The Influence of Glycans-Specific Bioconjugation on the FcγRI Binding and In vivo Performance of 89Zr-DFO-Pertuzumab

Abstract

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 C_H2 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.

Keywords: 89Zr; HER2; glycans; immunoPET; pertuzumab; radioimmunoconjugate; site-specific.

Figure 1

The construction of the pertuzumab immunoconjugates: DFO-^nsspertuzumab (top), DFO-^sspertuzumab-βGal (middle), and DFO-^sspertuzumab-EndoS (bottom).

Figure 2

Comparative SPR and ELISA analysis of the interaction between the pertuzumab immunoconjugates and recombinant human and mouse FcγRI. (A) ELISA data demonstrating the binding of the immunoconjugates to huFcγRI (10 μg/mL; left) and muFcγRI (10 μg/mL; right); (B) Sensorgrams showing robust dose-response curves and kinetic profiles for the binding of various concentrations of native pertuzumab and the DFO-bearing immunoconjugates to huFcγRI; (C) Bar graphs demonstrating the correlation between deglycosylation and binding affinity (K_D), on-rate (k_a), off-rate (k_d) and half-life (t_1/2). The binding affinity, kinetic constants and half-lives for each of the DFO-bearing immunoconjugates were compared with those obtained for unmodified pertuzumab; (D) Sensorgrams showing robust dose-response curves and kinetic profiles for the binding of various concentrations of native pertuzumab and the DFO-bearing immunoconjugates to muFcγRI. Statistically significant relationships are indicated with asterisks. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00001.

Figure 3

(A) Planar (left) and maximum intensity projection (MIP, right) PET images of athymic nude mice bearing subcutaneous BT474 xenografts injected with the three ⁸⁹Zr-DFO-pertuzumab radioimmunoconjugates (179 - 192 μCi, 6.6 - 7.1 MBq, 85 - 92 μg, in 200 μL 0.9% sterile saline). (B) Biodistribution data for athymic nude mice bearing HER2-expressing BT474 xenografts injected with ⁸⁹Zr-DFO-^nsspertuzumab, ⁸⁹Zr-DFO-^sspertuzumab-EndoS, or ⁸⁹Zr-DFO-^sspertuzumab-βGal (lateral tail vein injection, 15 - 20 µCi, 0.56 - 0.74 MBq, 6 - 9 µg).

Figure 4

(A) Planar (left) and maximum intensity projection (MIP, right) PET images of huNSG mice bearing subcutaneous BT474 xenografts collected between 24 and 144 h after the administration of the three radioimmunoconjugates (209 - 218 μCi, 7.7 - 8.1 MBq, 80 - 83 μg, in 200 μl 0.9% sterile saline); (B) Biodistribution data for ⁸⁹Zr-DFO-^nsspertuzumab, ⁸⁹Zr-DFO-^sspertuzumab-βGal, and ⁸⁹Zr-DFO-^sspertuzumab-EndoS 144 hours following administration in huNSG mice bearing subcutaneous HER2-expressing BT474 xenografts. T = tumor; L = liver; S = spleen; B = bone. * = p < 0.05.