Isolation and characterisation of antibodies against human and canine EphA2_ targets for comparative studies in brain cancer

Abstract

Glioblastoma multiforme (GBM) is one of the high-grade gliomas, with a patient median survival of around 15 months from the time of diagnosis. Current treatments for this brain cancer initially comprise of surgery and radiotherapy, followed by administration of Temozolomide. Despite many advances in preclinical studies of GBM in mice, the outcomes have not been translated to the clinical level for humans. This is because the mouse model of GBM does not recapitulate the human disease and is not a suitable model for comparative studies of spontaneous tumours in humans. The canine model represents a powerful, large animal model of gliomas, since canine brain tumours occur spontaneously, with an incidence rate and patient age profile quite similar to human populations.EphA2 is one of the multi-domain receptors of the most significant tyrosine kinase family of receptors, and is highly expressed in both human and canine glioblastoma. Overexpression of EphA2 has been shown to correlate with tumour stage, progression and patient survival. Since EphA2 is not expressed in normal brain tissue, but is over-expressed on GBM cells, it is potentially a highly useful receptor for antibody-based therapy of brain cancer in both humans and dogs. Thus, monoclonal antibodies (mAbs) which cross-react with both human and canine EphA2 would be valuable molecular entities with diagnostic and therapeutic potential.To generate mAbs specific for EphA2 with cross-reactivity to both canine and human EphA2 receptors, phage display technology was employed using various strategies for the presentation of the EphA2 antigen to the phage antibody library. This included biopanning against firstly recombinant whole extracellular domain (ECD) of both human and canine EphA2, secondly recombinant ligand-binding domain (LBD) of human and canine EphA2, and lastly against cells expressing the native EphA2.Biopanning against recombinant EphA2-ECD generated one promising antibody (mAb AGH001) which showed cross-reactivity to both recombinant human and canine EphA2-ECD. However, mAb AGH001 was shown to also bind to EphA2 Knock Down (KD) cells, suggesting mAb AGH001 may bind to a domain within EphA2-ECD, which is conserved among EphA family receptors, other than the unique LBD of EphA2. To isolate specific anti-EphA2 antibodies for the LBD on EphA2-ECD, the human and canine LBD was utilised as an antigen for a biopanning campaign.Utilising this strategy of biopanning against the LBD of EpahA2, mAb HuB1 was isolated and showed binding to both human and canine EphA2-LBD and EphA2-ECD as well; however, mAb HuB1 also showed binding to the negative EphA2 cell line. This could be due to partially overlapping epitopes on other Eph receptors.mAb AGH001 and a positive control antibody, murine mAb 4B3 did not bind to EphA2-ECD sub-domains. It can be concluded that the recombinant ECD sub-domains did not recapitulate the native conformation of EphA2. Furthermore, we demonstrated that mAb AGH001 has a conformational epitope, therefore relies on a structural conformation provided by the recombinant ECD. Cell-based biopanning was subsequently employed to isolate the EphA2 mAbs with specificity to native, membrane-bound EphA2. Initial biopanning against EphA2 KD cells was carried out as a depletion step, followed by biopanning of EphA2 positive cells; however, binding studies of phage antibody clones showed that the antibodies were not specific for EphA2. Although biopanning of a phage antibody library against both canine and human EphA2 was partially successful to the extent that antibodies were isolated, these antibodies were not specific for EphA2 and were not employed in further studies. In studies associated with demonstrating that antibodies against EphA2 and EphA3 may assist in the diagnosis of GBM, commercial antibodies were used, as well as a control mAb that binds EGFR. These antibody preparations were used to detect EphA2 and EphA3 receptors on characterised GBM patient-derived cell lines by conjugation to Raman reporters. Binding studies were consistent with the expected expression levels on these cell lines. Multiplexing of biomarkers can be used to detect GBM heterogeneity for diagnostic purposes, and the goal is to develop an assay that can be used to detect GBM circulating tumour cells (CTC) from blood samples.This thesis highlights the challenges associated with isolating specific antibodies for a particular member of a receptor family, where there are high levels of homology to other members of the same family or where there are shared domains with other receptor families. However, the potential benefits of such antibodies towards the diagnosis and therapy of cancers with a low life expectancy, such as GBM, warrants the development of innovative methods to isolate specific mAbs that bind EphA receptors. Encouragingly, this research has shown the utility of using antibodies that bind EphA2 and EphA3 receptors as new diagnostic tools that would facilitate the monitoring of treatment efficacy. This would be a major breakthrough, since serial biopsies to monitor treatment efficacy are challenging and also medical imaging can yield false negatives/positives, and cannot be relied upon to detect treatment response.