Abstract P4-12-14: Metal chelating polymer-conjugated trastuzumab labeled to high specific activity with indium-111 is cytotoxic to trastuzumab-sensitive and resistant breast cancer cells through the emission of auger electrons
Authors: Ngo Ndjock Mbong G , Lu Y , Chan C , Cai Z , Winnik MA , Reilly RM 
Published: August 2014

Purpose: Auger electrons are very low energy electrons emitted by Indium-111(111In) that cause lethal DNA damage when emitted in close to the cell nucleus. Our laboratory reported previously that trastuzumab modified with nuclear localizing sequence (NLS) peptides and labeled with 111In(111In-NLS-DTPA-trastuzumab) was internalized and transported to the nucleus of HER2-overexpressing breast cancer (BC) cells, reducing their clonogenic survival (CS) to ≤10%. However, a limitation was that at the specific activity (SA) achieved (0.24 MBq/μg) for 111In-DTPA-trastuzumab without or with NLS, only 1 in 50 molecules was radiolabeled. This reduced the potency for cells with low or intermediate HER2 density. Our purpose was to dramatically increase the SA of 111In-labeled trastuzumab by conjugation to a metal chelating polymer (MCP) that displays multiple DTPA chelators for 111In and compare its potency to 111In-NLS-trastuzumab on BC cells with a wide range of HER2 density including cells resistant to trastuzumab.

Methods: An MCP with a polyglutamide backbone, 29 DTPA groups, and a hydrazide reactive end-group was site-specifically linked to periodate-oxidized carbohydrates on the Fc domain of trastuzumab. HER2 immunoreactivity, internalization and nuclear importation were evaluated by cell binding and fractionation studies using HER2-overexpressing SK-Br-3 human BC cells. The SA achievable was assessed by radiolabeling 1, 5, 50 and 100 μg of the immunoconjugates with 9.25 MBq of 111In. The effect of high SA (5.5 MBq/μg) 111In-MCP-trastuzumab to on the CS of human BC cells with a wide range of HER2 density (5×105-1.3×106 HER2 receptors/cell) that were sensitive or resistant to trastuzumab was compared to low SA (0.37 MBq/μg) 111In-labeled trastuzumab conjugates. Studies are also in progress to assess DNA double-strand breaks caused by the Auger electrons emitted by 111In using the γH2AX assay.

Results: MCP-trastuzumab bound specifically to HER2 with high affinity (KD = 10.7±1.5nM) and was internalized and imported into the nucleus of HER2-positive BC cells. The smallest mass that could be labeled (labeling efficiency&gt;80%) with 111In for MCP-trastuzumab was 1 μg (SA = 8.9 MBq/μg) compared to 50 μg for DTPA-trastuzumab (SA = 0.08 MBq/μg). High SA 111In-MCP-trastuzumab reduced the CS of SK-Br-3 cells with high HER2 density to 1.8±1.3% and the CS of TrR1 trastuzumab-resistant BC cells with intermediate HER2 density to 17.1±1.6%. These represent a 4-5 and 5-6 fold greater potency compared to low SA 111In-trastuzumab conjugates on cells with high or intermediate HER2 expression, respectively.

Conclusion: 111In-MCP-trastuzumab was labeled to 37-fold higher SA than 111In-DTPA-trastuzumab. High SA increased its cytotoxicity potency for HER2-positive BC cells that were sensitive or resistant to trastuzumab. Auger electron radioimmunotherapy is a promising strategy for HER2-positive BC which may extend to trastuzumab-resistant tumours. Supported by a grant from the Canadian Breast Cancer Research Alliance with funds from the Canadian Cancer Society.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-14.