title: D-Amino acid peptide residualizing agents bearing N-hydroxysuccinimido- and maleimido-functional groups and their application for trastuzumab radioiodination.
abstract: BACKGROUND
  Proteins that undergo receptor-mediated endocytosis are subject to lysosomal degradation, requiring radioiodination methods that minimize loss of radioactivity from tumor cells after this process occurs. To accomplish this, we developed the residualizing radioiodination agent N(ϵ)-(3-[(*)I]iodobenzoyl)-Lys(5)-N(α)-maleimido-Gly(1)-D-GEEEK (Mal-D-GEEEK-[(*)I]IB), which enhanced tumor uptake but also increased kidney activity and necessitates generation of sulfhydryl moieties on the protein. The purpose of the current study was to synthesize and evaluate a new D-amino acid based agent that might avoid these potential problems.
  
  
  METHODS
  N(α)-(3-iodobenzoyl)-(5-succinimidyloxycarbonyl)-D-EEEG (NHS-IB-D-EEEG), which contains 3 D-glutamates to provide negative charge and a N-hydroxysuccinimide function to permit conjugation to unmodified proteins, and the corresponding tin precursor were produced by solid phase peptide synthesis and subsequent conjugation with appropriate reagents. Radioiodination of the anti-HER2 antibody trastuzumab using NHS-IB-D-EEEG and Mal-D-GEEEK-IB was compared. Paired-label internalization assays on BT474 breast carcinoma cells and biodistribution studies in athymic mice bearing BT474M1 xenografts were performed to evaluate the two radioiodinated D-peptide trastuzumab conjugates.
  
  
  RESULTS
  NHS-[(131)I]IB-D-EEEG was produced in 53.8%±13.4% and conjugated to trastuzumab in 39.5%±7.6% yield. Paired-label internalization assays with trastuzumab-NHS-[(131)I]IB-D-EEEG and trastuzumab-Mal-D-GEEEK-[(125)I]IB demonstrated similar intracellular trapping for both conjugates at 1h ((131)I, 84.4%±6.1%; (125)I, 88.6%±5.2%) through 24h ((131)I, 60.7%±6.8%; (125)I, 64.9%±6.9%). In the biodistribution experiment, tumor uptake peaked at 48 h (trastuzumab-NHS-[(131)I]IB-D-EEEG, 29.8%±3.6%ID/g; trastuzumab-Mal-D-GEEEK-[(125)I]IB, 45.3%±5.3%ID/g) and was significantly higher for (125)I at all time points. In general, normal tissue levels were lower for trastuzumab-NHS-[(131)I]IB-D-EEEG, with the differences being greatest in kidneys ((131)I, 2.2%±0.4%ID/g; (125)I, 16.9%±2.8%ID/g at 144 h).
  
  
  CONCLUSIONS
  NHS-[(131)I]IB-D-EEEG warrants further evaluation as a residualizing radioiodination agent for labeling internalizing antibodies/fragments, particularly for applications where excessive renal accumulation could be problematic.
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 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: Ganesan
authors.initials: G
authors.lastname: Vaidyanathan
authors.affiliation: Department of Radiology, Duke University Medical Center, Durham, NC, USA.
authors.forename: Satish
authors.initials: S
authors.lastname: Chitneni
authors.affiliation: Department of Radiology, Duke University Medical Center, Durham, NC, USA; Departments of Biomedical Engineering and Radiation Oncology, Duke University, Durham, NC, USA. Electronic address: zalut001@mc.duke.edu.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Zalutsky
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D001692:Biological Transport; D045744:Cell Line, Tumor; D004952:Esters; D005260:Female; D006801:Humans; D007457:Iodine Radioisotopes / Q000737:chemistry; D007553:Isotope Labeling; D051379:Mice; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D011854:Radiochemistry; D000068878:Trastuzumab
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D004952:Esters; D007457:Iodine Radioisotopes; C545084:N(epsilon)-(3-iodobenzoyl)-Lys(5)-Nalpha-maleimido-Gly(1)-glycyl-glutamyl-glutamyl-gluamyl-lysine; D009842:Oligopeptides; D000068878:Trastuzumab
keywords: Radioiodination; Radionuclide therapy; Residualizing agents; Trastuzumab; d-amino acid peptide