Radoimmunoimaging of a murine mammary carcinoma with a monoclonal antibody to vascular endothelial growth factor receptor-2

GABRIEL L. FISZMAN; ERNESTO G. D'ORIO; ADRIÁN GÓNGORA; ALEJANDRO MLADOVAN; MARÍA A. JASNIS; ALBERTO BALDI

Washington DC, USA

Congreso; American Association for Cancer Research; 2006

American Association for Cancer Research

The vascular endothelial growth factor (VEGF) has been identified as the major tumor angiogenic factor. One of its receptors, the VEGF receptor-2 (VEGFR-2, KDR in humans or flk1 in mice) is overexpressed in tumor and neovascular endothelial cells.The aim of the present work was to evaluate if VEGFR-2 could be used as a target for tumor radioimmunoimaging with monoclonal antibodies (Mabs).The extracellular domain of human recombinant KDR was amplified by RT-PCR from human umbilical vein endothelial cells (HUVEC) RNA, and cloned into an expression vector containing the Fc sequences from either human or murine IgG1. The fusion protein KDR-Fc was transiently expressed in COS-7 mammalian cells, and purified by protein G chromatography. For Mab production, KDR fused to murine Fc was used as immunogen, while KDR fused to human Fc was used for screening by solid phase ELISA. Hybridomas were also screened by cell-ELISA and cell proliferation assay using HUVEC. After cell fusion and selection, we isolated the hybridoma secreting a Mab that recognized KDR but unable to block VEGF binding to HUVEC.In order to test the Mab in vivo, we used a murine mammary adenocarcinoma cell line (LM3) which secrets VEGF. Cryostat sections of LM3 tumors showed cell membrane and diffuse cytoplasmic Immunofluorescence staining with the Mab. LM3 cells were injected sc in the flank of female BALB/c mice (4 x 105 cells). When tumors reached 5-8 mm in diameter, mice were injected with the Mab labeled with 131I by Iodogen method (6-9 ug representing 1.2-1.8 x106 cpm/mouse). Irrelevant mouse 131I-IgG (NIgG) was used as control. Mabs biodistribution and Gamma Camera images were evaluated at days 1, 2, 5 and 9. A mínimum of three mice was used for each time point, and the experiment was repeated three times.Scintigraphy images showed optimal tumor-background Mab uptake ratio, and were easily visible since day 1. Biodistribution, expressed as % of injected dose/tissue gram (%ID/g), showed Mab tumor uptake of 7, 5.12, 6.19 and 3.65 % vs NIgG tumor uptake of 5.15, 3.99, 2.90 and 1.52 %, at days 1, 2, 5 and 9 (p< 0.05) respectively. Levels of radiotracer were higher in tumors than in all of other tissues: at day 9 it was almost two folds higher in tumor than in lungs and 13- folds higher than in intestine. The ratio between %ID/g in tumor and %ID/g in the different organs (liver, intestine, spleen, lungs and kidneys) was always >1. At day 5, NIgG began to clear from the different tissues, while Mab showed slower tumor elution kinetics.Our Mab anti VEGFR-2 might be very useful as a tracer for in vivo tumor localization and eventually for the follow up of the disease.