Preclinical Evaluation of an Anti-EphA2 Minibody-Based ImmunoPET Agent as a Diagnostic Tool For Cancer

In this study, we report the development and characterization of a copper-64 (⁶⁴Cu) radiolabeled anti-EphA2 minibody (Mb) for pre-treatment characterization of antigen expression via Positron Emission Tomography (PET). Minibodies, ≈85 kDa molecular weight antibody fragments, are advantageous as targeting molecules due to accelerated serum clearance which enables imaging at earlier time points relative to the parent IgG. As EphA2, a tyrosine kinase receptor, is overexpressed in various cancer types with minimal expression in normal tissue, rapid quantification of EphA2 expression could be beneficial for patient stratification.

Recombinantly produced anti-EphA2-Mb was evaluated for purity, stability, affinity, and in vivo target localization. Following bifunctional chelator conjugation, radiolabeling with ⁶⁴Cu and evaluating purity, stability and immunoreactivity of resultant radioimmunoconjugate, [⁶⁴Cu]Cu-NOTA-anti-EphA2-Mb, 11.1 MBq (300 μCi) and 0.2 MBq (5 μCi) doses were administered to HT1080-fibrosarcoma-bearing nude mice for in-vivo PET imaging and ex-vivo biodistribution analyses respectively at 4 and 24 h post-injection (p.i.). Antigen-specificity was assessed via a blocked control group which received the dose co-administered with non-radiolabeled anti-EphA2-Mb.

Anti-EphA2-Mb produced via recombinant protein expression was pure, stable and had high binding affinity to human EphA2 antigen (K_D = 0.63 ± 0.24 nM). When labeled with ⁶⁴Cu via NOTA, [⁶⁴Cu]Cu-NOTA-anti-EphA2-Mb had high purity, in-vitro stability in PBS and mouse serum up to 24 h, and high immunoreactivity. On administering to tumor-bearing mice, [⁶⁴Cu]Cu-NOTA-anti-EphA2-Mb exhibited rapid tumor targeting with 25.53±2.92%ID/g at 4 h, and 22.13±7.68%ID/g at 24 h p.i. Competitive inhibition reduced tumor uptake (11.24±0.88%D/g, 24 h p.i., p = 0.0286). There was minimal uptake of the radiotracer in non-target tissues, except kidney and liver, and fast clearance from the blood, with high tumor to blood ratios. Tumor SUV_mean values obtained from region of interest (ROI) Quantification of the PET images were 1.13±0.03 and 1.08±0.06 at 4 and 24 h respectively.

Our findings demonstrate that anti-EphA2-Mb is an excellent targeting molecule, and [⁶⁴Cu]Cu-NOTA-anti-EphA2-Mb is a promising immunoPET agent with potential for use for other theranostic applications.