title: Dual-Receptor-Targeted Radioimmunotherapy of Human Breast Cancer Xenografts in Athymic Mice Coexpressing HER2 and EGFR Using 177Lu- or 111In-Labeled Bispecific Radioimmunoconjugates.
abstract: One mechanism of resistance to trastuzumab in human epidermal growth factor receptor-2 (HER2)-positive breast cancer (BC) is increased epidermal growth factor receptor (EGFR) expression. We have developed (111)In-labeled bispecific radioimmunoconjugates (bsRICs) that bind HER2 and EGFR on BC cells by linking trastuzumab Fab fragments through a polyethylene glycol (PEG24) spacer to epidermal growth factor (EGF). We hypothesized that tumors coexpressing HER2 and EGFR could be treated by dual-receptor-targeted radioimmunotherapy with these bsRICs labeled with the β-particle emitter (177)Lu or the Auger electron-emitter (111)In.
  
  
  METHODS
  The binding of (177)Lu-DOTA-Fab-PEG24-EGF to tumor cells (MDA-MB-231, SK-OV-3, MDA-MB-231/H2N, or TrR1) coexpressing HER2 and EGFR was assessed in competition assays. The clonogenic survival of these cells was measured after exposure to (177)Lu-DOTA-Fab-PEG24-EGF or (111)In-DTPA-Fab-PEG24-EGF or to monospecific (177)Lu- or (111)In-labeled trastuzumab Fab or EGF. The tumor and normal tissue biodistribution of (177)Lu-DOTA-Fab-PEG24-EGF was studied at 48 h after injection in athymic mice bearing subcutaneous MDA-MB-231/H2N tumors. Radiation-absorbed doses to tumors and normal tissues were estimated and compared for (111)In- and (177)Lu-labeled bsRICs. The maximum injected amount of (177)Lu-DOTA-Fab-PEG24-EGF that caused no observable adverse effects (NOAEL) was identified in BALB/c mice. Athymic CD1 nu/nu mice bearing subcutaneous trastuzumab-sensitive MDA-MB-231/H2N or trastuzumab-resistant TrR1 tumors were treated with (177)Lu-DOTA-Fab-PEG24-EGF or (111)In-DTPA-Fab-PEG24-EGF at the NOAEL, or with unlabeled immunoconjugates or normal saline. Tumor growth was evaluated over a period of 49 d.
  
  
  RESULTS
  (177)Lu-DOTA-Fab-PEG24-EGF bound specifically to HER2 and EGFR on tumor cells. Monospecific (177)Lu- and (111)In-labeled trastuzumab Fab or EGF killed tumor cells that predominantly expressed HER2 or EGFR, respectively, whereas bsRICs were cytotoxic to cells that displayed either HER2 or EGFR or both receptors. bsRICs were more effective than monospecific agents. (177)Lu-DOTA-Fab-PEG24-EGF was more cytotoxic than (111)In-DTPA-Fab-PEG24-EGF. The tumor uptake of (177)Lu-DOTA-Fab-PEG24-EGF was 2-fold greater than (177)Lu-DOTA-trastuzumab Fab or (177)Lu-DOTA-EGF. The NOAEL for (177)Lu-DOTA-Fab-PEG24-EGF was 11.1 MBq (10 μg). Trastuzumab-sensitive MDA-MB-231/H2N and trastuzumab-resistant TrR1 tumors were growth-inhibited by (177)Lu-DOTA-Fab-PEG24-EGF or (111)In-DTPA-Fab-PEG24-EGF. Unlabeled immunoconjugates had no effect on tumor growth. (177)Lu-DOTA-Fab-PEG24-EGF inhibited tumor growth more effectively than (111)In-DTPA-Fab-PEG24-EGF because of a 9.3-fold-higher radiation-absorbed dose (55.0 vs. 5.9 Gy, respectively).
  
  
  CONCLUSIONS
  These results are encouraging for further development of these bsRICs for dual-receptor-targeted radioimmunotherapy of BC coexpressing HER2 and EGFR, including trastuzumab-resistant tumors.
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Eva J
authors.initials: EJ
authors.lastname: Razumienko
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Jason C
authors.initials: JC
authors.lastname: Chen
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cai
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Conrad
authors.initials: C
authors.lastname: Chan
authors.affiliation: Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada Department of Medical Imaging, University of Toronto, Toronto, Ontario, Canada Toronto General Research Institute, Toronto, Ontario, Canada; and Joint Department of Medical Imaging, University Health Network, Toronto, Ontario, Canada raymond.reilly@utoronto.ca.
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Reilly
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics* / Q000532:radiotherapy / Q000628:therapy*; D066246:ErbB Receptors / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D018796:Immunoconjugates / Q000009:adverse effects / Q000627:therapeutic use*; D007205:Indium Radioisotopes / Q000009:adverse effects / Q000627:therapeutic use; D008187:Lutetium / Q000009:adverse effects / Q000627:therapeutic use; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D016499:Radioimmunotherapy / Q000379:methods*; D011868:Radioisotopes / Q000009:adverse effects / Q000627:therapeutic use; D011874:Radiometry; D019275:Radiopharmaceuticals / Q000009:adverse effects / Q000627:therapeutic use*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics*; D014018:Tissue Distribution; D023041:Xenograft Model Antitumor Assays
chemical_list: D066246:ErbB Receptors; D018796:Immunoconjugates; D007205:Indium Radioisotopes; D008187:Lutetium; D011868:Radioisotopes; D019275:Radiopharmaceuticals; D018719:Erb-b2 Receptor Tyrosine Kinases; C512478:EGFR protein, human; C508053:ERBB2 protein, human
keywords: 111In; 177Lu; EGFR; HER2; bispecific radioimmunoconjugates