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.

原理：绝大多数的放射免疫偶联物是通过随机偶联方法产生的，该偶联方法是将双功能螯合剂与抗体的赖氨酸连接。但是，这种方法不可避免地产生定义不清和异质性的免疫偶联物，因为抗体的整个结构中都分布有多个赖氨酸。为了规避此问题，我们先前已经开发了一种化学化学生物共轭策略，该策略可将位点特异性地将货物附加到与免疫球蛋白Fc区C _H 2域相连的双天线重链聚糖上。在当前的研究中，我们探索了这种方法对位点特异性生物缀合对Fc受体结合和放射免疫缀合物的体内行为的影响。

方法：我们基于靶向HER2的抗体帕妥珠单抗合成了三种去铁氧胺（DFO）标记的免疫偶联物：一种使用随机生物偶联方法（DFO- ^nss pertuzumab），两种使用我们的化学酶学方案变体（DFO - ^ss pertuzumab-EndoS和DFO- ^ss帕妥珠单抗-βGal）。随后，我们表征了这些构建体并评估了它们结合HER2，人FcγRI（huFcγRI）和小鼠FcγRI（muFcγRI）的能力。用Zrconium-89放射性标记免疫缀合物后，我们在两种表达HER2的乳腺癌小鼠模型中进行了PET成像和生物分布研究。

结果：MALDI-ToF和SDS-PAGE分析证实了生物缀合的位点特异性，流式细胞仪和表面等离振子共振（SPR）表明，所有三种免疫缀合物均与天然帕妥珠单抗一样有效地结合HER2。然而，至关重要的是，SPR实验还阐明，与天然培妥珠单抗（4.7±0.2 nM），DFO- ^nss培妥珠单抗（4.1±0.1 nM）相比，DFO - ^ss帕妥珠单抗-EndoS对huFcγRI的结合亲和力减弱（17.4±0.3 nM），和DFO- ^SS帕妥珠单抗-的βGal（4.7±0.2纳米）。携带表达HER2的BT474人乳腺癌异种移植物的无胸腺裸鼠的ImmunoPET和生物分布实验在体内没有明显差异放射免疫缀合物的行为。然而，在荷瘤人源化NSG小鼠中进行的实验表明，⁸⁹ Zr-DFO- ^ss pertuzumab-EndoS在肿瘤中产生较高的活性浓度（111.8±39.9％ID / g），而在肝脏和脾脏中产生较低的活性浓度（4.7±0.8％） ID / g和分别为13.1±4.0％ID / g）（其非位点特异性标记的表亲），我们认为这种现象源于前者与huFcγRI的结合改变。

结论：这些数据强调，这种针对位点的生物缀合方法不仅比传统方法产生更均一且定义明确的放射免疫缀合物，而且还可以通过减少与FcγRI的结合来改善其在小鼠模型中的体内性能。