Immunotherapeutic Targeting of EphA3

Abstract

Eph receptors form the largest family of receptor tyrosine kinases. Eph receptors interact with cell-surface ephrin ligands to direct cell growth and migration. High expression of Eph receptors in a number of cancers has been linked to progression through facilitation of invasiveness and metastatic spread. EphA3 protein, which was originally described in leukemia, has also reported to be expressed in sarcomas, lung cancer, prostate cancer, melanoma and glioblastoma.The EphA3-specific monoclonal antibody, IIIA4, binds and activates human and mouse EphA3 with similar affinities. Binding is followed by internalization of receptor-antibody complexes. High expression of EphA3 has been reported on LK63 pre-B acute lymphoblastic leukemia (ALL) cell line and contrasts with lack of expression of EphA3 in the Reh, a similar pre-B ALL cell line. We investigated the effect of IIIA4 monoclonal antibody treatment in LK63 and Reh NOD/SCID xenograft models. In the LK63 xenograft model, administration of the IIIA4 antibody led to inhibition of tumor growth and decreased spread from bone marrow to the spleen and other organs with a corresponding increase in survival, however no reduction in engraftment was observed in Reh xenograft model, suggesting that the effect was directed against the leukemic cells rather than the stromal and vascular elements in these tumors.To confirm the tumor-specific effect of IIIA4 therapy, LK63 EphA3-knockdown and Reh EphA3-expressing cell lines were developed and tested in the xenograft model of leukemia. Similar to LK63 xenograft model, upon treatment with IIIA4 monoclonal antibody, the EphA3-expressing Reh xenografts showed reduction in the bone marrow engraftment and slowing of the disease progression. Consistent with the effect of EphA3 monoclonal antibody treatment on Reh xenograft model, the LK63 EphA3-knockdown xenografts showed minimal difference between treated and control group but had a notably significant reduction in the splenic engraftment compared to the normal LK63 model.To understand the mechanism leading to therapeutic effect of IIIA4 on EphA3-expressing leukemic xenografts, the effect of EphA3 activation on the LK63 and LK63 EphA3-knockdown cells was examined. This analysis showed elevated level of Src kinase activity upon stimulation with either IIIA4 or ephrinA5-Fc. Src kinases have been implicated in Eph-mediated signalling in other systems, and its activation appears to target adhesive mechanisms, particularly integrins, and cell motility functions consistent with the observed effects of antibody on cell movement.Given the capacity of the antibody to induce internalisation of EphA3, the possibility of linking a cytotoxic payload to IIIA4 was examined. The alpha-particle emitting radio-active isotope bismuth-213 was linked to IIIA4 and tested in the LK63 model with a considerably enhanced therapeutic effect specifically a significant increase in survival. More importantly, no toxicity to normal tissues was observed, as EphA3 is expressed at very low level or is entirely absent on normal tissues.A Humaneered® form (FA225-C10) of IIIA4 antibody has been generated for clinical development. This antibody binds to EphA3 with an improved affinity and has retained the direct apoptotic activity of mouse IIIA4 antibody. In the xenograft model of leukemia the antibody directly affects the tumor cells with no effect on stromal and vascular elements. In human tumor xenografts of prostate cell line DU-145 in nude mice, FA225-C10 antibody produced a significant inhibition of tumor growth. In this model the tumor cells do not express EphA3 but the tumor stroma and neovasculature express EphA3 and treatment with antibody reduced both tumor stroma and new vessel density.In Summary targeting EphA3 tumor cells and/or tumor stromal fibroblasts may therefore constitute a novel approach to treating solid tumors as well as hematologic malignancies. The Phase 2 component of a clinical study in acute myeloid leukemia (AML), myelodysplastic syndrom (MDS) and myelofibrosis (MF) patients is ongoing in which the activity of KB004 will be characterized. Future clinical studies might be extended to other cancers including acute lymphoblastic leukaemia (ALL) and other solid tumors. DisclosuresYarranton:Glaxo: Equity Ownership; Stemline Therapeutics: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; KaloBios: Employment.