Trastuzumab Labeled to High Specific Activity with 111In 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 In-111. In-111 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 In-111 for comparison (trastuzumab-NH-Bn-DTPA-In-111). The maximum specific activity (SA) for labeling trastuzumab-Hy-MCP with In-111 was 90-fold greater than for trastuzumab-NH-Bn-DTPA-In-111 [8.9 MBq/mu g (1.5 x 10(6) MBq/mu mol) vs 0.1 MBq/mu g (1.2 x 10(4) MBq/mu mol)]. Trastuzumab-Hy-MCP-In-111 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/mu g) than low SA (0.37 MBq/mu g) radioimmunoconjugates. At 20 nmol/L, high SA trastuzumab-Hy-MCP-In-111 was 6-fold more effective at reducing the clonogenic survival (CS) of HER2 overexpressed and HER2 gene-amplified SK-BR-3 cells (1.3 x 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-In-111 (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 x 10(5) receptors/cell) but without HER2 gene amplification was reduced to 20.5 +/- 4.3% by high SA trastuzumab-Hy-MCP-In-111 (20 nmol/L). The CS of HER2-overexpressed (5 x 10(5) HER2/cell) but trastuzumab-resistant TrR1 cells was decreased to 17.1 +/- 1.6% by high SA trastuzumab-Hy-MCP-In-111. Unlabeled trastuzumab (20 nmol/L) was 18-fold less potent than high SA trastuzumab-Hy-MCP-In-111 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 In-111 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.