Department of Nuclear Medicine and PET, Post Graduate Institute of Medical Education and Research.
Department of General Surgery, Post Graduate Institute of Medical Education and Research.
Nucl Med Commun. 2022 Apr 1;43(4):458-467. doi: 10.1097/MNM.0000000000001521.
Receptors on breast cancer cells play a crucial role in the management of patients. Trastuzumab is a widely used drug for the treatment of HER2/neu expressing tumors. ImmunoPET with trastuzumab is not feasible due to slow pharmacokinetics. Fragment of antigen-binding (Fab) radiolabeled with positron emitters can be used for immunoPET.
Fab has been generated by papain digestion and conjugated with the bifunctional chelating agent NOTA. The SDS-PAGE and MALDI-TOF were used to see the integrity of Fab and conjugated Fab. In-vitro stability and target specificity for HER2/neu receptors were performed in plasma and receptor binding with bio-layer interferometry (BLI) techniques. Radiolabeling was standardized with 68GaCl3 and PET imaging was performed in seven patients showing 18F fluorodeoxyglucose (18F-FDG) uptake and correlated with HER2/neu expression by immunohistochemistry.
Fab production was optimized at molar ratio 23:1 of trastuzumab and papain at 37 °C with a constant stirrer at 850 rpm for 22-24 h, at pH 8. Conjugation with NOTA was standardized at molar ratio 1:25 of trastuzumab Fab and NOTA. Molecular mass of trastuzumab Fab-NOTA was found approximately 46.3 kDa (~1/3 of intact antibody). Trastuzumab Fab-NOTA showed radiolabelling efficiency of 48-70% with incubation time 15 min at 37-40 °C and pH 4.5-5.0. BLI demonstrated the affinity of trastuzumab, trastuzumab Fab and trastuzumab Fab-NOTA towards HER2/neu receptor with KD of <1pM, ~0.5nM and ~20nM, respectively. All immunohistochemistry proven patients showed uptake in primary breast lesion and lymph nodes.
Trastuzumab Fab-NOTA is suitable for radiolabelling with 68Ga and ImmunoPET imaging of HER2/neu receptor.
乳腺癌细胞上的受体在患者管理中起着至关重要的作用。曲妥珠单抗是一种广泛用于治疗 HER2/neu 表达肿瘤的药物。由于药代动力学缓慢，曲妥珠单抗的免疫 PET 不可行。用正电子发射体标记抗原结合片段 (Fab) 可用于免疫 PET。
通过木瓜蛋白酶消化生成 Fab，并与双功能螯合剂 NOTA 连接。使用 SDS-PAGE 和 MALDI-TOF 观察 Fab 和连接的 Fab 的完整性。在等离子体中进行体外稳定性和针对 HER2/neu 受体的靶向特异性研究，并使用生物层干涉技术 (BLI) 进行受体结合研究。用 68GaCl3 进行放射性标记，并对 7 名表现出 18F 氟脱氧葡萄糖 (18F-FDG) 摄取的患者进行 PET 成像，并通过免疫组织化学与 HER2/neu 表达相关联。
在 37°C 下，木瓜蛋白酶与曲妥珠单抗的摩尔比为 23:1，恒定搅拌速度为 850rpm，持续 22-24 小时，pH 值为 8，优化了 Fab 的生产。NOTA 的偶联标准化为曲妥珠单抗 Fab 和 NOTA 的摩尔比为 1:25。曲妥珠单抗 Fab-NOTA 的分子量约为 46.3kDa（完整抗体的 1/3）。曲妥珠单抗 Fab-NOTA 在 37-40°C 和 pH 4.5-5.0 下孵育 15 分钟，放射性标记效率为 48-70%。BLI 证明了曲妥珠单抗、曲妥珠单抗 Fab 和曲妥珠单抗 Fab-NOTA 对 HER2/neu 受体的亲和力，其 KD 值分别为<1pM、0.5nM 和~20nM。所有经免疫组织化学证实的患者均在原发性乳房病变和淋巴结中显示摄取。
曲妥珠单抗 Fab-NOTA 适合用 68Ga 标记，并可用于 HER2/neu 受体的免疫 PET 成像。
