Abstract 4553: Novel bispecific 111In-labeled immunoconjugates (bsICs) for imaging HER2/HER3 heterodimerization in breast cancer

Abstract

Abstract Objective: Our objective was to construct novel bsICs capable of binding HER2 and HER3 labeled with 111In for imaging HER2-HER3 heterodimerization in breast cancer (BC) possibly through higher avidity for dimeric vs. monomeric complexes. Methods: bsICs were composed of trastuzumab Fab recognizing HER2 linked to a 7.5 kDa HER3-binding peptide of heregulin-β1 (pHRG). Fab were produced by digestion of trastuzumab IgG with papain. Fab were modified with Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) or related analogues with polyethyleneglycol (PEG) spacers (SM-PEG12-NHS and SM-PEG24-NHS) to introduce maleimide groups for cross-linking to pHRG thiolated with Traut's reagent. bsICs were derivatized with diethylenetriaminepentaacetic acid (DTPA) for labeling with 111In. The ability to independently bind HER2 or HER3 was determined in competition assays using 111In-bsICs on cells expressing HER2, HER3 or both receptors competed with a 25-fold excess of Fab, pHRG or combined ligands. Results: Conjugation of Fab to pHRG was confirmed by SDS-PAGE, Western blot and size-exclusion HPLC. bsICs were labeled with 111In to a radiochemical purity of 98.9 ± 0.2% (specific activity 375 ± 14 MBq/mg). Insertion of a PEG spacer between Fab and pHRG was required for displacement of binding of 111In-bsICs to HER3 but not HER2. For 111In-bsICs with the longest spacer (PEG24), the binding to SKBR-3 cells (HER2+/HER3+) was reduced to 32.7 ± 0.7%, 85.9 ± 1.0% or 30.4 ± 1.6%, respectively by Fab, pHRG or both ligands. The binding to SKOV-3 cells (HER2+/HER3-) was reduced to 16.7 ± 0.8%, 88.8 ± 5.3% or 13.4 ± 0.1% by Fab, pHRG or both ligands. The binding to MDA-MB-468 cells (HER2-/HER3+) was reduced to 98.5 ± 1.5%, 68.8 ± 0.3% or 64.8 ± 4.5% by Fab, pHRG or both ligands. Less efficient competition for HER3 was observed for the PEG12 spacer and no competition for 111In-bsICs without a spacer. Conclusion: 111In-bsICs composed of trastuzumab Fab and pHRG bound specifically to HER2 and HER3 when an appropriate length PEG spacer was inserted between these two binding moieies. These 111In-bsICs may be useful for imaging HER2-HER3 heterodimerization in BC. Supported by the Ontario Institute of Cancer Research (1 mm Challenge) and the Canadian Breast Cancer Foundation (Ontario Branch). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4553.