EphA3 Gene Editing    

The Eph receptors constitute the largest subfamily of receptor tyrosine kinases (RTKs), and interact with cell membrane-bound ligands, called ephrins. Based on sequence similarity and ligand binding specificity, receptor and ligand families are divided into two categories: A and B. Ephrin-As are glycosyl phosphatidyl inositol (GPI) anchored while ephrin-Bs span the plasma membrane. In the human genome, there are nine EphAs and five EphBs, which usually preferentially bind five ephrin-A and three ephrin-B ligands, respectively. The Eph receptor and their ligands are often overexpressed in a variety of cancers and affect tumor growth, angiogenesis and metastasis.

Figure 1. Diagram showing domain structure of A- and B-type Ephs and their cognate ephrin ligands. (Janes P W, et al., 2014)

EphA3 was first identified as a surface antigen on a pre-B lymphoblastic leukemia cell line (LK63) in Andrew Boyd's laboratory by affinity separation with a monoclonal antibody (IIIA4) raised against the cells. Then, it was separately identified as an antigen on tumor cells from a melanoma patient, recognized by a lytic CD4+ T-cell immune response that is believed to promote tumor rejection. Therefore, from its identification, it has been suggested to play a role in cancer, a characteristic now understood to be true of many other Eph receptors.

EphA3 in Cancer

Similar to other protein families involved in the development, Eph receptors often re-emerge in cancer. For EphA3, many were initially found in tumor cell lines. EphA3 was first characterized in hematologic cancers, and was later identified as having a role in multiple hematologic disorders. Guan et al. collected 617 bone marrow samples from various types of hematologic malignancies and observed a significant association between the copy number variations of EphA3 and acute lymphoblastic leukemia, multiple myeloma, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, and myelodysplastic syndrome. EphA3 was identified in melanoma as a tumor antigen targeted by a lytic T-cell response in a patient, and high EphA3 levels were found in a series of melanomas, particularly metastatic tumors. Recent genomic screening of somatic copy number alterations in melanoma cell lines also identified EphA3 as both amplified and over-expressed. EphA3 over-expression is also reported in epithelial tumors including those of the lung and kidney, and in fact, integrated gene expression data suggests high expression in subsets of lung, breast, colorectal and gastric cancers. High levels of EphA3 expression are related to the high invasive capacity and poor overall survival in hepatocellular carcinoma, and with angiogenesis and poor prognosis in gastric cancer. In colorectal cancer (CRC), high expression in a subset of cancers positively correlates with tumor size and grade, infiltration and metastasis.

Therapeutic Targeting of EphA3

In view of the roles of EphA3 and other Ephs in the various mechanisms of tumor progression described above, a lot of effort has gone into the development of Eph inhibitors, including the use of kinase inhibitors, recombinant Eph or ephrin ECD fusion proteins, and most particularly monoclonal antibodies, because of their potential for high affinity and specificity. For EphA3, the main therapeutic candidate is the monoclonal antibody IIIA4. Similar to ligands, pre-clustered IIIA4 effectively triggers EphA3 activation, contraction of the cytoskeleton and cell rounding, and unclustered IIIA4 also enhances ligand activation in vitro. In addition, IIIA4 localizes to, and is internalized rapidly into, EphA3-positive human tumor cells in mouse xenografts, leading to significant inhibition of tumor growth. Furthermore, in a GBM mouse model, treatment with radio-labeled IIIA4 blocked tumor growth and markedly increased survival, indicating its potential as a highly specific tumor targeting agent. The IIIA4 antibody has also been used in the mouse model of blood disease. A recombinant chimeric version of IIIA4, composed of the mouse variable regions grafted onto a human framework, produced a humanized, non-fucosylated mAb initially termed KB004, and later named as the INN nomenclature of Ifabotuzumab. In vivo, KB004 exhibited significant inhibition of EphA3 positive tumor growth in a prostate cancer xenograft model. In addition, mAb KB004 activity in Pre-B-ALL xenografts in mice led to antileukemic effector responses that included an Ab-mediated reduction in metastatic site occurrences. The function of KB004 was also included in a phase-I clinical trial of treating patients with hematologic malignancies.

EPHA3 Gene Editing Services

CRISPR/Cas9 Platform^CB, one of the leading biotechnological companies specializing in gene editing, is dedicated to offering comprehensive CRISPR/Cas9 gene-editing services to a wide range of genomics researchers. Based on our platform, we can help you effectively EPHA3 gene deleted, inserted or point mutated in cells or animals by CRISPR/Cas9 technology.

• EPHA3 Gene Knockout: We offer EPHA3 gene knockout cell line and knockout animal model generation service with high quality. Typically, we develop CRISPR-mediated gene editing cell lines including HEK239T, Hela, HepG2, U87, but we can use other cell lines according to your requirements. Our one-stop KO animal model generation service covers from sgRNA design and construction, pronuclear microinjection to Founders genotyping and breeding.
• EPHA3 Gene Knockin: CRISPR/Cas9 Platform^CB provides the one-stop EPHA3 knock-in cell line and knockout animal model generation services, including point mutation and gene insertion. Our expert staff has succeeded in dozens of EPHA3 knock-in cell line generation projects, including stem cells, tumor cells and even difficult-to-handle cells. We also have extensive experience in incorporating CRISPR/Cas9 technology into animal models, which have been fully recognized by our clients.

If you have any questions, please feel free to contact us.

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