Novel antibodies directed against the human erythropoietin receptor: creating a basis for clinical implementation

Abstract

SummaryRecombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been controversial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that several of these specifically recognize EPOR by Western blot, immunoprecipitation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment.

Widespread Expression of Erythropoietin Receptor in Brain and Its Induction by Injury

“…Against a common background mainly consisting of chaperone proteins, human EPOR protein was detected in eluates from ctEPOR-AB beads, but not from control beads. The identification of 11 EPOR-derived peptides with high mass accuracy at both precursor and fragment ion level resulted in sequence coverage of 22.6% ( Figures 2B, C), basically in line with recent LC-MS data on EPOR immunoprecipitates (37). Taken together, our results show that ctEPOR-AB indeed binds specifically to full-length EPOR.…”

“…The great need for specific EPOR-AB in the field is also reflected by a very recent study of Drorit Neumann and colleagues (37). This group of authors published specific mouse and rat monoclonal EPOR-AB that detect EPOR expression in human cancer cells and tissues (37).…”

“…This group of authors published specific mouse and rat monoclonal EPOR-AB that detect EPOR expression in human cancer cells and tissues (37). Complementary to this approach and with particular focus on the brain, we have developed a highly specific and sensitive polyclonal rabbit AB, ctEPOR-AB, suitable for applications not only in human but also in murine material.…”

Immunoreactivities for hepcidin, ferroportin, and hephaestin in astrocytes and choroid plexus epithelium of human brains

“…The brain samples were embedded in paraffin and sectioned at a thickness of 4 μm. The primary rabbit antibodies used in this study were antibodies against hepcidin (ab30760; Abcam, Cambridge, UK), ferroportin (SLC40A1) (ab78066; Abcam), hephaestin (NBP1‐85483; Novus Biologicals, Centennial, CO, USA), ferritin heavy chain (ab75972, Abcam), and early endosome antigen 1 (EEA1) (NBP1‐30914); Novus), while the primary mouse antibodies were those against ferroportin (ab239583; Abcam), glial fibrillary acidic protein (GFAP) (ab10062; Abcam), and EEA1 (NBP2‐36568; Novus). Information on the antibodies is summarized in Table .…”

Erythropoietin increases macrophage-mediated T cell suppression

“…Thus, like VEGF, EPO can exhibit pleiotropic functions that particular cancers can commandeer. With disagreement regarding erythropoietin receptor (EPOR) expression by various cancer cells [19, 27–35], the question remains as to how EPO might promote cancer progression. Evidence for leucocytes expressing the EPOR inform models for how EPO might temper immunity within TMEs [36–40].…”