Rationale: The overwhelming majority of radioimmunoconjugates are produced via random conjugation methods predicated on attaching bifunctional chelators to the lysines of antibodies. However, this approach inevitably produces poorly defined and heterogeneous immunoconjugates because antibodies have several lysines distributed throughout their structure. To circumvent this issue, we have previously developed a chemoenzymatic bioconjugation strategy that site-specifically appends cargoes to the biantennary heavy chain glycans attached to CH2 domains of the immunoglobulin's Fc region. In the study at hand, we explore the effects of this approach to site-specific bioconjugation on the Fc receptor binding and in vivo behavior of radioimmunoconjugates. Methods: We synthesized three desferrioxamine (DFO)-labeled immunoconjugates based on the HER2-targeting antibody pertuzumab: one using random bioconjugation methods (DFO-nsspertuzumab) and two using variants of our chemoenzymatic protocol (DFO-sspertuzumab-EndoS and DFO-sspertuzumab-βGal). Subsequently, we characterized these constructs and evaluated their ability to bind HER2, human FcγRI (huFcγRI), and mouse FcγRI (muFcγRI). After radiolabeling the immunoconjugates with zirconium-89, we conducted PET imaging and biodistribution studies in two different mouse models of HER2-expressing breast cancer. Results: MALDI-ToF and SDS-PAGE analysis confirmed the site-specific nature of the bioconjugation, and flow cytometry and surface plasmon resonance (SPR) revealed that all three immunoconjugates bind HER2 as effectively as native pertuzumab. Critically, however, SPR experiments also illuminated that DFO-sspertuzumab-EndoS possesses an attenuated binding affinity for huFcγRI (17.4 ± 0.3 nM) compared to native pertuzumab (4.7 ± 0.2 nM), DFO-nsspertuzumab (4.1 ± 0.1 nM), and DFO-sspertuzumab-βGal (4.7 ± 0.2 nM). ImmunoPET and biodistribution experiments in athymic nude mice bearing HER2-expressing BT474 human breast cancer xenografts yielded no significant differences in the in vivo behavior of the radioimmunoconjugates. Yet experiments in tumor-bearing humanized NSG mice revealed that 89Zr-DFO-sspertuzumab-EndoS produces higher activity concentrations in the tumor (111.8 ± 39.9 %ID/g) and lower activity concentrations in the liver and spleen (4.7 ± 0.8 %ID/g and 13.1 ± 4.0 %ID/g, respectively) than its non-site-specifically labeled cousin, a phenomenon we believe stems from the altered binding of the former to huFcγRI. Conclusion: These data underscore that this approach to site-specific bioconjugation not only produces more homogeneous and well-defined radioimmunoconjugates than traditional methods but may also improve their in vivo performance in mouse models by reducing binding to FcγRI.

Galectin-3 binding protein (Gal-3BP) is a glycoprotein that is overexpressed and secreted by several cancers and has been implicated as a marker of both tumor progression and poor prognosis in melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and breast cancer. The expression of Gal-3BP by a variety of neoplasms makes it an enticing target for both diagnostics and therapeutics, including immuno-positron emission tomography (immunoPET) probes and antibody-drug conjugates (ADCs). Herein, we report the development, in vitro characterization, and in vivo evaluation of a pair of Gal-3BP-targeting radioimmunoconjugates for 89Zr-immunoPET. A humanized anti-Gal-3BP antibody, 1959, and its corresponding ADC, 1959-sss/DM4 (DM4 = ravtansine), were modified with desferrioxamine (DFO) to yield DFO-1959 and DFO-1959-sss/DM4 immunoconjugates bearing 1-2 DFO/monoclonal antibody. Both DFO-modified immunoconjugates retained their affinity for Gal-3BP in enzyme-linked immunosorbent assay experiments. The chelator-bearing antibodies were radiolabeled with zirconium-89 (t1/2 ≈ 3.3 d) to produce radioimmunoconjugates ─ [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 ─ with high specific activity (>444 MBq/mg, >12 mCi/mg) and stability (>80% intact after 168 h in human serum at 37 °C). In mice bearing subcutaneous Gal-3BP-secreting A375-MA1 xenografts, [89Zr]Zr-DFO-1959 clearly delineated tumor tissue, reaching a maximum tumoral activity concentration (54.8 ± 15.8%ID/g) and tumor-to-background contrast (tumor-to-blood = 8.0 ± 4.6) at 120 h post-injection. The administration of [89Zr]Zr-DFO-1959 to mice bearing subcutaneous Gal-3BP-expressing melanoma patient-derived xenografts produced similarly promising results. [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 exhibited nearly identical pharmacokinetic profiles in the mice bearing A375-MA1 tumors, though the latter produced higher uptake in the spleen and kidneys. Both [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 effectively visualized Gal-3BP-secreting tumors in murine models of melanoma. These results suggest that both probes could play a role in the clinical imaging of Gal-3BP-expressing malignancies, particularly as companion theranostics for the identification of patients likely to respond to Gal-3BP-targeted therapeutics such as 1959-sss/DM4.

The acyclic ligands H4C3octapa and p-SCN-Bn-H4C3octapa were synthesized for the first time, using nosyl protection chemistry. These new ligands were compared to the previously studied ligands H4octapa and p-SCN-Bn-H4octapa to determine the extent to which the addition of a single carbon atom to the backbone of the ligand would affect metal coordination, complex stability, and, ultimately, utility for in vivo radiopharmaceutical applications. Although only a single carbon atom was added to H4C3octapa and the metal donor atoms and denticity were not changed, the solution chemistry and radiochemistry properties were drastically altered, highlighting the importance of careful ligand design and radiometal-ligand matching. It was found that [In(C3octapa)](-) and [Lu(C3octapa)](-) were substantially different from the analogous H4octapa complexes, exhibiting fluxional isomerization and a higher number of isomers, as observed by (1)H NMR, VT-NMR, and 2D COSY/HSQC-NMR experiments. Past evaluation of the DFT structures of [In(octapa)](-) and [Lu(octapa)](-) revealed very symmetric complexes; in contrast, the [In(C3octapa)](-) and [Lu(C3octapa)](-) complexes were much less symmetric, suggesting lower symmetry and less rigidity than that of the analogous H4octapa complexes. Potentiometric titrations revealed the formation constants (log K(ML), pM) were ~2 units lower for the In(3+) and Lu(3+) complexes of H4C3octapa when compared to that of the more favorable H4octapa ligand (~2 orders of magnitude less thermodynamically stable). The bifunctional ligands p-SCN-Bn-H4C3octapa and p-SCN-Bn-H4octapa were conjugated to the antibody trastuzumab and radiolabeled with (111)In and (177)Lu. Over a 5 day stability challenge experiment in blood serum, (111)In-octapa- and (111)In-C3octapa-trastuzumab immunoconjugates were determined to be ~91 and ~24% stable, respectively, and (177)Lu-octapa- and (177)Lu-C3octapa-trastuzumab, ~89% and ~4% stable, respectively. This work suggests that 5-membered chelate rings are superior to 6-membered chelate rings for large metal ions like In(3+) and Lu(3+), which is a crucial consideration for the design of bifunctional chelates for bioconjugation to targeting vectors for in vivo work.