Trastuzumab Labeled to High Specific Activity with (111)In by Site-Specific Conjugation to a Metal-Chelating Polymer Exhibits Amplified Auger Electron-Mediated Cytotoxicity on HER2-Positive Breast Cancer Cells.
Our objective was to evaluate the cytotoxicity toward HER2-positive human breast cancer (BC) cells of trastuzumab modified site-specifically with a metal-chelating polymer (MCP) that presents multiple DTPA chelators for complexing (111)In. (111)In emits subcellular range Auger electrons that induce multiple lethal DNA double-strand breaks (DSBs) in cells. MCPs were synthesized with a polyglutamide backbone with 24 or 29 pendant DTPA groups, with or without nuclear translocation sequence (NLS) peptide modification and a terminal hydrazide group for reaction with aldehydes generated by sodium periodate (NaIO4)-oxidation of glycans on the Fc-domain of trastuzumab. Trastuzumab was site-specifically modified with two DTPA and labeled with (111)In for comparison (trastuzumab-NH-Bn-DTPA-(111)In). The maximum specific activity (SA) for labeling trastuzumab-Hy-MCP with (111)In was 90-fold greater than for trastuzumab-NH-Bn-DTPA-(111)In [8.9 MBq/μg (1.5 × 10(6) MBq/μmol) vs 0.1 MBq/μg (1.2 × 10(4) MBq/μmol)]. Trastuzumab-Hy-MCP-(111)In was bound, internalized, and imported into the nucleus of SK-BR-3 cells. NLS peptide modification of MCPs did not increase nuclear importation. A greater density of DNA DSBs was found for BC cells exposed to high SA (5.5 MBq/μg) than low SA (0.37 MBq/μg) radioimmunoconjugates. At 20 nmol/L, high SA trastuzumab-Hy-MCP-(111)In was 6-fold more effective at reducing the clonogenic survival (CS) of HER2 overexpressed and HER2 gene-amplified SK-BR-3 cells (1.3 × 10(6) receptors/cell) than low SA MCP-radioimmunoconjugates (CS = 1.8 ± 1.3 vs 10.9 ± 0.7%; P = 0.001). Low SA trastuzumab-NH-Bn-DTPA-(111)In (20 nmol/L) reduced the CS of SK-BR-3 cells to 15.8 ± 2.1%. The CS of ZR-75-1 cells with intermediate HER2 density (4 × 10(5) receptors/cell) but without HER2 gene amplification was reduced to 20.5 ± 4.3% by high SA trastuzumab-Hy-MCP-(111)In (20 nmol/L). The CS of HER2-overexpressed (5 × 10(5) HER2/cell) but trastuzumab-resistant TrR1 cells was decreased to 17.1 ± 1.6% by high SA trastuzumab-Hy-MCP-(111)In. Unlabeled trastuzumab (20 nmol/L) was 18-fold less potent than high SA trastuzumab-Hy-MCP-(111)In at reducing the CS of SK-BR-3 cells (CS = 37.0 ± 5.3%) and 3-fold less effective against Zr-75-1 cells (CS = 53.1 ± 9.8%). Unlabeled trastuzumab had no effect on the survival of TrR1 cells. We conclude that increasing the SA for labeling with (111)In by site-specific conjugation of MCPs to trastuzumab greatly amplified the cytotoxic potency against HER2-overexpressed and gene-amplified BC cells and extended its cytotoxicity to cells with intermediate HER2 expression but without gene amplification and to cells that are HER2 overexpressed but trastuzumab-resistant.
†Department of Pharmaceutical Sciences, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada.
Ghislaine
G
Ngo Ndjock Mbong
‡Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6, Canada.
Yijie
Y
Lu
†Department of Pharmaceutical Sciences, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada.
Conrad
C
Chan
†Department of Pharmaceutical Sciences, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada.
Zhongli
Z
Cai
‡Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6, Canada.
Peng
P
Liu
†Department of Pharmaceutical Sciences, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada.
Amanda J
AJ
Boyle
‡Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6, Canada.
Mitchell A
MA
Winnik
†Department of Pharmaceutical Sciences, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada.|§Department of Medical Imaging, University of Toronto, 263 McCaul Street, Toronto, ON M5T 1W7, Canada.|∥Toronto General Research Institute, University Health Network, 200 Elizabeth Street, Toronto, ON M5G 2C4, Canada.
Raymond M
RM
Reilly
D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D005260:Female; D006801:Humans; D007205:Indium Radioisotopes / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D000068878:Trastuzumab / Q000737:chemistry* / Q000494:pharmacology
D007205:Indium Radioisotopes; D011108:Polymers; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab
Auger electrons; breast cancer; indium-111; metal-chelating polymers; trastuzumab