Abstract
Currently, the most applied (89)Zr-immuno-PET platform is the [(89)Zr]Zr-deferoxamine (DFO)-monoclonal antibody (mAb) constructs, where the investigational agent is obtained through combining [(89)Zr]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 (177)Lu or (161)Tb and the instability of the [(89)Zr]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 [(89)Zr]Zr-DTPA-mAb complexes using [(89)Zr]Zr-oxalate have been unsuccessful. However, [(89)Zr]ZrCl(4), which exists as [Zr(4)(OH)(8)(OH(2))(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 [(89)Zr]Zr-DTPA-mAb and [(177)Lu]Lu/[(161)Tb]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 [(89)Zr]ZrCl(4) 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 [(89)Zr]Zr-DTPA-pertuzumab was assessed in BT-474 tumor mouse models and compared with biodistribution of [(89)Zr]Zr-DFO-pertuzumab and [(161)Tb]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 (89)Zr. In formulation, DTPA-pertuzumab exhibited greater chemical stability, and the radioaggregate formation was lower in [(89)Zr]Zr-DTPA-pertuzumab than in [(89)Zr]Zr-DFO-pertuzumab. [(89)Zr]Zr-DTPA-pertuzumab was also stable in ascorbate-containing formulations. In human serum, the drop in radiomonomer content for [(89)Zr]Zr-DTPA-pertuzumab was smaller than for [(89)Zr]Zr-DFO-pertuzumab. Compared with [(89)Zr]Zr-DFO-pertuzumab, [(89)Zr]Zr-DTPA-pertuzumab biodistribution exhibited lower liver and higher blood and tumor uptake and was more consistent with the biodistribution of [(161)Tb]Tb-DTPA-pertuzumab. Conclusion: The ability to radiolabel CHX-A″-DTPA-mAbs with (89)Zr has been demonstrated, allowing for the generation of (89)Zr/(177)Lu/(161)Tb-based true radiotheranostic pairs. On the basis of our biodistribution data, [(89)Zr]Zr-DTPA-mAbs may be better suited as a companion diagnostic to radiotherapeutic DTPA-mAb analogs than is [(89)Zr]Zr-DFO-mAbs.