Imaging Very Late Antigen‑4 on MOLT4 Leukemia Tumors with Cysteine Site-Specific ⁸⁹Zr-Labeled Natalizumab Immuno-Positron Emission Tomography

Very late antigen-4 (VLA4; CD49d) is a promising immune therapy target in treatment-resistant leukemia and multiple myeloma, and there is growing interest in repurposing the humanized monoclonal antibody (Ab), natalizumab, for this purpose. Positron emission tomography with radiolabeled Abs (immuno-PET) could facilitate this effort by providing information on natalizumab’s in vivo pharmacokinetic and target delivery properties. In this study, we labeled natalizumab with ⁸⁹Zr specifically on sulfhydryl moieties via maleimide–deferoxamine conjugation. High VLA4-expressing MOLT4 human T cell acute lymphoblastic leukemia cells showed specific ⁸⁹Zr–natalizumab binding that was markedly blocked by excess Ab. In nude mice bearing MOLT4 tumors, ⁸⁹Zr–natalizumab PET showed high-contrast tumor uptake at 7 days postinjection. Biodistribution studies confirmed that uptake was the highest in MOLT4 tumors (2.22 ± 0.41%ID/g) and the liver (2.33 ± 0.76%ID/g), followed by the spleen (1.51 ± 0.42%ID/g), while blood activity was lower at 1.12 ± 0.21%ID/g. VLA4-specific targeting in vivo was confirmed by a 58.1% suppression of tumor uptake (0.93 ± 0.15%ID/g) when excess Ab was injected 1 h earlier. In cultured MOLT4 cells, short-term 3 day exposure to the proteasome inhibitor bortezomib (BTZ) did not affect the α4 integrin level, but BTZ-resistant cells that survived the treatment showed increased α4 integrin expression. When the effects of BTZ treatment were tested in mice, there was no change of the α4 integrin level or ⁸⁹Zr–natalizumab uptake in MOLT4 leukemia tumors, which underscores the complexity of tumor VLA4 regulation in vivo. In conclusion, ⁸⁹Zr–natalizumab PET may be useful for noninvasive monitoring of tumor VLA4 and may assist in a more rational application of Ab-based therapies for hematologic malignancies.