Annals of the Russian academy of medical sciencesAnnals of the Russian academy of medical sciencesВестник Российской академии медицинских наук0869-60472414-3545"Paediatrician" Publishers LLC111310.15690/vramn1113MOLECULAR MEDICINE AND GENETICS: CURRENT ISSUESАКТУАЛЬНЫЕ ВОПРОСЫ ГЕНЕТИКИ И МОЛЕКУЛЯРНОЙ МЕДИЦИНЫResearch ArticleGenome-editing techniques to increase the therapeutic efficacy of monoclonal antibodiesГеномное редактирование для оптимизации синтеза и повышения терапевтической эффективности рекомбинантных моноклональных антителhttps://orcid.org/0000-0002-9898-98949166-9151FirstovaVictoria V.ФирстоваВиктория Валерьевна
Russian Federation

PhD

д.б.н.

victoria1@mail.ruhttps://orcid.org/0000-0001-9667-16743180-1459ShemyakinIgor G.ШемякинИгорь Георгиевич
Russian Federation

PhD, Professor

д.м.н., профессор

shemyakin@obolensk.orghttps://orcid.org/0000-0003-1078-45856567-8380DyatlovIvan A.ДятловИван Алексеевич
Russian Federation

MD, PhD, Professor

д.м.н., профессор, академик РАН

dyatlov@obolensk.orgState Research Center for Applied Microbiology and BiotechnologyГосударственный научный центр прикладной микробиологии и биотехнологии24122019211220197463783872504201906112019Copyright ©; 2019, "Paediatrician" Publishers LLCCopyright ©; 2019, Издательство "Педиатръ"2019"Paediatrician" Publishers LLCИздательство "Педиатръ"https://vestnikramn.spr-journal.ru/jour/article/view/1113

The review presents a general description of therapeutic monoclonal antibodies, cell lines used to obtain them, characterizes the reasons for the immunogenicity of recombinant antibodies, and approaches used to eliminate the side effects of therapeutic monoclonal antibodies. The focus is on resolving the immunogenicity problems of fully human therapeutic monoclonal antibodies. The most attention are concentrated on the data of antibody-producing cell genomic editing to increase the yield of the product, the stability of expression of the recombinant protein and reduce its immunogenicity. Modern methods of site-directed modification (zinc finger method, TALEN and CRISPR/CAS9) for editing the genome of the CHO cell line are analyzed. The strategies of genomic editing choice carrying out taking into account the advances of omix technologies are discussed. Approaches to increase the life span of producer cells are considered, including an increase in the expression of anti-apoptotic signals and the deletion of proapoptotic genes, an increase in the duration of the cell cycle of cells in the G0/G1 phase. The approaches used to regulate the posttranslational modification of monoclonal antibodies are considered. Significant part of the review are devoted to the discussion of the spesificity and differences of glycosylation, galactosylation and sialization of monoclonal antibodies in different expression systems and the associated different degree of immunogenicity of monoclonal antibodies. The main approaches to the regulation of the synthesis of monoclonal antibodies at the stage of translation using non-coding RNA are considered.

В обзоре приведены общая характеристика терапевтических моноклональных антител; рассмотрены клеточные линии, используемые для их получения; охарактеризованы причины иммуногенности рекомбинантных антител и подходы, используемые для устранения побочных эффектов терапевтических моноклональных антител. Суммированы литературные данные о геномном редактировании клеток-продуцентов моноклональных антител с целью повышения выхода продукта, стабильности экспрессии рекомбинантного белка и снижения его иммуногенности. Проанализированы современные методы сайт-направленной модификации (метод «цинковых пальцев», TALEN и CRISPR/CAS9) для редактирования генома клеточной линии СНО. Проведен анализ выбора стратегии геномного редактирования с учетом достижений омиксных технологий. Рассматриваются подходы к увеличению продолжительности жизни клеток-продуцентов, включая увеличение экспрессии антиапоптотических сигналов и делецию проапоптотических генов, увеличение длительности клеточного цикла клеток в G0/G1-фазе. Описаны подходы, используемые для регулирования посттрансляционной модификации моноклональных антител. Значительное место в обзоре отведено обсуждению особенностей гликозилирования, галактозилирования и сиалирования моноклональных антител в разных системах экспрессии и связанной с ними степенью иммуногенности моноклональных антител. Рассмотрены основные подходы регуляции синтеза моноклональных антител на стадии трансляции с использованием некодирующих РНК.

monoclonal antibodiesCHO cell linegenomic editingCRISPR/CAS9non-coding RNAмоноклональные антителаклеточная линия СНОгеномное редактированиеCRISPR/CAS9некодирующие РНКThe work was carried out in the framework of Agreement No. 075-15-2019-1671 of October 31, 2019 with the Ministry of Education and Science of Russia (task 2.8. Development of recombinant human monoclonal antibodies for the treatment of diseases caused by pathogenic microorganisms, bacterial and plant toxins).Работа выполнена в рамках Соглашения № 075-15-2019-1671 от 31 октября 2019 г. с Минобрнауки России (задача 2.8 Разработка рекомбинантных человеческих моноклональных антител для лечения заболеваний, вызванных патогенными микроорганизмами, бактериальными и растительными токсинами).Grilo AL, Mantalaris A. The increasingly human and profitable monoclonal antibody market. Sci Soc. 2019;37(1):9−16. doi: 10.1016/j.tibtech.2018.05.014.Jefferis R. Glycosylation as a strategy to improve antibody’based therapeutics. Nat Rev Drug Discov. 2009;8(3):226−234. doi: 10.1038/nrd2804.Puck TT. Development of the Chinese Hamster Ovary (CHO) cell for use in somatic cell genetics. In: Gottesman M.M., ed. Molecular cell genetics. John Wiley and Sons: New York, NY, USA; 1985. Р. 37–64.Eigen M, Schuster P. The hypercycle. A principle of natural self-organization. Part A: Emergence of the hypercycle. Naturwissenschaften. 1977;64(11):541−565. doi: 10.1007/bf00450633.Wurm FM, Wurm MJ. Cloning of CHO cells, productivity and genetic stability ― a discussion. Processes. 2017;5(20):1−13. doi: 10.3390/pr5020020.Wang W, Zheng W, Hu F, et al. Enhanced biosynthesis performance of heterologous proteins in CHO-K1 cells using CRISPR-Cas9. ACS Synth Biol. 2018;7(5):1259−1268. doi: 10.1021/acssynbio.7b00375.Lieu PT, Machleidt T, Thyagarajan B, et al. Generation of site-specific retargeting platform cell lines for drug discovery using phiC31 and R4 integrases. J Biomol Screen. 2009;14(10):1207−1215. doi: 10.1177/1087057109348941.Kito M, Itami S, Fukano Y, et al. Construction of engineered CHO strains for high-level production of recombinant proteins. Appl Microbiol Biotechnol. 2002;60(4):442−448. doi: 10.1007/s00253-002-1134-1.Huang Y, Li Y, Wang YG, et al. An efficient and targeted gene integration system for high-level antibody expression. J Immunol Methods. 2007;322(1−2):28−39. doi: 10.1016/j.jim.2007.01.022Jia YL, Guo X, Lu JT, et al. CRISPR/Cas9-mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long-term stability. J Cell Mol Med. 2018;22(9):4106−4116. doi: 10.1111/jcmm.13687.Borth N, Mattanovich D, Kunert R, Katinger H. Effect of increased expression of protein disulfide isomerase and heavy chain binding protein on antibody secretion in a recombinant CHO cell line. Biotechnol Prog. 2005;21(1):106–111. doi: 10.1021/bp0498241.Tan HK, Lee MM, Yap MG, Wang DI. Overexpression of cold-inducible RNA-binding protein increases interferon-gamma production in Chinese-hamster ovary cells. Biotechnol Appl Biochem. 2008;49(4):247−257. doi: 10.1042/BA20070032.Zhang P, Woen S, Wang T, et al. Challenges of glycosylation analysis and con’ trol: an integrated approach to producing optimal and consistent therapeutic drugs. Drug Discov. Today. 2016;21:740–765. doi: 10.1016/j.drudis.2016.01.006.Toussaint C, Henry O, Durocher Y. Metabolic engineering of CHO cells to alter lactate metabolism during fed-batch cultures. J Biotechnol. 2016;217:122−131. doi: 10.1016/j.jbiotec.2015.11.010.Zhou M, Crawford Y, Ng D, et al. Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases. J Biotechnol. 2011;153(1−2):27−34. doi: 10.1016/j.jbiotec.2011.03.003.Ibarra N, Watanabe S, Bi JX, et al. Modulation of cell cycle for enhancement of antibody productivity in perfusion culture of NS0 cells. Biotechnol Prog. 2003;19(1):224−228. doi: 10.1021/bp025589f.Kunert R, Reinhart D. Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol. 2016;100:3451–3461. doi: 10.1007/s00253-016-7388-9.Pybus LP, Dean G, West NR, et al. Model-directed engineering of “difficult-to-express” monoclonal antibody production by Chinese hamster ovary cells. Biotechnol Bioeng. 2014;111(2):372−385. doi: 10.1002/bit.25116.Jadhav V, Hackl M, Bort JA, et al. A screening method to assess biological effects of microRNA overexpression in Chinese hamster ovary cells. Biotechnol Bioeng. 2012;109(6):1376−1385. doi: 10.1002/bit.24490.Hackl M, Jadhav V, Klanert G, et al. Analysis of microRNA transcription and post-transcriptional processing by Dicer in the context of CHO cell proliferation. J Biotechnol. 2014;190:76–84. doi: 10.1016/j.jbiotec.2013.12.018.Druz A, Betenbaugh M, Shiloach J. Glucose depletion activates mmu-miR-466h-5p expression through oxidative stress and inhibition of histone deacetylation. Nucleic Acids Research. 2012;40(15):7291–7302. doi: 10.1093/nar/gks452.Chung S, Quarmby V, Gao X, et al. Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities. MAbs. 2012;4(3):326−340. doi: 10.4161/mabs.19941.Ronda C, Pedersen LE, Hansen HG, et al. Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool. Biotechnol Bioeng. 2014;111(8):1604−1616. doi: 10.1002/bit.25233.Chung CY, Wang Q, Yang S, et al. Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells. Biotechnol Bioeng. 2017;114(12):2848−2856. doi: 10.1002/bit.26375.Chusainow J, Yang YS, Yeo JH, et al. A study of monoclonal antibody‐producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng. 2009;102(4):1182−1196. doi: 10.1002/bit.22158.Osterlehner A, Simmeth S, Göpfert U. Promoter methylation and transgene copy numbers predict unstable protein production in recombinant Chinese hamster ovary cell lines. Biotechnol Bioeng. 2011;108(11):2670‐2681. doi: 10.1002/bit.23216.Du Z, Treiber D, McCarter JD, et al. Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures. Biotechnol Bioeng. 2015;112(1):141–155. doi: 10.1002/bit.25332.Zhang X, Han L, Zong H, et al. Enhanced production of anti-PD1 antibody in CHO cells through transient co-transfection with anti-apoptotic genes Bcl-x L and Mcl-1. Bioprocess Biosyst Eng. 2018;41(5):633−640. doi: 10.1007/s00449-018-1898-z.Pasquinelli AE. MicroRNAs and their targets: Recognition, regulation and an emerging reciprocal relationship. Nat Rev Genet. 2012;13(4):271–282. doi: 10.1038/nrg3162.Barron N, Sanchez N, Kelly P, Clynes M. MicroRNAs: tiny targets for engineering CHO cell phenotypes? Biotechnol Lett. 2011;33(1):11–21. doi: 10.1007/s10529-010-0415-5.Hack M, Borth N, Grillari J. MiRNAs ― pathway engineering of CHO cell factories that avoids translational burdening. Trends Biotechnol. 2012;30:405–406. doi: 10.1016/j.tibtech.2012.05.002.Burroughs AM, Ando Y, de Hoon MJ, et al. Deep-sequencing of human Argonaute-associated small RNAs provides insight into miRNA sorting and reveals Argonaute association with RNA fragments of diverse origin. RNA Biol. 2011;8(1):158–177. doi: 10.4161/rna.8.1.14300.Keam SP, Hutvagner G. tRNA-derived fragments (tRFs): emerging new roles for an ancient RNA in the regulation of gene expression. Life. 2015;5(4):1638–1651. doi: 10.3390/life5041638.Dietrich A, Wallet C, Iqbal RK, et al. Organellar non-coding RNAs: emerging regulation mechanisms. Biochimie. 2015;117:48–62. doi: 10.1016/j.biochi.2015.06.027.Ro S, Ma HY, Park C, et al. The mitochondrial genome encodes abundant small noncoding RNAs. Cell Res. 201;23(6):759–774. doi: 10.1038/cr.2013.37.Stiefel F, Fischer S, Sczyrba A, et al. miRNA profiling of high, low and non-producing CHO cells during biphasic fed-batch cultivation reveals process relevant targets for host cell engineering. J Biotechnol. 2016;225:31–43. doi: 10.1016/j.jbiotec.2016.03.028.Klanert G, Jadhav V, Shanmukam V, et al. A signature of 12 microRNAs is robustly associated with growth rate in a variety of CHO cell lines. J Biotechnol. 2016;235:150–161. doi: 10.1016/j.jbiotec.2016.03.022Lim SM, Park SH, Lee JH, et al. Differential expression of microRNAs in recombinant Chinese hamster ovary cells treated with sodium butyrate using digital RNA counting. J Biotechnol. 2018;283:37−42. doi: 10.1016/j.jbiotec.2018.07.018.Bort JA, Hackl M, Hoflmayer H, et al. Dynamic mRNA and miRNA profiling of CHO-K1 suspension cell cultures. J Biotechnol. 2012;7(4):500–515. doi: 10.1002/biot.201100143.Betancur JG. Pervasive lncRNA binding by epigenetic modifying complexes: the challenges ahead. Biochim Biophys Acta. 2016;1859(1):93−101. doi: 10.1016/j.bbagrm.2015.10.009.Trimarchi T, Bilal E, Ntziachristos P, et al. Genome-wide mapping and characterization of Notch-regulated long noncoding RNAs in acute leukemia. Cell. 2014;158(3):593−606. doi: 10.1016/j.cell.2014.05.049.Gonzalez I, Munita R, Agirre E, et al. A lncRNA regulates alternative splicing via establishment of a splicing-specific chromatin signature. Nat Struct Mol Biol. 2015;22(5):370−376. doi: 10.1038/nsmb.3005.Loayza-Puch F, Agamia R. Lncing protein translation to metastasis. EMBO J. 2013;32(20):2657–2658. doi: 10.1038/emboj.2013.210.Geisler S, Coller J. RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts. Nat Rev Mol Cell Biol. 2013;14(11):699−712. doi: 10.1038/nrm3679.Tay Y, Rinn J, Pandolfi PP. The multilayered complexity of ceRNA crosstalk and competition. Nature. 2014;505(7483):344−352. doi: 10.1038/nature12986.Chujo T, Yamazaki T, Hirose T. Architectural RNAs (arcRNAs): a class of long noncoding RNAs that function as the scaffold of nuclear bodies. Biochim Biophys Acta. 2016;1859(1):139−146. doi: 10.1016/j.bbagrm.2015.05.007.Gomez JA, Wapinski OL, Yang YW, et al. The NeST long ncRNA controls microbial susceptibility and epigenetic activation of the interferon-γ locus. Cell. 2013;152(4):743−754. doi: 10.1016/j.cell.2013.01.015.Vito D, Smales CM. The long non-coding RNA transcriptome landscape in CHO cells under batch and fed-batch conditions. Biotechnol J. 2018;13(10):e1800122. doi: 10.1002/biot.201800122.Patrucco L, Chiesa A, Soluri MF, et al. Engineering mammalian cell factories with SINEUP noncoding RNAs to improve translation of secreted proteins. Gene. 2015;569(2):287−293. doi: 10.1016/j.gene.2015.05.070.Zucchelli S, Patrucco L, Persichetti F, et al. Engineering translation in mammalian cell factories to increase protein yield: the unexpected use of long non-coding SINEUP RNAs. Comput Struct Biotechnol J. 2016;14:404−410. doi: 10.1016/j.csbj.2016.10.004.Xu X, Nagarajan H, Lewis NE, et al. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat Biotechnol. 2011;29(8):735−741. doi: 10.1038/nbt.1932.Becker J, Hackl M, Rupp O, et al. Unraveling the Chinese hamster ovary cell line transcriptome by next-generation sequencing. J Biotechnol. 2011;156(3):227−235. doi: 10.1016/j.jbiotec.2011.09.014.Plomp R, Dekkers G, Rombouts Y, et al. Hinge region O-glycosylation of human immunoglobulin G3 (IgG3). Mol Cell Proteomics. 2015;14(5):1373–1384. doi: 10.1074/mcp.M114.047381.Popp O, Moser S, Zielonka J, et al. Development of a pre-glycoengineered CHO-K1 host cell line for the expression of antibodies with enhanced Fc mediated effector function. MAbs. 2018;10(2):290−303. doi: 10.1080/19420862.2017.1405203.Grav LM, Lee JS, Gerling S, et al. One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment. Biotechnol J. 2015;10(9):1446−1456. doi: 10.1002/biot.201500027.Mimura Y, Kelly RM, Unwin L, et al. Enhanced sialylation of a human chimeric IgG1 variant produced in human and rodent cell lines. J Immunol Methods. 2016;428:30–36. doi: 10.1016/j.jim.2015.11.009.Kurogochi M, Mori M, Osumi K, et al. Glycoengineered monoclonal antibodies with homogeneous glycan (M3, G0, G2, and A2) using a chemoenzymatic approach have different affinities for Fc-gamma RIIIa and variable antibody-dependent cellular cytotoxicity activities. PloS one. 2015;10:e0132848. doi: 10.1371/journal.pone.0132848.