Developing and Characterizing the Tumor-Targeting Efficiency of an Anti-EphA2-CD11b Bispecific Antibody

Targeting molecules, such as antibodies and peptides, play a key role in the precise delivery of cytotoxic payloads to tumor sites by binding to specific tumor-associated antigens or other proteins within the tumor microenvironment. This investigation evaluates the potential therapeutic application of a bispecific antibody (BsAb), which simultaneously targets EphA2, a tumor-associated antigen, and CD11b, a protein expressed by tumor-associated macrophages and myeloid-derived suppressor cells (TAMCs). Recombinantly produced anti-EphA2-CD11b-BsAb was conjugated to a bifunctional chelator, NOTA-SCN, and then radiolabeled with copper-64 (⁶⁴Cu). The [⁶⁴Cu]Cu-NOTA-anti-EphA2-CD11b-BsAb radioimmunoconjugate was subsequently administered to HT1080-fibrosarcoma-bearing nude mice via tail vein injection. Positron Emission Tomography (PET) and ex vivo biodistribution analyses were performed to determine tumor uptake and pharmacokinetic localization. At 4, 24, and 48 h postinjection (p.i.), the percent injected dose per gram (%ID/g) of [⁶⁴Cu]Cu-NOTA-anti-EphA2-CD11b-BsAb in HT1080 xenografts were 5.35 ± 2.24, 4.44 ± 1.90, and 4.10 ± 0.60, respectively. There was high uptake in the liver as well as in CD11b-expressing organs, including the spleen, bone marrow, and lung. Binding in these CD11b-rich organs was significantly reduced by coadministering the dose with nonradiolabeled anti-CD11b-IgG and anti-EphA2-CD11b-BsAb, with a concurrent increase in tumor uptake compared to nonblocked mice (8.39 ± 1.37%ID/g for blocked and 4.44 ± 1.90%ID/g for nonblocked at 24 h p.i., <i>p</i> = 0.0175). Further optimization studies showed that at lower molar activity (3.7 MBq/nmol, 100 μCi/nmol), there were significantly higher tumor accumulations and reduced uptake in CD11b-expressing organs compared to higher molar activity (22.2 MBq/nmol, 600 μCi/nmol). Anti-EphA2-CD11b-BsAb is a functional targeting molecule and would require optimization through molar activity or blocking with nonradiolabeled antibody to maximize tumor targeting.