Abstract：
Denileukin Diftitox (DD), comprising fragments of diphtheria toxin (DT) and interleukin-2 (IL2), was developed for the treatment of lymphoma and has been approved for marketing in Japan. Toxicological evaluation including pharmacokinetics and immunogenicity in preclinical animals is important for drug development and thus the assays of DD and anti-drug antibody (ADA) were developed by electrochemiluminescence (ECL) detection. For the DD assay, ruthenium-labeled anti-DT Ab and biotinylated anti-IL2 Ab were mixed with serum samples and the mixture was captured by streptavidin-coated wells for ECL detection. For the ADA assay, signals of immuno-complex of biotinylated DD, ruthenium-labeled DD, and ADA, bound to streptavidin plate were determined. DD was quantifiable from 10 ng/mL. Accuracy and precision of quality control samples were within ±20% and 20%, respectively, and stability of DD in rat serum was successfully assessed. Precision of positive control samples of ADA was within the acceptance criteria and cut point values for ADA detection in the screening and confirmatory assay were determined by statistical analysis. Drug-induced ADA was detected by screening assay followed by confirmatory assay. The developed method was successfully applied to assess pharmacokinetics and immunogenicity to support toxicity studies in rats.
摘要：
DenileukinDiftitox（DD），包含白喉毒素（DT）和白介素2（IL2）的片段，是为治疗淋巴瘤而开发的，并已在日本批准上市。包括临床前动物中的药代动力学和免疫原性的毒理学评估对于药物开发很重要，因此通过电化学发光（ECL）检测开发了DD和抗药物抗体（ADA）的测定法。对于DD测定，将钌标记的抗DTAb和生物素化的抗IL2Ab与血清样品混合,并通过链霉抗生物素蛋白包被的孔捕获混合物用于ECL检测。对于ADA测定，生物素化DD的免疫复合物信号，钌标记的DD，还有ADA,确定与链霉亲和素平板的结合。DD可从10ng/mL定量。质量控制样品的准确度和精密度在±20%和20%以内，分别,并成功评估了大鼠血清中DD的稳定性。ADA的阳性对照样品的精确度在接受标准内,并且通过统计分析确定筛选和确证测定中ADA检测的切点值。药物诱导的ADA通过筛选测定，然后进行确认测定。开发的方法已成功用于评估药代动力学和免疫原性，以支持大鼠的毒性研究。
