Effective Tumor Targeting by EphA2-Agonist-Biotin-Streptavidin Conjugates.

Parima Udompholkul,Maurizio Pellecchia,Robert M Hoffman,Ming Zhao,Carlo Baggio,Luca Gambini,Yu Sun

doi:10.3390/molecules26123687

Abstract

We recently reported on a potent synthetic agent, 135H11, that selectively targets the receptor tyrosine kinase, EphA2. While 135H11 possesses a relatively high binding affinity for the ligand-binding domain of EphA2 (Kd~130 nM), receptor activation in the cell required the synthesis of dimeric versions of such agent (namely 135H12). This was expected given that the natural ephrin ligands also need to be dimerized or clustered to elicit agonistic activity in cell. In the present report we investigated whether the agonistic activity of 135H11 could be enhanced by biotin conjugation followed by complex formation with streptavidin. Therefore, we measured the agonistic EphA2 activity of 135H11-biotin (147B5) at various agent/streptavidin ratios, side by side with 135H12, and a scrambled version of 147B5 in pancreatic- and breast-cancer cell lines. The (147B5)n-streptavidin complexes (when n = 2, 3, 4, but not when n = 1) induced a strong receptor degradation effect in both cell lines compared to 135H12 or the (scrambled-147B5)4-streptavidin complex as a control, indicating that multimerization of the targeting agent resulted in an increased ability to cause receptor clustering and internalization. Subsequently, we prepared an Alexa-Fluor-streptavidin conjugate to demonstrate that (147B5)4-AF-streptavidin, but not the scrambled equivalent complex, concentrates in pancreatic and breast cancers in orthotopic nude-mouse models. Hence, we conclude that these novel targeting agents, with proper derivatization with imaging reagents or chemotherapy, can be used as diagnostics, and/or to deliver chemotherapy selectively to EphA2-expressing tumors.

Highlights

The receptor tyrosine kinase of the Eph family controls various cellular functions including cell–cell communications that are mediated by their interactions with cell surface ephrin ligands [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]
To characterize the binding properties of the agents, we developed a heterogeneous assay based on the Dissociation-Enhanced Lanthanide Fluorescent Immunoassay (DELFIA) [35] platform, where an earlier-generation biotinylated EphA2-binding peptide is captured on the surface of streptavidin-coated 96-well plates
For the current studies with agents that were biotinylated (145B5 and its scrambled version; Table 1), we could not run the DELFIA assay as the agents would be captured by the streptavidin-coated plate, we opted to measure direct binding affinities between the agents and EphA2-LBD via isothermal titration calorimetry (ITC)

Summary

Introduction

The receptor tyrosine kinase of the Eph family controls various cellular functions including cell–cell communications that are mediated by their interactions with cell surface ephrin ligands [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. Aberrant EphA2 overexpression causes an excess of unbound receptor and it contributes to the spread of certain solid tumors including prostate cancer [1,17], melanoma [13,18], breast cancer [2,3], brain cancer [10,19], ovarian cancer [20], urinary bladder cancer [11], pancreatic cancer [5,6,21,22], esophageal cancer [23], lung cancer [24], stomach cancer [25], and several types of leukemia [15,16]. An extremely aggressive tumor which accounts for about 7% of all cancer deaths in the United States, EphA2 overexpression is inversely correlated with patient survival [5,21]. Our recent studies in a variety of pancreatic cancer cell lines, or primary pancreatic cancer tissues, demonstrated elevated EphA2 levels in all studied cases [22]

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Results

Conclusion