Abstract
Some types of immunotherapy of malignant tumours are aimed at restoration of T-cells’ ability to recognize and eliminate cancer. Programmed cell death ligand-1 (PD-L1) overexpression is characteristic of many human tumours and is associated with poor prognosis for patients. The development of monoclonal antibodies (mAbs) specific for PD-L1 or PD-1 is a promising area of immunotherapy of malignant tumours. However, before a therapeutic antibody-based product enters the market, it is necessary to ensure its safety and efficacy, i.e. perform a full scope of preclinical and clinical studies.The aim of the study was to develop and validate a bioanalytical method that does not require additional labeling and that could be used for determination of mAbs specific for human PD-L1 in the blood serum of a biological test system during preclinical studies.Materials and methods: an antigen in the form of a dimer of PD-1 extra-cellular domain covalently bonded to the Fc-fragment of human IgG (R&D Systems, USA) was used in the study. The antigen was immobilised on Dip and Read™ Protein A biosensors (Fortebio, USA). The therapeutic anti-PD-1 antibody GNR-051 was developed and produced by IBC “Generium” (Russia). The healthy cynomolgus monkey serum samples used as matrix were obtained from the Research Institute of Medical Primatology (Sochi, Russia). The assessment of binding was performed using Octet® QKe interferometer (Fortebio, USA) by real-time analysis of the dose-dependent rate of the antigen-antibody complex formation.Results: the paper presents experimental data on the development and validation of the test method for determination of the therapeutic PD-1-binding mAb concentration in cynomolgus monkey serum in the antibody concentration range from 2 to 2500 µg/mL. The authors assessed the calibration curve reliability, between-run and within-run precision and accuracy, dilution linearity, specificity and selectivity of the test method.Conclusions: the authors developed and validated the biolayer interferometry-based method for determination of therapeutic mAbs concentration. The method was shown to comply with the Eurasian Economic Union’s regulatory requirements in terms of the main validation parameters: analytical range, accuracy, precision, and selectivity.
Highlights
Разработка и валидация методики определения концентрации антитела человека, блокирующего связывание PD-1 с лигандами, в сыворотке крови яванского макака методом биослойной интерферометрии
Some types of immunotherapy of malignant tumours are aimed at restoration of T-cells’ ability to recognize and eliminate cancer
Programmed cell death ligand-1 (PD-L1) overexpression is characteristic of many human tumours and is associated with poor prognosis for patients
Summary
Для разработки и валидации методики использовали антиген (кат. No 1086-PD, R&D Systems, США) в виде димера внеклеточного домена рецептора PD-1 человека, ковалентно связанного с Fc-фрагментом иммуноглобулина человека. Метод определения концентрации GNR-051 основан на непосредственном интерферометрическом измерении в реальном времени кинетики специфического взаимодействия антигена PD-1, иммобилизованного на биосенсоре, с антителом GNR-051 в исследуемом образце сыворотки крови яванского макака. Испытания выполняли с использованием интерферометра Octet® QKe (ForteBio, США), определяли дозозависимую скорость увеличения специфического биослоя (нм) на рабочей поверхности сенсора. Все модельные и градуировочные образцы готовили на основе пулированной, неразведенной сыворотки крови яванского макака, содержащей антитело, специфичное к рецептору PD-1, в известных концентрациях. Семь градуировочных растворов готовили путем внесения GNR-051 в неразведенную пулированную сыворотку крови яванского макака до конечной концентрации GNR-051 1000, 333,3, 111,1, 37,0, 12,4, 4,1, 1,4 мкг/мл. Для оценки правильности разрабатываемой методики готовили одиннадцать модельных образцов путем внесения GNR-051 (от 2 до 2500 мкг/мл) в неразведенную пулированную сыворотку крови яванского макака. Образец GNR-051 с концентрацией 2500 мкг/мл испытывали в разведениях 1:20 и 1:40, остальные образцы — в разведениях 1:10 и 1:20
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