Dual-Receptor-Targeted Radioimmunotherapy of Human Breast Cancer Xenografts in Athymic Mice Coexpressing HER2 and EGFR Using 177Lu- or 111In-Labeled Bispecific Radioimmunoconjugates.

UNLABELLED: 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 ß-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). CONCLUSION: 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.

Small-animal SPECT/CT of HER2 and HER3 expression in tumor xenografts in athymic mice using trastuzumab Fab-heregulin bispecific radioimmunoconjugates.

UNLABELLED: Heterodimerization of human epidermal growth factor receptor 2 (HER2) with HER3 initiates aberrant downstream growth-signaling pathways in tumors. Our objective was to construct bispecific radioimmunoconjugates (bsRICs) that recognize HER2 and HER3 and evaluate their ability to image tumors in athymic mice that express one or both receptors using small-animal SPECT/CT. METHODS: bsRICs were constructed by reacting the maleimide-derivatized trastuzumab Fab fragments that bind HER2 with a thiolated form of the HER3-binding peptide of heregulin-ß1 (HRG) with or without a 12- or 24 mer polyethylene glycol (PEG) spacer. bsRICs were derivatized with diethylenetriaminepentaacetic acid for labeling with (111)In. The ability of (111)In-bsRICs to bind HER2 or HER3 was determined in competition assays with unlabeled Fab or HRG on cells expressing one or both receptors. Tumor and normal-tissue uptake were examined in CD1 athymic mice bearing subcutaneous tumor xenografts that expressed HER2, HER3, or both receptors, with or without the preadministration of unlabeled Fab or HRG to determine the specificity of uptake. RESULTS: Conjugation of Fab to HRG was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis-Western blot and size-exclusion high-performance liquid chromatography. Improved HER2 and HER3 binding and greater displacement of binding by competitors was found for (111)In-bsRICs that incorporated a PEG spacer, with the PEG(24) spacer being optimal. The highest uptake of (111)In-bsRICs (7.8% ± 2.1% injected dose per gram [%ID/g]) in BT-474 human breast cancer xenografts (HER2-positive/HER3-positive) occurred at 48 h after injection. The preadministration of trastuzumab Fab decreased uptake in SK-OV-3 (HER2-positive/HER3-negative) human ovarian cancer xenografts from 7.0 ± 1.2 to 2.6 ± 1.5 %ID/g (P < 0.001). The preadministration of an excess of HRG decreased uptake in MDA-MB-468 (HER2-negative/HER3-positive) human breast cancer xenografts from 4.4 ± 0.9 to 2.6 ± 0.5 %ID/g (P < 0.05). All tumors were imaged by small-animal SPECT/CT. CONCLUSION: (111)In-bsRICs composed of trastuzumab Fab and HRG exhibited specific binding in vitro to tumor cells displaying HER2 or HER3 and were taken up specifically in vivo in tumors expressing one or both receptors, permitting tumor visualization by small-animal SPECT/CT. These agents could be useful for imaging heterodimerized HER2 and HER3 receptors because their bivalent properties may result in preferential binding to the heterodimerized forms. The approach may also be extended to constructing bsRICs for visualizing other peptide growth factor receptors.