Diabodies Targeting Epithelial Membrane Protein 2 Reduce Tumorigenicity of Human Endometrial Cancer Cell Lines

Abstract

Purpose Endometrial cancer (EC) is the most common gynecologic malignancy. One promising biomarker is epithelial membrane protein-2 (EMP2), and its expression is an independent prognostic indicator for tumors with poor clinical outcome expression. The present study assesses the suitability of EMP2 as a therapeutic target. Experimental Design Human monovalent anti-EMP2 antibody fragments were isolated from a human phage display library, and engineered as bivalent antibody fragments (diabodies) with specificity and avidity to both EMP2 peptides and native cell-surface EMP2 protein. Diabodies were assessed using cell death and apoptosis assays. In addition, the efficacy of EMP2 diabodies on EC tumors was determined using mouse xenograft models. Results Treatment of human endometrial adenocarcinoma cell lines with anti-EMP2 diabodies induced significant cell death and caspase 3 cleavage in vitro. These responses correlated with cellular EMP2 expression, and were augmented by progesterone (which physiologically induces EMP2 expression). In vivo, treatment of subcutaneous human xenografts of HEC-1A cell lines with anti-EMP2 diabodies suppressed tumor growth, and induced striking xenograft cell death. Conclusions These findings suggest that EMP2 may be a potential pharmacological target for human EC.

Rationale and Preclinical Efficacy of a Novel Anti-EMP2 Antibody for the Treatment of Invasive Breast Cancer

“…In the present study, we examined the protein expression and frequency of EMP2 in human invasive breast cancers with an emphasis on triple negative disease. Given its high membrane expression, we hypothesized that EMP2 may serve as novel target for therapy, and consistent with this idea, anti-EMP2 antibody fragments (diabodies) resulted in reduced tumor growth in both endometrial and ovarian cancer models (7, 20). To further the use of anti-EMP2 therapy, we created a novel fully human anti-EMP2 IgG1 antibody and characterized its therapeutic potential.…”

“…For studies described here, we constructed a fully human anti-EMP2 IgG1 antibody. To do this, the Db variable (V) region sequences of KS49 were obtained by PCR (20) and then cloned into the pCR-II-TOPO vector (Life Technologies, Carlsbad, CA). The cloning was confirmed by sequencing.…”

“…Biotinylated 24 amino acid peptides corresponding to the extracellular loop of human EMP2 (20) were coated onto streptavidin-coated 96-well plates (Roche Applied Science, Indianapolis, IN). ELISA was performed as described previously (20).…”

Epithelial Membrane Protein-2 (EMP2) Activates Src Protein and Is a Novel Therapeutic Target for Glioblastoma

“…Anti-EMP2 diabodies and control diabodies have been detailed previously (11,26), and the variable regions were recently cloned to produce a fully human IgG1 (12). Both cell lines were also tested for murine pathogens, including mycoplasma by the Division of Laboratory Animal Medicine at UCLA prior to injection.…”

“…In contrast to native IgG1, diabodies biodistribute more quickly, penetrate target tissues efficiently, and clear more rapidly from circulation. Both diabody and native IgG1 reagents bind to the second extracellular loop of EMP2 (26), and these immunoglobulin variants show cross-reactivity for mouse and human EMP2 as detected by flow cytometry and IHC (26). To determine whether the anti-EMP2 diabody or IgG1 could induce cell death, a panel of GBM cells such as U87MG, U87/EGFR VIII, U373, T98, and GM5 were incubated with EMP2-specific immunoglobulin reagents or a vehicle control.…”