Актуальные подходы к оценке биологической активности иммуноцитокинов в условиях in vitro

В настоящем обзоре рассматриваются существующие подходы к оценке биологической активности иммуноцитокинов in vitro в ходе доклинических исследований: с использованием монослойных клеточных культур, репортерных клеточных систем, совместных культур, трехмерных (3D) моделей опухолей. Монослойных культур достаточно для подтверждения используемого в дизайне иммуноцитокина механизма действия его отдельных участков, и  тест-системы «золотого стандарта» для определения специфической биологической активности цитокина и эффекторных функций антительного участка остаются востребованными. Коммерческие репортерные клеточные системы являются альтернативным вариантом оценки биологической активности цитокинового и антительного участков на уровне активации сигнальных путей. Совместные культуры опухолевых и эффекторных клеток позволяют оценить цитотоксический и иммуномодулирующий эффекты антительного и цитокинового участков без использования методов 3D-культивирования. Использование 3D-моделей опухолей позволяет заменить несколько тестов на биологическую активность отдельных участков иммуноцитокина, проводимых на монокультурах и совместных культурах, на одно комплексное исследование, однако такие модели требуют значительных временных и материальных затрат.

1. Berraondo P., Sanmamed M.F., Ochoa M.C. et al. Cytokines in clinical cancer immunotherapy. Br J Cancer 2019;120(1):6–15. DOI: 10.1038/s41416-018-0328-y

2. Conlon K.C., Miljkovic M.D., Waldmann T.A. Cytokines in the treatment of cancer. J Interferon Cytokine Res 2019;39(1):6–21. DOI: 10.1089/jir.2018.0019

3. Hutmacher C., Neri D. Antibody-cytokine fusion proteins: biopharmaceuticals with immunomodulatory properties for cancer therapy. Adv Drug Deliv Rev 2019;141:67–91. DOI: 10.1016/j.addr.2018.09.002

4. Collins S., Joshi A., Shen L. et al. 357 TAK-573, an anti-CD38-attenuated interferon alpha (IFNα) fusion protein (AttenukineTM), has demonstrated IFNα receptor (IFNAR) pathway modulation in patients with relapsed/refractory multiple myeloma. J Immunother Cancer 2020;8:Suppl 3. DOI: 10.1136/jitc-2020-SITC2020.0357

5. Runbeck E., Crescioli S., Karagiannis S.N., Papa S. Utilizing immunocytokines for cancer therapy. Antibodies (Basel) 2021;10(1):10. DOI: 10.3390/antib10010010

6. Kaufman H.L., Mehnert J.M., Cuillerot J.-M. et al. Targeted modified IL-2 (NHS-IL2, MSB0010445) combined with stereotactic body radiation in advanced melanoma patients after progression on ipilimumab: assessment of safety, clinical, and biologic activity in a phase 2a study. J Clin Oncol 2014;32(15 suppl):TPS9107. DOI: 10.1200/jco.2014.32.15_suppl.tps9107

7. Overgaard N.H., Fan T.M., Schachtschneider K.M. et al. Of mice, dogs, pigs, and men: choosing the appropriate model for immuno-oncology research. ILAR J 2018;59(3):247–62. DOI: 10.1093/ilar/ily014

8. Neri D. Antibody-cytokine fusions: versatile products for the modulation of anticancer immunity. Cancer Immunol Res 2019:7(3):348–54. DOI: 10.1158/2326-6066.CIR-18-0622

9. Young P.A., Morrison S.L., Timmerman J.M. Antibody-cytokine fusion proteins for treatment of cancer: engineering cytokines for improved efficacy and safety. Semin Oncol 2014;41(5):623–36. DOI: 10.1053/j.seminoncol.2014.08.002

10. Gillies S.D., Lan Y., Lo K.M. et al. Improving the efficacy of antibody-interleukin 2 fusion proteins by reducing their interaction with Fc receptors. Cancer Res 1999;59(9):2159–66.

11. Wahab S., Hussain A. Cytokines as targets for immunomodulation. Int J Pharm Pharm Sci 2013;5:60–4.

12. Zou G.M., Tam Y.K. Cytokines in the generation and maturation of dendritic cells: recent advances. Eur Cytokine Netw 2002;13(2):186–99.

13. Gillies S.D., Lan Y., Hettmann T. et al. A low-toxicity IL-2-based immunocytokine retains antitumor activity despite its high degree of IL-2 receptor selectivity. Clin Cancer Res 2011;17(11):3673–85. DOI: 10.1158/1078-0432.CCR-10-2921

14. Pogue S.L., Taura T., Bi M. et al. Targeting attenuated interferon-α to myeloma cells with a CD38 antibody induces potent tumor regression with reduced off-target activity. PLoS ONE 2016;11(9):e0162472. DOI: 10.1371/journal.pone.0162472

15. Klein C., Waldhauer I., Nicolini V.G. et al. Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines. Oncoimmunology 2017;6(3):e1277306 DOI: 10.1080/2162402x.2016.1277306

16. Klein C., Inja W., Nicolini V. et al. Abstract PR8: Novel tumortargeted, engineered IL-2 variant (IL-2v)-based immunocytokines for immunotherapy of cancer. Cancer Res 2013;73:PR8.

17. Hemmerle T., Neri D. The dose-dependent tumor targeting of antibody-IFNγ fusion proteins reveals an unexpected receptor-trapping mechanism in vivo. Cancer Immunol Res 2014;2(6):559–67. DOI: 10.1158/2326-6066.cir-13-0182

18. Kaspar M., Trachsel E., Neri D. The antibody-mediated targeted delivery of interleukin-15 and GM-CSF to the tumor neovasculature inhibits tumor growth and metastasis. Cancer Res 2007;67(10):4940–8. DOI: 10.1158/0008-5472.CAN-07-0283

19. Tzeng A., Kwan B.H., Opel C.F. et al. Antigen specificity can be irrelevant to immunocytokine efficacy and biodistribution. Proc Natl Acad Sci U S A 2015;112(11):3320–5. DOI: 10.1073/pnas.1416159112

20. Gillies S.D. A new platform for constructing antibody-cytokine fusion proteins (immunocytokines) with improved biological properties and adaptable cytokine activity. Protein Eng Des Sel 2013;26(10):561–9. DOI: 10.1093/protein/gzt045

21. Mock J., Stringhini M., Villa A. et al. An engineered 4-1BBL fusion protein with “activity on demand”. Proc Natl Acad Sci U S A 2020;117(50):31780–8. DOI: 10.1073/pnas.2013615117

22. Venetz D., Koovely D., Weder B., Neri D. Targeted reconstitution of cytokine activity upon antigen binding using split cytokine antibody fusion proteins. J Biol Chem 2016;291(35):18139–47. DOI: 10.1074/jbc.M116.737734

23. Кособокова Е.Н., Шешукова Е.В., Пинюгина М.В. и др. Разработка технологии получения слитного белка на основе моноклонального гуманизированного антитела против опухолевого антигена HER2 и интерферона-α-2b человека из Nicotiana benthamiana. Биотехнология 2019;35(2):49–57. DOI: 10.21519/0234-2758-2019-35-2-49-57

24. Mire-Sluis A.R., Page L., Thorpe R. Quantitative cell line based bioassays for human cytokines. J Immunol Methods 1995;187(2):191–9. DOI: 10.1016/0022-1759(95)00220-1

25. Dakhel S., Lizak C., Matasci M. et al. An attenuated targetedTNF localizes to tumors in vivo and regains activity at the site of disease. Int J Mol Sci 2021;22(18):10020. DOI: 10.3390/ijms221810020

26. Jiang X.R., Song A., Bergelson S. et al. Advances in the assessment and control of the effector functions of therapeutic antibodies. Nat Rev Drug Discov 2011;10(2):101–10. DOI: 10.1038/nrd3365

27. Fenerty K.E., Padget M., Wolfson B. et al. Immunotherapy utilizing the combination of natural killer- and antibody dependent cellular cytotoxicity (ADCC)-mediating agents with poly (ADP-ribose) polymerase (PARP) inhibition. J Immunother Cancer 2018;6(1): 133. DOI: 10.1186/s40425-018-0445-4

28. Xi B., Ye P.F., Golubovskaya V., Abassi Y. In vitro functional assay using real-time cell analysis for assessing cancer immunotherapeutic agents. In: Immuno-oncology. Cellular and translational approaches. Ed. by S.L. Tan. Humana, N.Y., 2020. P. 35–50. DOI: 10.1007/978-1-0716-0171-6_3

29. Li Z., Zhu Y., Li C. et al. Anti-VEGFR2-interferon-α2 regulates the tumor microenvironment and exhibits potent antitumor efficacy against colorectal cancer. Oncoimmunology 2017;6(3):e1290038. DOI: 10.1080/2162402X.2017.1290038

30. Wang L., Yu C., Wang J. Development of reporter gene assays to determine the bioactivity of biopharmaceuticals. Biotechnol Adv 2020;39:107466. DOI: 10.1016/j.biotechadv.2019.107466

31. Liu J., Liu R., Gray P. et al. Development of a luciferase reporter Jurkat cell line under the control of endogenous interleukin-2 promoter. J Immunol Methods 2017;451:48–53. DOI: 10.1016/j.jim.2017.08.006

32. Mock J., Pellegrino C., Neri D. A universal reporter cell line for bioactivity evaluation of engineered cytokine products. Sci Rep 2020;10(1):3234. DOI: 10.1038/s41598-020-60182-4

33. Brancato V., Oliveira J.M., Correlo V.M. et al. Could 3D models of cancer enhance drug screening? Biomaterials 2020;232:119744. DOI: 10.1016/j.biomaterials.2019.119744

34. Bregenzer M.E., Horst E.N., Mehta P. et al. Integrated cancer tissue engineering models for precision medicine. PLoS ONE 2019;14(5):e0216564. DOI: 10.1371/journal.pone.0216564

35. Dhiman N., Kingshott P., Sumer H. et al. On-chip anticancer drug screening – recent progress in microfluidic platforms to address challenges in chemotherapy. Biosens Bioelectron 2019;137:236–54. DOI: 10.1016/j.bios.2019.02.070

36. Herter S., Morra L., Schlenker R. et al. A novel threedimensional heterotypic spheroid model for the assessment of the activity of cancer immunotherapy agents. Cancer Immunol Immunother 2017;66(1):129–40. DOI: 10.1007/s00262-016-1927-1

37. Soerensen M.M., Ros W., Rodriguez-Ruiz M.E. et al. Safety, PK/PD, and anti-tumor activity of RO6874281, an engineered variant of interleukin-2 (IL-2v) targeted to tumor-associated fibroblasts via binding to fibroblast activation protein (FAP). J Clin Oncol 2018;36(15 suppl):e15155. DOI: 10.1200/JCO.2018.36.15_suppl.e15155

38. Ayuso J.M., Truttschel R., Gong M.M. et al. Evaluating natural killer cell cytotoxicity against solid tumors using a microfluidic model. Oncoimmunology 2019;8(3):1553477. DOI: 10.1080/2162402X.2018.1553477

39. Gladkov O., Biakhov M., Ramlau R. et al. Phase II trial of huKSIL2 with cyclophosphamide (CTX) in patients with extensive disease small-cell lung cancer (ED-SCLC). J Clin Oncol 2012;30(15 Suppl):7090. DOI: 10.1200/jco.2012.30.15_suppl.7090

40. Jaekel A., Kehler P., Lischke T. et al. Preclinical characterization of GT-00A x IL15: a novel IL-15-based immunocytokine with unique tumor targeting properties. J Immunother Cancer 2021;9(Suppl 2):A741. DOI: 10.1136/jitc-2021-SITC2021.712