Radiolabeling of CHX-A″-DTPA-Antibody Conjugates with [⁸⁹Zr]ZrCl₄

Currently, the most applied ⁸⁹Zr-immuno-PET platform is the [⁸⁹Zr]Zr-deferoxamine (DFO)-monoclonal antibody (mAb) constructs, where the investigational agent is obtained through combining [⁸⁹Zr]Zr-oxalate with mAbs conjugated to the bifunctional chelator p-SCN-Bn-DFO. This approach struggles with several limitations, including the inability of DFO to incorporate lanthanide-based radiometals such as ¹⁷⁷Lu or ¹⁶¹Tb and the instability of the [⁸⁹Zr]Zr-DFO complex in ascorbate-containing formulations. Conversely, whereas pentetic acid (DTPA)-based bifunctional chelators have been extensively applied to generate clinical β-therapeutic mAb constructs, the previous efforts to create stable [⁸⁹Zr]Zr-DTPA-mAb complexes using [⁸⁹Zr]Zr-oxalate have been unsuccessful. However, [⁸⁹Zr]ZrCl₄, which exists as [Zr₄(OH)₈(OH₂)₁₆]⁸⁺ in aqueous solutions, is chemically more accessible than its commercially available oxalate form, enabling the direct labeling of p-SCN-Bn-CHX-A″-DTPA. The methodology described here allows for the generation of [⁸⁹Zr]Zr-DTPA-mAb and [¹⁷⁷Lu]Lu/[¹⁶¹Tb]Tb-DTPA-mAb radiotheranostic pairs, where the targeting vector in the diagnostic and the therapeutic analogs is identical. Methods: Pertuzumab was selected for proof-of-concept studies and was conjugated to p-SCN-Bn-CHX-A″-DTPA. Radiolabeling of DTPA-pertuzumab with [⁸⁹Zr]ZrCl₄ involved a 10-min incubation in acetate buffer (pH 4.5), followed by PD-10 desalting gel column purification. The in-formulation radiochemical purity and pooled human serum stability were assessed using size-exclusion high-performance liquid chromatography, and radioimmunoreactivity was evaluated using the stationary antigen magnetic bead-based method. Biodistribution of [⁸⁹Zr]Zr-DTPA-pertuzumab was assessed in BT-474 tumor mouse models and compared with biodistribution of [⁸⁹Zr]Zr-DFO-pertuzumab and [¹⁶¹Tb]Tb-DTPA-pertuzumab. Results: Conjugated batches consistently produced DTPA-pertuzumab with acceptable chelate-to-mAb ratios and chemical purity. DTPA-pertuzumab was radiolabeled with up to 3.4 GBq (92 mCi) of ⁸⁹Zr. In formulation, DTPA-pertuzumab exhibited greater chemical stability, and the radioaggregate formation was lower in [⁸⁹Zr]Zr-DTPA-pertuzumab than in [⁸⁹Zr]Zr-DFO-pertuzumab. [⁸⁹Zr]Zr-DTPA-pertuzumab was also stable in ascorbate-containing formulations. In human serum, the drop in radiomonomer content for [⁸⁹Zr]Zr-DTPA-pertuzumab was smaller than for [⁸⁹Zr]Zr-DFO-pertuzumab. Compared with [⁸⁹Zr]Zr-DFO-pertuzumab, [⁸⁹Zr]Zr-DTPA-pertuzumab biodistribution exhibited lower liver and higher blood and tumor uptake and was more consistent with the biodistribution of [¹⁶¹Tb]Tb-DTPA-pertuzumab. Conclusion: The ability to radiolabel CHX-A″-DTPA-mAbs with ⁸⁹Zr has been demonstrated, allowing for the generation of ⁸⁹Zr/¹⁷⁷Lu/¹⁶¹Tb-based true radiotheranostic pairs. On the basis of our biodistribution data, [⁸⁹Zr]Zr-DTPA-mAbs may be better suited as a companion diagnostic to radiotherapeutic DTPA-mAb analogs than is [⁸⁹Zr]Zr-DFO-mAbs.