Identification and characterization of Nanobodies targeting the EphA4 receptor.

The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders. Therefore, the aim of our study was to generate potent and selective Nanobodies against the ligand-binding domain of the human EphA4 receptor. We identified two Nanobodies, Nb 39 and Nb 53, that bind EphA4 with affinities in the nanomolar range. These Nanobodies were most selective for EphA4, with residual binding to EphA7 only. Using Alphascreen technology, we found that both Nanobodies displaced all known EphA4-binding ephrins from the receptor. Furthermore, Nb 39 and Nb 53 inhibited ephrin-induced phosphorylation of the EphA4 protein in a cell-based assay. Finally, in a cortical neuron primary culture, both Nanobodies were able to inhibit endogenous EphA4-mediated growth-cone collapse induced by ephrin-B3. Our results demonstrate the potential of Nanobodies to target the ligand-binding domain of EphA4. These Nanobodies may deserve further evaluation as potential therapeutics in disorders in which EphA4-mediated signaling plays a role.

Development of a Trispecific Antibody Designed to Simultaneously and Efficiently Target Three Different Antigens on Tumor Cells.

Targeting Eph (erythropoietin producing hepatoma) receptors with monoclonal antibodies is being explored as therapy for several types of cancer. To test whether simultaneous targeting of EphA2, EphA4, and EphB4 would be an effective approach to cancer therapy, we generated a recombinant trispecific antibody using the variable domain genes of anti-EphA2, anti-EphA4, and anti-EphB4 monoclonal antibodies. A multidisciplinary approach combining biochemical, biophysical, and cellular-based assays was used to characterize the trispecific antibody in vitro and in vivo. Here we demonstrate that the trispecific antibody is expressed at high levels by mammalian cells, monodispersed in solution, thermostable, capable of simultaneously binding the three receptors, and able to activate the three targets effectively as evidenced by receptor internalization and degradation both in vitro and in vivo. Furthermore, pharmacokinetic analysis using tumor-bearing nude mice showed that the trispecific antibody remains in the circulation similarly to its respective parental antibodies. These results indicate that simultaneous blockade of EphA2, EphA4, and EphB4 could be an attractive approach to cancer therapy.

EphA4 receptor tyrosine kinase is a modulator of onset and disease severity of experimental autoimmune encephalomyelitis (EAE).

The EphA4 receptor tyrosine kinase is a major regulator of axonal growth and astrocyte reactivity and is a possible inflammatory mediator. Given that multiple sclerosis (MS) is primarily an inflammatory demyelinating disease and in mouse models of MS, such as experimental autoimmune encephalomyelitis (EAE), axonal degeneration and reactive gliosis are prominent clinical features, we hypothesised that endogenous EphA4 could play a role in modulating EAE. EAE was induced in EphA4 knockout and wildtype mice using MOG peptide immunisation and clinical severity and histological features of the disease were then compared in lumbar spinal cord sections. EphA4 knockout mice exhibited a markedly less severe clinical course than wildtype mice, with a lower maximum disease grade and a slightly later onset of clinical symptoms. Numbers of infiltrating T cells and macrophages, the number and size of the lesions, and the extent of astrocytic gliosis were similar in both genotypes; however, EphA4 knockout mice appeared to have decreased axonal pathology. Blocking of EphA4 in wildtype mice by administration of soluble EphA4 (EphA4-Fc) as a decoy receptor following induction of EAE produced a delay in onset of clinical symptoms; however, most mice had clinical symptoms of similar severity by 22 days, indicating that EphA4 blocking treatment slowed early EAE disease evolution. Again there were no apparent differences in histopathology. To determine whether the role of EphA4 in modulating EAE was CNS mediated or due to an altered immune response, MOG primed T cells from wildtype and EphA4 knockout mice were passively transferred into naive recipient mice and both were shown to induce disease of equivalent severity. These results are consistent with a non-inflammatory, CNS specific, deleterious effect of EphA4 during neuroinflammation that results in axonal pathology.

[Preparation and analysis of monoclonal antibody against EphA4 peptide].

OBJECTIVE: To prepare monoclonal antibody (MAb) against carboxy-terminus of E-phA4 and to analyze the immunological characteristics and significance of the antibody. METHODS: Mice were immunized with a chemically synthesized peptide that had been conjugated to KLH (keyhole limpet hemocyamin). A panel of antibodies specifically against EphA4 were obtained by hybridoma technique. The immunological properties and significance of the MAb were analyzed by immunological and immunochemistry techniques. RESULTS: A hybridoma cell line secreting anti-EphA4 MAb was established. The MAb reacted specifically with human, chicken and mouse EphA4, but did not cross-react with EphA5 and EphA7. The antibody could be used for immunochemical techniques such as ELISA, immunoprecipitation, Western blot and immunohistochemistry. CONCLUSION: The anti-EphA4 MAb generated by immunizing a synthetic peptide possesses excellent immunological properties. MAb can be applied for immunological and immunohistochemical purpose and will become an important tool in the study of EphA4 and its ligand.