyrosine Kinases; C438937:N-succinimidyl 4-guanidinomethyl-3-iodobenzoate; C508053:ERBB2 protein, human
keywords: Internalization; Monoclonal antibody; Radioiodination; Residualizing label