phkineticaФармакокинетика и ФармакодинамикаPharmacokinetics and Pharmacodynamics2587-78362686-8830ООО «Издательство ОКИ»10.37489/2587-7836-2026-3-69-83NIMRBAphkinetica-552Research ArticleМЕТОДЫ РАЗРАБОТКИ НОВЫХ ЛЕКАРСТВЕННЫХ СРЕДСТВMETHODS FOR DEVELOPMENT OF NEW DRUGSРазработка репортерной клеточной линии, экспрессирующей рецептор фактора роста эндотелия сосудов 2 и репортерный ген люциферазы светлячка на основе клеток СНО-К1 (CHO-KDR/LUC)Development of a reporter cell line expressing vascular endothelial growth factor receptor 2 and a firefly luciferase reporter gene based on CHO-K1 cells (CHO-KDR/LUC)https://orcid.org/0009-0003-5762-2193ШубинаС. С.ShubinaS. S.

Шубина Софья Сергеевна — н. с. лаборатории стабильных клеточных линий отдела клеточной биологии

пос. Вольгинский, городской округ Покров 

Sofia S. Shubina — Research Associate at the Laboratory of Stable Cell Lines, Department of Cell Biology

Volginsky settlement, Pokrov urban district 

shubina.sofa22@gmail.comhttps://orcid.org/0000-0002-5895-9463ГладченкоЮ. А.GladchenkoYu. A.

Гладченко Юрий Андреевич — с. н. с. лаборатории молекулярной биологии отдела молекулярной биологии и биохимии

пос. Вольгинский, городской округ Покров 

Yury A. Gladchenko — Senior Researcher, Molecular Biology Laboratory, Department of Molecular Biology and Biochemistry

Volginsky settlement, Pokrov urban district 

yagladchenko@generium.ruhttps://orcid.org/0009-0005-2975-9111ПолупановаА. А.PolupanovaA. A.

Полупанова Анна Алексеевна — н. с. лаборатории стабильных клеточных линий отдела клеточной биологии

пос. Вольгинский, городской округ Покров 

Anna A. Polupanova — Researcher, Laboratory of Stable Cell Lines, Cell Biology Department

Volginsky settlement, Pokrov urban district 

aapolupanova@generium.ruhttps://orcid.org/0009-0003-9518-2942АнфилатоваА. И.AnfilatovaA. I.

Анфилатова Анастасия Ивановна — н. с. лаборатории стабильных клеточных линий отдела клеточной биологии

пос. Вольгинский, городской округ Покров 

Anastasia I. Anfilatova — Researcher, Laboratory of Stable Cell Lines, Cell Biology Department

Volginsky settlement, Pokrov urban district 

anfilatova@generium.ruhttps://orcid.org/0000-0003-2733-7458ТимоноваС. С.TimonovaS. S.

Тимонова Софья Сергеевна — к. б. н., начальник лаборатории стабильных клеточных линий отдела клеточной биологии

пос. Вольгинский, городской округ Покров 

Sofia S. Timonova — PhD, Cand. Sci. (Biol.), Head of the Laboratory of Stable Cell Lines, Cell Biology Department

Volginsky settlement, Pokrov urban district 

timonova1993@yandex.ruАО «Генериум»РоссияJSC "Generium"Russian Federation202616102026036983Copyright © Шубина С.С., Гладченко Ю.А., Полупанова А.А., Анфилатова А.И., Тимонова С.С., 20262026Шубина С.С., Гладченко Ю.А., Полупанова А.А., Анфилатова А.И., Тимонова С.С.Shubina S.S., Gladchenko Y.A., Polupanova A.A., Anfilatova A.I., Timonova S.S.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.pharmacokinetica.ru/jour/article/view/552

Актуальность. Анти-VEGF-агенты доказали эффективность в терапии офтальмологических и онкологических заболеваний, а потенциал их применения продолжает расти. Ограничения доступности готовых коммерческих систем биоанализа для определения биологической активности антиVEGF препаратов обуславливают необходимость разработки собственной независимой репортерной клеточной линии.Цель. Получение стабильной репортерной клеточной линии с рецептором-2 фактора роста эндотелия сосудов (KDR) и репортерным геном люциферазы светлячка (LUC) на основе суспензионной линии клеток CHO-К1.Методы. СНО-К1 (ГосНИИгенетика, Россия) трансфицировали двумя плазмидами, несущими ген KDR и ген LUC, в различных соотношениях. Влияние выбранного соотношения плазмид оценивали по уровню люциферазной активности с помощью наборов Promega Bio-Glo (Promega, США) или буферов, содержащих субстрат D-люциферин (Молекта, Россия). Для активации репортерного ответа клеточной линии добавляли препарат VEGF165 (АО «Генериум», Россия). Результаты. Путём ко-трансфекции клеток СНО-К1 плазмидами, кодирующими ген репортер (LUC) и ген рецептора VEGFR-2 человека (KDR) была получена репортерная клеточная линия CHO-KDR/LUC. Максимальная индукция люциферазы была достигнута при соотношении плазмид KDR 25/75 LUC (%) и составляла 5,5х105 RLU при выходе на плато, ЕС50 составляла 35,19 нг/мл. Анализ люциферазного ответа с использованием гомологичных лигандов показал специфичность по отношению к VEGF165. С увеличением количества клеточных генераций ростовые характеристики и люциферазный ответ на стимуляцию VEGF165 оставались стабильными.Заключение. Репортерная клеточная линия СНО-KDR/LUC подходит для анализа биологической активности белков, применяемых в анти-VEGF-терапии. Дальнейшая валидация и применение позволит повысить эффективность разработки новых терапевтических белков VEGF-терапии и биоаналогов.

Background. Anti-VEGF agents have proven effective in the treatment of ophthalmological and oncological diseases, and their potential applications continue to grow. Limited availability of commercial bioassay systems for determining the biological activity of anti-VEGF drugs necessitates the development of a proprietary, independent reporter cell line.Objective. To generate a stable reporter cell line expressing vascular endothelial growth factor receptor-2 (KDR) and a firefly luciferase (LUC) reporter gene based on the CHO-K1 suspension cell line.Methods. CHO-K1 (GosNIIGenetika, Russia) was transfected with two plasmids carrying the KDR gene and the LUC gene in varying ratios. The effect of plasmid ratio was assessed by measuring luciferase activity using Promega Bio-Glo kits (Promega, USA) or buffers containing the D-luciferin substrate (Molecta, Russia). VEGF165 (JSC "Generium", Russia) was added to activate the cell line's reporter response.Results. The CHO-KDR/LUC reporter cell line was generated by co-transfecting CHO-K1 cells with plasmids encoding the reporter gene (LUC) and the human VEGFR-2 receptor gene (KDR). Maximum luciferase induction was achieved with a KDR plasmid ratio of 25/75 LUC (%) and reached a plateau of 5x105 RLU; the EC50 was 35.19 ng/mL. Analysis of the luciferase response using homologous ligands demonstrated specificity for VEGF165. With increasing cell generations, growth characteristics and the luciferase response to VEGF165 stimulation remained stable.Conclusion. The CHO-KDR/LUC reporter cell line is suitable for analyzing the biological activity of proteins used in anti-VEGF therapy. Further validation and application will improve the efficiency of developing new therapeutic proteins and biosimilars for VEGF therapy.

рецептор 2 фактор роста эндотелия сосудов (VEGFR-2)анти-VEGF агентлюцифераза светлячкарепортерный анализбиолюминесценцияклеточная линияCHOvascular endothelial growth factor receptor 2 (VEGFR-2)anti-VEGF agentluciferase fireflyreporter assaybioluminescencecell lineCHOДанное исследование выполнено в рамках разработки в АО «Генериум».This research was conducted as part of a development project at JSC "Generium".ReferencesSiebring-van Olst E, van Beusechem VW. High-Throughput Firefly Luciferase Reporter Assays. Methods Mol Biol. 2018;1755:19-29. doi: 10.1007/978-1-4939-7724-6_2.Siebring-van Olst E, van Beusechem VW. High-Throughput Firefly Luciferase Reporter Assays. Methods Mol Biol. 2018;1755:19-29. doi: 10.1007/978-1-4939-7724-6_2.Walhart T, Isaacson-Wechsler E, Ang KH, et al. A Cell-Based Renilla Luminescence Reporter Plasmid Assay for High-Throughput Screening to Identify Novel FDA-Approved Drug Inhibitors of HPV-16 Infection. SLAS Discov. 2020 Jan;25(1):79-86. doi: 10.1177/2472555219860771.Walhart T, Isaacson-Wechsler E, Ang KH, et al. A Cell-Based Renilla Luminescence Reporter Plasmid Assay for High-Throughput Screening to Identify Novel FDA-Approved Drug Inhibitors of HPV-16 Infection. SLAS Discov. 2020 Jan;25(1):79-86. doi: 10.1177/2472555219860771.Li H, Wang YG, Ma ZC, et al. A high-throughput cell-based gaussia luciferase reporter assay for measurement of CYP1A1, CYP2B6, and CYP3A4 induction. Xenobiotica. 2021 Jul;51(7):752-763. doi: 10.1080/00498254.2021.1918800.Li H, Wang YG, Ma ZC, et al. A high-throughput cell-based gaussia luciferase reporter assay for measurement of CYP1A1, CYP2B6, and CYP3A4 induction. Xenobiotica. 2021 Jul;51(7):752-763. doi: 10.1080/00498254.2021.1918800.Vlachou A, Nchioua R, Regensburger K, et al. A Gaussia luciferase reporter assay for the evaluation of coronavirus Nsp5/3CLpro activity. Sci Rep. 2024 Sep 5;14(1):20697. doi: 10.1038/s41598-024-71305-6.Vlachou A, Nchioua R, Regensburger K, et al. A Gaussia luciferase reporter assay for the evaluation of coronavirus Nsp5/3CLpro activity. Sci Rep. 2024 Sep 5;14(1):20697. doi: 10.1038/s41598-024-71305-6.Zhang JW, Wang H, Liu J, et al. Generation of a stable GFP-reporter Zika virus system for high-throughput screening of Zika virus inhibitors. Virol. Sin. 2021;36:476-489. doi: 10.1007/s12250-020-00316-0.Zhang JW, Wang H, Liu J, et al. Generation of a stable GFP-reporter Zika virus system for high-throughput screening of Zika virus inhibitors. Virol. Sin. 2021;36:476-489. doi: 10.1007/s12250-020-00316-0.Lupitha SS, Chandrasekhar L, Varadarajan SN, et al. A reporter cell line for real-time imaging of autophagy and apoptosis. Toxicol Lett. 2020 Jun 15;326:23-30. doi: 10.1016/j.toxlet.2020.02.011.Lupitha SS, Chandrasekhar L, Varadarajan SN, et al. A reporter cell line for real-time imaging of autophagy and apoptosis. Toxicol Lett. 2020 Jun 15;326:23-30. doi: 10.1016/j.toxlet.2020.02.011.Sellmyer MA, Richman SA, Lohith K, et al. Imaging CAR T Cell Trafficking with eDHFR as a PET Reporter Gene. Mol Ther. 2020 Jan 8;28(1):42-51. doi: 10.1016/j.ymthe.2019.10.007.Sellmyer MA, Richman SA, Lohith K, et al. Imaging CAR T Cell Trafficking with eDHFR as a PET Reporter Gene. Mol Ther. 2020 Jan 8;28(1):42-51. doi: 10.1016/j.ymthe.2019.10.007.Jung J, Hwang S, Seol H, et al. Generation of Brachyury-mCherry knock-in reporter human pluripotent stem cell line (SNUe003-A-2) using CRISPR/CAS9 nuclease. Stem Cell Res. 2021 May;53:102321. doi: 10.1016/j.scr.2021.102321.Jung J, Hwang S, Seol H, et al. Generation of Brachyury-mCherry knock-in reporter human pluripotent stem cell line (SNUe003-A-2) using CRISPR/CAS9 nuclease. Stem Cell Res. 2021 May;53:102321. doi: 10.1016/j.scr.2021.102321.Auld DS, Narahari J, Ho PI, et al. Characterization and Use of TurboLuc Luciferase as a Reporter for High-Throughput Assays. Biochemistry. 2018 Aug 7;57(31):4700-4706. doi: 10.1021/acs.biochem.8b00290.Auld DS, Narahari J, Ho PI, et al. Characterization and Use of TurboLuc Luciferase as a Reporter for High-Throughput Assays. Biochemistry. 2018 Aug 7;57(31):4700-4706. doi: 10.1021/acs.biochem.8b00290.Phonbuppha J, Tinikul R, Chaiyen P. Use of Bacterial Luciferase as a Reporter Gene in Eukaryotic Systems. Methods Mol Biol. 2021;2274:53-65. doi: 10.1007/978-1-0716-1258-3_6.Phonbuppha J, Tinikul R, Chaiyen P. Use of Bacterial Luciferase as a Reporter Gene in Eukaryotic Systems. Methods Mol Biol. 2021;2274:53-65. doi: 10.1007/978-1-0716-1258-3_6.Ghalehbandi S, Yuzugulen J, Pranjol MZI, Pourgholami MH. The role of VEGF in cancer-induced angiogenesis and research progress of drugs targeting VEGF. Eur J Pharmacol. 2023 Jun 15;949:175586. doi: 10.1016/j.ejphar.2023.175586.Ghalehbandi S, Yuzugulen J, Pranjol MZI, Pourgholami MH. The role of VEGF in cancer-induced angiogenesis and research progress of drugs targeting VEGF. Eur J Pharmacol. 2023 Jun 15;949:175586. doi: 10.1016/j.ejphar.2023.175586.Callan A, Heckman J, Tah G, et al. VEGF in Diabetic Retinopathy and Age-Related Macular Degeneration. Int J Mol Sci. 2025 May 22;26(11):4992. doi: 10.3390/ijms26114992.Callan A, Heckman J, Tah G, et al. VEGF in Diabetic Retinopathy and Age-Related Macular Degeneration. Int J Mol Sci. 2025 May 22;26(11):4992. doi: 10.3390/ijms26114992.Shi Z, Kuai M, Li B, et al. The role of VEGF in Cancer angiogenesis and tumorigenesis: Insights for anti-VEGF therapy. Cytokine. 2025 May;189:156908. doi: 10.1016/j.cyto.2025.156908.Shi Z, Kuai M, Li B, et al. The role of VEGF in Cancer angiogenesis and tumorigenesis: Insights for anti-VEGF therapy. Cytokine. 2025 May;189:156908. doi: 10.1016/j.cyto.2025.156908.Doggrell SA. Pegaptanib: the first antiangiogenic agent approved for neovascular macular degeneration. Expert Opin Pharmacother. 2005 Jul;6(8):1421-3. doi: 10.1517/14656566.6.8.1421.Doggrell SA. Pegaptanib: the first antiangiogenic agent approved for neovascular macular degeneration. Expert Opin Pharmacother. 2005 Jul;6(8):1421-3. doi: 10.1517/14656566.6.8.1421.Takeda AL, Colquitt J, Clegg AJ, Jones J. Pegaptanib and ranibizumab for neovascular age-related macular degeneration: a systematic review. Br J Ophthalmol. 2007 Sep;91(9):1177-82. doi: 10.1136/bjo.2007.118562.Takeda AL, Colquitt J, Clegg AJ, Jones J. Pegaptanib and ranibizumab for neovascular age-related macular degeneration: a systematic review. Br J Ophthalmol. 2007 Sep;91(9):1177-82. doi: 10.1136/bjo.2007.118562.Rasmussen A, Sander B, Larsen M, et al. Neovascular age-related macular degeneration treated with ranibizumab or aflibercept in the same large clinical setting: visual outcome and number of injections. Acta Ophthalmol. 2017 Mar;95(2):128-132. doi: 10.1111/aos.13233.Rasmussen A, Sander B, Larsen M, et al. Neovascular age-related macular degeneration treated with ranibizumab or aflibercept in the same large clinical setting: visual outcome and number of injections. Acta Ophthalmol. 2017 Mar;95(2):128-132. doi: 10.1111/aos.13233.Kanadani T, Rabelo N, Takahashi D, et al. Comparison of antiangiogenic agents (ranibizumab, aflibercept, bevacizumab and zivaflibercept) in the therapeutic response to the exudative form of age-related macular degeneration according to the treat-and-extend protocol- true head-to-head study. Int J Retina Vitreous. 2024 Feb 2;10(1):13. doi: 10.1186/s40942-024-00537-5.Kanadani T, Rabelo N, Takahashi D, et al. Comparison of antiangiogenic agents (ranibizumab, aflibercept, bevacizumab and zivaflibercept) in the therapeutic response to the exudative form of age-related macular degeneration according to the treat-and-extend protocol- true head-to-head study. Int J Retina Vitreous. 2024 Feb 2;10(1):13. doi: 10.1186/s40942-024-00537-5.Avery RL, Castellarin AA, Steinle NC, et al. Systemic pharmacokinetics following intravitreal injections of ranibizumab, bevacizumab or aflibercept in patients with neovascular AMD. Br J Ophthalmol. 2014 Dec;98(12):1636-41. doi: 10.1136/bjophthalmol-2014-305252.Avery RL, Castellarin AA, Steinle NC, et al. Systemic pharmacokinetics following intravitreal injections of ranibizumab, bevacizumab or aflibercept in patients with neovascular AMD. Br J Ophthalmol. 2014 Dec;98(12):1636-41. doi: 10.1136/bjophthalmol-2014-305252.Wang L, Liu WQ, Broussy S, et al. Recent advances of anti-angiogenic inhibitors targeting VEGF/VEGFR axis. Front Pharmacol. 2024 Jan 4;14:1307860. doi: 10.3389/fphar.2023.1307860.Wang L, Liu WQ, Broussy S, et al. Recent advances of anti-angiogenic inhibitors targeting VEGF/VEGFR axis. Front Pharmacol. 2024 Jan 4;14:1307860. doi: 10.3389/fphar.2023.1307860.Ge P, Reyila A, Li XY, et al. Efficacy and safety of aflibercept plus chemotherapy in metastatic colorectal cancer: A systematic review and PRISMA-Compliant single-arm Meta-Analysis of noncomparative clinical studies and randomized controlled trials. J Clin Pharm Ther. 2022 Jun;47(6):798-808. doi: 10.1111/jcpt.13610.Ge P, Reyila A, Li XY, et al. Efficacy and safety of aflibercept plus chemotherapy in metastatic colorectal cancer: A systematic review and PRISMA-Compliant single-arm Meta-Analysis of noncomparative clinical studies and randomized controlled trials. J Clin Pharm Ther. 2022 Jun;47(6):798-808. doi: 10.1111/jcpt.13610.Лаксолан® Препараты компании Генериум | Лекарства — описания и характеристики. Доступно по: https://www.generium.ru/products/laksolan/. Ссылка активна на 21.09.2026.Laksolan® Preparaty kompanii Generium | Lekarstva — opisaniya i kharakteristiki. (In Russ). Доступно по: https://www.generium.ru/products/laksolan/. Ссылка активна на 21.09.2026.Holz FG, Oleksy P, Ricci F, et al; COLUMBUS-AMD Study Group. Efficacy and Safety of Biosimilar FYB201 Compared with Ranibizumab in Neovascular Age-Related Macular Degeneration. Ophthalmology. 2022 Jan;129(1):54-63. doi: 10.1016/j.ophtha.2021.04.031.Holz FG, Oleksy P, Ricci F, et al; COLUMBUS-AMD Study Group. Efficacy and Safety of Biosimilar FYB201 Compared with Ranibizumab in Neovascular Age-Related Macular Degeneration. Ophthalmology. 2022 Jan;129(1):54-63. doi: 10.1016/j.ophtha.2021.04.031.Woo SJ, Veith M, Hamouz J, et al. Efficacy and Safety of a Proposed Ranibizumab Biosimilar Product vs a Reference Ranibizumab Product for Patients With Neovascular Age-Related Macular Degeneration: A Randomized Clinical Trial. JAMA Ophthalmol. 2021 Jan 1;139(1):68-76. doi: 10.1001/jamaophthalmol.2020.5053.Woo SJ, Veith M, Hamouz J, et al. Efficacy and Safety of a Proposed Ranibizumab Biosimilar Product vs a Reference Ranibizumab Product for Patients With Neovascular Age-Related Macular Degeneration: A Randomized Clinical Trial. JAMA Ophthalmol. 2021 Jan 1;139(1):68-76. doi: 10.1001/jamaophthalmol.2020.5053.Woo SJ, Bradvica M, Vajas A, et al. Efficacy and Safety of the Aflibercept Biosimilar SB15 in Neovascular Age-Related Macular Degeneration: A Phase 3 Randomized Clinical Trial. JAMA Ophthalmol. 2023 Jul 1;141(7):668-676. doi: 10.1001/jamaophthalmol.2023.2260.Woo SJ, Bradvica M, Vajas A, et al. Efficacy and Safety of the Aflibercept Biosimilar SB15 in Neovascular Age-Related Macular Degeneration: A Phase 3 Randomized Clinical Trial. JAMA Ophthalmol. 2023 Jul 1;141(7):668-676. doi: 10.1001/jamaophthalmol.2023.2260.Yu L, Jia C, Yao W, et al. Development and Validation of a ReporterCell-Line-Based Bioassay for Therapeutic Soluble gp130-Fc. Molecules. 2019 Oct 25;24(21):3845. doi: 10.3390/molecules24213845.Yu L, Jia C, Yao W, et al. Development and Validation of a ReporterCell-Line-Based Bioassay for Therapeutic Soluble gp130-Fc. Molecules. 2019 Oct 25;24(21):3845. doi: 10.3390/molecules24213845.Human vascular endothelial growth factor receptor 2 reporter assay system. Indigo Biosciences. Available from: https://indigobiosciences.com/product/human-vegfr-reporter-assay-kit/. Accessed September 21, 2026.Human vascular endothelial growth factor receptor 2 reporter assay system. Indigo Biosciences. Available from: https://indigobiosciences.com/product/human-vegfr-reporter-assay-kit/. Accessed September 21, 2026.NFAT-Luc/KDR Stable Cell Line - HEK293 (CSC-RR0555). Creative Biogene. Available from: https://www.creative-biogene.com/nfatluc-kdr-stable-cell-line-hek293t-item-csc-rr0541-475126.HTML. Accessed September 21, 2026.NFAT-Luc/KDR Stable Cell Line - HEK293 (CSC-RR0555). Creative Biogene. Available from: https://www.creative-biogene.com/nfatluc-kdr-stable-cell-line-hek293t-item-csc-rr0541-475126.HTML. Accessed September 21, 2026.NFAT-RE-luc2P HEK293 Cell Line. Promega Corporation. Available from: https://worldwide.promega.com/products/luciferase-assays/geneticreporter-vectors-and-cell-lines/gloresponse-nfat-re-luc2p-hek293-cellline/?tabset0=0. Accessed September 21, 2026.NFAT-RE-luc2P HEK293 Cell Line. Promega Corporation. Available from: https://worldwide.promega.com/products/luciferase-assays/geneticreporter-vectors-and-cell-lines/gloresponse-nfat-re-luc2p-hek293-cellline/?tabset0=0. Accessed September 21, 2026.VEGFR2/NFAT reporter Hek293 recombinant cell line. BPS Bioscience. Available from: https://bpsbioscience.com/vegfr2-nfat-hek293-cell-line-79387. Accessed September 21, 2026.VEGFR2/NFAT reporter Hek293 recombinant cell line. BPS Bioscience. Available from: https://bpsbioscience.com/vegfr2-nfat-hek293-cell-line-79387. Accessed September 21, 2026.VEGF Reporter 293 Cell Line. AcceGen Biotechnology. Available from: https://www.accegen.com/product/vegf-reporter-293-cell-line-abcrc154f/. Accessed September 21, 2026.VEGF Reporter 293 Cell Line. AcceGen Biotechnology. Available from: https://www.accegen.com/product/vegf-reporter-293-cell-line-abcrc154f/. Accessed September 21, 2026.Human VEGF R2 (Luc) HEK293 Reporter Cell. ACROBiosystems. Available from: https://www.acrobiosystems.com/P4553-Human-VEGFR2-%28Luc%29-HEK293-Reporter-Cell.html?srsltid=AfmBOop6HW1IMLL8A5OnVPgBSSg2CaoEhnEsSGz4JOXcUtUuk1iENOYx. Accessed September 21, 2026.Human VEGF R2 (Luc) HEK293 Reporter Cell. ACROBiosystems. Available from: https://www.acrobiosystems.com/P4553-Human-VEGFR2-%28Luc%29-HEK293-Reporter-Cell.html?srsltid=AfmBOop6HW1IMLL8A5OnVPgBSSg2CaoEhnEsSGz4JOXcUtUuk1iENOYx. Accessed September 21, 2026.

The authors declare that there are no conflicts of interest present.