Fab fragments of monoclonal antibodies directed against HER2/neu or tumor-associated glycoprotein-72 for radioimmunodetection of breast cancer

Description

Overexpressed surface receptors/antigens on cancer cells can be targeted specifically by antibodies for radioimmunoimaging or intraoperative radioimmunodetection of cancer. In breast cancer, HER2/neu and tumor-associated glycoprotein-72 (TAG-72) are overexpressed in 30% and 80% of patients, respectively. Their overexpression has been associated with a poor prognosis and more aggressive form of disease. The goal of this research was to construct Fab fragments of monoclonal antibodies directed against HER2/neu or TAG-72 for radioimmunoimaging and intraoperative radioimmunodetection of breast cancer. Fab fragments of the anti-HER2/neu monoclonal antibody trastuzumab (Herceptin ʾ ) were prepared by proteolysis of intact trastuzumab IgG and subsequently radiolabeled with 99m Tc or 111 In for tumor localization studies in athymic mice bearing subcutaneous HER2/neu-positive BT-474 breast cancer xenografts. Trastuzumab Fab was immunoreactive toward HER2/neu with a 2-4 fold higher K d compared with intact trastuzumab IgG (1.4-3.6 10 -8 M vs. 4.7-14 10 -9 M, respectively). Both 99m Tc- and 111 In-trastuzumab Fab localized avidly and specifically in BT-474 xenografts. 99m Tc-trastuzumab Fab was useful for imaging HER2/neu overexpression at early time points, i.e. up to 24 hours post-injection. Small BT-474 tumor xenografts (approximately 3-5 mm) were clearly visualized by gamma-scintigraphy as early as 2-6 hours after injection of 99m Tc-trastuzumab Fab. 111 In-trastuzumab Fab was useful for visualizing the BT-474 xenografts at later times, i.e. up to 72 hours post-injection. At this point, tumor uptake and the tumor-to-blood (T/B) ratio reached 7.8% ID/g and 25:1, respectively. Immunoreactive recombinant Fab (rFab) of anti-TAG-72 monoclonal antibody CC49 was produced by cloning the genes of Fab from CC49 hybridoma cells and subsequent expression in Pichia pastoris. rFab was purified by affinity chromatography to homogeneity. 123 I-rFab accumulated specifically in TAG-72-positive LS 174T colon cancer xenografts in athymic mice, evidenced by its 18-fold greater tumor uptake of 123 I-rFab in LS 174T xenografts than in TAG-72-negative A375 tumors (6.0 vs. 0.3% ID/g at 24 hours post-injection). 123 I-rFab was rapidly cleared from the blood and normal tissues, allowing LS 174T xenografts to be visualized by gamma-scintigraphy as early as 2 hours post-injection. These promising results suggest that trastuzumab Fab and CC49 rFab may be useful for radioimmunoimaging or intraoperative radio immunodetection of breast cancer.

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