The development and evaluation of novel monoclonal antibodies directed against folate pathway components

Abstract

Folates are essential in the de novo nucleotide biosynthetic pathway, folate receptor alpha (FR-α) is a folate transporter which is an attractive target for anticancer drug design due to its limited expression in normal tissues. It has great potential to direct therapies toward pathologic cells whilst minimizing toxicity in normal tissues. The enzyme folylpolyglutamate synthetase (FPGS) polyglutamates folates and folate analogues, trapping them within the cell and increasing their affinity as substrates for subsequent enzymatic reactions. Expression of folate biochemical pathway components, such as FR-α and FPGS, may be indicators of malignancy and also determine response to antifolate chemotherapeutics and other folate pathway targeted therapies currently being evaluated. Knowledge of their expression in tumours may enable optimal use by identifying responsive subgroups of patients. In spite of their importance in the diagnosis and treatment of cancer, the lack of monoclonal anti-FR-α and FPGS antibodies suitable for immunohistochemistry (IHC) analysis of formalin fixed, paraffin embedded biopsy samples or Western blot analysis has limited research in this area. The aim of this project was to generate and fully characterize monoclonal antibodies to detect specific expression of these proteins in formalin fixed, paraffin embedded tissues for use on archival tissue and samples collected prospectively in connection with clinical trials of antifolates. Novel monoclonal antibodies with specificity for sensitive detection of FR-α and FPGS in paraffin-embedded tissues were developed. ELISA and Western blot analysis confirmed specific reactivity of both antibodies to the recombinant protein, a single 40kD protein in whole cell lysates from cell lines known to express FR-α, and a single 60kD protein from cells expressing FPGS. Epitope mapping experiments confirmed specificity restricted to a linear epitope present in the protein target sequences. IHC analysis of FR-α in a panel of normal tissues demonstrated predominantly membrane with cytoplasmic staining limited to some ovarian epithelia (inclusion cysts), placental trophoblasts and proximal kidney tubules; FPGS demonstrated a wider pattern of expression. FR-α analysis of a panel of malignant and benign tissues demonstrated limited expression with variable intensities of staining and patterns of membrane and cytoplasmic Development and Evaluation of Novel Monoclonal Antibodies to FR-α and FPGS VI reactivity between cases. In the majority of malignant ovarian tumours, including an archival tissue microarray (TMA) of 167 tumour samples, membrane staining was observed in 89% of cases. FPGS analysis on a panel of benign and malignant tissues demonstrated frequent and high cytoplasmic expression in a range of tumours compared to normal adjacent tissue. The archival ovarian cancer TMA analysis showed a significant association between high expression of FR-α and poor patient survival (p=0.012, Cox regression) indicating a role for FR-α as a prognostic marker and potential therapeutic target for ovarian cancer and other cancers with expression of FR-α, detectable via the use of our antibody on FFPE tumour samples.