EPHA3 as a Novel Therapeutic Target in the Hematological Malignancies

Niamh Keane; Ciara Freeman; Ronan Swords; Francis J Giles


Expert Rev Hematol. 2012;5(3):325-340. 

In This Article

Preclinical Data & Translational Advances

With a high level of interclass binding, targeting specific Eph extracellular domains is difficult.[98] IIIA4, an IgG1κ mono-clonal antibody targeting EPHA3, was raised from BALB/c mice inoculated with LK63 pre-B ALL cell lines, and the RTK was first isolated during efforts to screen for novel RTKs.[15,99] IIIA4 targets a site closely adjacent to the heterotetramerization site on the N-terminal of EPHA3's extracellular domain adjacent to the ligand-binding site and has a high affinity for EPHA3.[16] Mutations of EPHA3 that alter the specific interaction site of IIIA4 or areas closely adjacent (G187R and S46F) and sushi domain and EGF-like domain mutations were recently shown to prevent IIIA4 binding to EPHA3.[12]

IIIA4 functions as an agonist of EPHA3 and, in ex vivo studies, binds with subnanomolar affinity (kD of 5 × 10−10 mol/l).[21] IIIA4 binds to and activates EPHA3, resulting in the same sequence of events that occurs following binding of its natural ligand ephrin-A5;[21] in other words, activation of EPHA3 kinase domain and cell morphology change with cell rounding, loss of cellular protrusions and cytoskeletal contraction.[21] IIIA4 must be preclustered in order to elicit these changes, or ephrin-A5 Fc administered in combination,[21] with synergy observed with this pairing.[21]

These data lend support to targeting a monoclonal antibody to EPHA3 as a therapeutic measure. The effect of IIIA4 in vivo against EPHA3 using mouse xenograft models of LK63 pre-B ALL, HEK293 cell line and SK-MEL28 melanoma cell lines was assessed. Rapid internalization of the monoclonal antibody and ephrin-A5 is observed on administration of IIIA4 in mice tumor xenograft models and the mouse leukemia model.[21] Further efforts then focused on refining the antibody for improved receptor targeting with clinical use in mind. IIIA4 has been modified using antibody humaneering technology to produce KB004, which has now entered clinical stage development. Humaneering technology results in production of monoclonal antibodies likely to exhibit low immunogenicity on repeated administration, having replaced nonhuman antibody V-segments with human sequences.[85] A nonfucosylated antibody was selected for, with that knowledge that this would enhance CD16-mediated antibody-dependent cell cytotoxicity.[85]

As well as effecting antibody-dependent cell-mediated cytotoxicity (ADCC) and inducing apoptosis, targeting of EPHA3 receptor by KB004 is likely to act through many of the effects of EPHA3 receptor activation as the antibody has agonist activity on EPHA3. Effects include loss of cell–matrix adhesion and alteration of cell phenotype reminiscent of mesenchymal–epithelial transition,[55] thus contributing to reduced invasiveness, activating RhoA, which contributes to the latter effects as well as bypassing prosurvival pathways outlined, and disruption of a myriad of interacting pathways and alteration of bone marrow microenvironment.[55] Activation of Eph RTK has a role in alteration of the surrounding stroma and, in particular, assists with downregulation of ephrin ligands[53] and inhibits bidirectional signaling in the ephrin-bearing cell, with consequent loss of promalignant pathways in that cell via src signaling.[54] With emerging data that EPHA3 is upregulated in the tumor microenvironment and supports survival of malignant cells, it is likely that KB004 will alter the microenvironment and render it hostile for tumor growth and proliferation.[100] The net effect is suppression of the malignant phenotype of the EPHA3-bearing cell, loss of support of survival pathways and alteration of the tumor microenvironment such that it is less conducive with promotion of hematologic malignancy. Furthermore, preclinical data have indicated that KB004 affects killing of cells via ADCC and apoptosis following KB004 binding (Figure 5).[85,101] Disruption of the malignant cells' microenvironment and compromised blood supply following this may have a role in mediating cell death.

Figure 5.

EPHA3-expressing cell pre- and post-binding of IIIA4/KB004. The cell phenotype and microenvironment of EPHA3-expressing hematologic malignancy cells is represented prior to the binding of the monoclonal antibody. The cells are flattened and adhere to surfaces. EPHA3 is upregulated in bone marrow in response to hypoxia and has been found to be expressed on bone marrow vasculature in acute myeloid leukemia samples. The promalignant features pertaining to this phenotype are outlined in Figure 3 and, in addition, the vascularity of the bone marrow promotes malignancy too. Binding of IIIA4 in preclinical models resulted in an agonist effect with activation of the EPHA3 kinase domain, recruitment of CrkII and activation of RhoA and downstream mediators. The antiproliferative effects of agonist binding of EPHA3 are represented in Figure 4. Cells change morphology to a less invasive phenotype. Antibody targeting of the stroma and blood vessels makes the bone marrow environment more hostile to EPHA3 cells. Apoptosis has been clearly demonstrated in EPHA3 cells targeted by KB004 (derived from IIIA4). Additionally, preclinical studies indicated that ADCC targeting EPHA3 cells is by CD16+ effector cells.

With regard to leukemia of myeloid origin, KB004 stimulated apoptosis following binding to EPHA3.[85] In cells from patients with CML refractory to imatinib, KB004 induced cell death via apoptosis and ADCC in malignant cells only.[85] In patients with AML, LSCs expressing CD123 coexpressed EPHA3, with specific targeting of the LSCs by KB004 and inhibition of myeloid leukemia colony formation following treatment with KB004[101] and normal CD34+ hematopoietic stem cells were not affected.[85] Other preclinical investigations extended analysis to include myeloma as well as CML, AML, myeloproliferative neoplasms and myelodysplastic syndrome.[101] Again, at least 50% of samples were EPHA3 positive.[101]

A Phase I clinical study entitled 'Study of the anti-EPHA3 monoclonal antibody KB004 in subjects with EPHA3-expressing hematological malignancies' is ongoing.[201] This safety study is nonrandomized with single group assignment and four treatment arms assigning patients to different doses of KB004. Given the promising preclinical data and the many possible roles for KB004 in treatment of hematological malignancies, results from the trial are keenly awaited with the hope that targeting EPHA3 may radically alter the treatment and prognosis of a number of malignancies.


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