Abstract B32: Microenvironmental distribution of trastuzumab in metastases and xenograft models is highly heterogeneous and decreases sharply when administered in combination with bevacizumab

Despite significant success, the response of Her2+ patients to trastuzumab (TzMAb, Herceptin ®) is varied, with many still experiencing tumor progression. We have used 3D tissue, tumor xenograft and metastatic models to examine the microregional distribution of TzMAb when administered alone or in combination with bevacizumab (BvMAb). Methods: Her2 positive (Her2+) SKOV-3 ovarian and MDA361, JIMT-1, BT474 mammary cancer cells were grown as 3D tissue discs, spheroids and as xenografts. Her2 expression was ranked as SKOV3 > BT474 > JIMT-1 > MDA361. Variable concentrations (25-100 µg/mL for in vitro; 2.5-10 mg/kg ip q3d for in vivo) of TzMAb, BvMAb or isotype IgG control antibodies were administered and tissues collected. Multiplexed immunohistochemistry generated maps of whole tissue sections for quantitative and qualitative analysis. In addition to direct visualization of fluorescent-tagged antibody therapeutics, features including Her2 expression (Her2), hypoxia (pimonidazole), blood vessels (CD31), vascular perfusion (carbocyanine fluorescent dye), pericytes (SMA, desmin), basal lamina (CIV) and tight junctions (ZO-1) were mapped relative to each other. Findings: In vitro: The rate of TzMAb distribution through 3D Her2+ tissue models was not significantly different to that of BvMAb or an isotype control (IgG). Inter-model variability was not correlated with the degree of Her2 expression. In vivo: All the xenograft models exhibited highly heterogeneous distribution of TzMAb, with variation at inter-vessel, inter-tumor and intra-tumor levels. There was no discernible pattern in the deposition of TzMAb; it was not limited to tumor margins or the central core, and was often bound up to 200 µm away from the nearest blood vessels. Other areas containing perfused vessels had little to no binding. Areas of limited TzMAb binding persisted after repeat dosing. SKOV-3 and BT474 metastatic lesions collected from liver and lung tissues had on average greater TzMAb staining intensity than was seen in subcutaneous tumors. However, there was a significant range in the ability of TzMAb to access and bind Her2+ve cells. Lesions ranged from less than 150 µm to several mm in diameter, with some metastases showing bound TzMAb on every cell and others showing no observable bound drug despite containing perfused vessels or being surrounded by highly perfused normal lung or liver tissue. For both subcutaneous tumors and metastatic lesions, no consistent, quantifiable difference o