Kameswaran Mythili, Pandey Usha, Dash Ashutosh, Samuel Grace, Venkatesh Meera
Isotope Production & Applications Division, Bhabha Atomic Research Centre, Mumbai, India.
Indian J Med Res. 2016 Jan;143(1):57-65. doi: 10.4103/0971-5916.178593.
BACKGROUND & OBJECTIVES: Radioimmunotherapy is extensively being used for the treatment of non-Hodgkin's lymphoma (NHL). Use of rituximab, a chimeric anti-CD20 antibody directed against the CD20 antigen in combination with suitable beta emitters is expected to result in good treatment response by its cross-fire and bystander effects. The present work involves the conjugation of p-isothiocyanatobenzyl DOTA (p-SCN-Bn-DOTA) to rituximab, its radiolabelling with [90] Y and in vitro and in vivo evaluation to determine its potential as a radioimmunotherapeutic agent.
Rituximab was conjugated with p-SCN-Bn-DOTA at 1:1 antibody: DOTA molar ratio. The number of DOTA molecules linked to one molecule of rituximab was determined by radioassay and spectroscopic assay. Radiolabelling of rituximab with 90 Y was carried out and its in vitro stability was evaluated. In vitro cell binding studies were carried out in Raji cells expressing CD20 antigen. Biodistribution studies were carried out in normal Swiss mice.
Using both radioassay and spectroscopic method, it was determined that about five molecules of DOTA were linked to rituximab. Radiolabelling of the rituximab conjugate with [90] Y and subsequent purification on PD-10 column gave a product with radiochemical purity (RCP) > 98 per cent which was retained at > 90 per cent up to 72 h when stored at 37°C. In vitro cell binding experiments of 90 Y-DOTA-rituximab with Raji cells exhibited specific binding of 20.7 ± 0.1 per cent with [90] Y-DOTA-rituximab which reduced to 15.5 ± 0.2 per cent when incubated with cold rituximab. The equilibrium constant K d for 90 Y-DOTA-Rituximab was determined to be 3.38 nM. Radiolabelled antibody showed clearance via hepatobiliary and renal routes and activity in tibia was found to be quite low indicating in vivo stability of [90] Y-DOTA-rituximab.
INTERPRETATION & CONCLUSIONS: p-SCN-Bn-DOTA was conjugated with rituximab and radiolabelling with 90 Y was carried out. In vitro studies carried out in Raji cells showed the specificity of the radiolabelled conjugate suggesting the potential uitability of the formulation as a radiopharmaceutical for therapy of NHL.
放射免疫疗法广泛应用于非霍奇金淋巴瘤（NHL）的治疗。将利妥昔单抗（一种针对CD20抗原的嵌合抗CD20抗体）与合适的β发射体联合使用，有望通过其交叉火力和旁观者效应产生良好的治疗反应。本研究涉及对异硫氰酸对苯苄基二乙三胺五乙酸（p-SCN-Bn-DOTA）与利妥昔单抗进行偶联，用[90]Y对其进行放射性标记，并进行体外和体内评估，以确定其作为放射免疫治疗剂的潜力。
以1:1的抗体与DOTA摩尔比将利妥昔单抗与p-SCN-Bn-DOTA偶联。通过放射性测定和光谱测定确定与一个利妥昔单抗分子相连的DOTA分子数量。用90Y对利妥昔单抗进行放射性标记，并评估其体外稳定性。在表达CD20抗原的Raji细胞中进行体外细胞结合研究。在正常的瑞士小鼠中进行生物分布研究。
通过放射性测定和光谱法均确定约有五个DOTA分子与利妥昔单抗相连。用[90]Y对利妥昔单抗偶联物进行放射性标记，随后在PD-10柱上纯化，得到放射化学纯度（RCP）>98%的产物，当在37°C储存时，该产物在72小时内保持>90%。90Y-DOTA-利妥昔单抗与Raji细胞的体外细胞结合实验显示，[90]Y-DOTA-利妥昔单抗的特异性结合为20.7±0.1%，与冷利妥昔单抗孵育时降至15.5±0.2%。90Y-DOTA-利妥昔单抗的平衡常数Kd被确定为3.38 nM。放射性标记的抗体通过肝胆和肾脏途径清除，胫骨中的活性相当低，表明[90]Y-DOTA-利妥昔单抗在体内的稳定性。
p-SCN-Bn-DOTA与利妥昔单抗偶联，并用90Y进行放射性标记。在Raji细胞中进行的体外研究显示了放射性标记偶联物的特异性，表明该制剂作为治疗NHL的放射性药物具有潜在适用性。
