ساخت وکتور دوسیسترونی مبتنی بر دومین فیوژن IntF2A برای بیان همزمان زنجیره‌های پلی‌پپتیدی آنتی‌بادی بواسیزوماب و بررسی تظاهر موقت آن در گیاه توتون

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری، گروه مهندسی تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه ارومیه، ارومیه، ایران

2 دانشیار، گروه مهندسی تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه ارومیه، ارومیه، ایران

چکیده

هدف: بواسیزوماب (با نام تجاری آوستین) یک آنتی‌بادی مونوکلونال (mAb) انسانی شده است که به‌طور گسترده‌ای در درمان سرطان‌های مختلفی کاربرد دارد. این دارو جزء گرانقیمت‌ترین و پرفروش‌ترین داروهای دنیا است. بواسیزوماب از طریق تکنولوژی DNA نوترکیب در سیستم بیان پستانداری، تخمدان همستر چینی تولید می‌شود. در سال‌های اخیر، سیستم‌های بیان گیاهی به دلیل مزایای مختلف برای تولید پروتئین‌های نوترکیب دارویی، توجه جهانی را به خود جلب کرده‌اند. تولید موفقیت‌آمیز mAbها به تاشدگی و گردایش مناسب زنجیره سبک (HC) و زنجیره سنگین (LC) بستگی دارد. بیان همزمان کارآمد و کنترل شده این زنجیره‌های پپتیدی یک ملاحظه مهم در طراحی وکتورهای بیان آنتی‌بادی است. هدف این تحقیق ساخت وکتور دو‌سیسترونی برای بیان همزمان زنجیره‌های پپتیدی HC و LC آنتی‌بادی بواسیزوماب با استفاده از دومین فیوژنIntF2A و ارزیابی آن از طریق بیان موقت در گیاه توتون است.

مواد و روش‌ها: به منظور ساخت وکتور نوترکیب، توالی ژن‌های رمزکننده بواسیزوماب و IntF2A از نظر کدونی بهینه‌سازی شد و برای بیان در گیاه توتون به‌طور مصنوعی سنتز شدند. وکتور نوترکیب دوسیسترونی بواسطه IntF2A با روش معمول هضم آنزیمی-اتصال ساخته شد. در نهایت، سازه نوترکیب برای بیان گذرا بواسیزوماب در گیاهان توتون از طریق روش آگروفیلتراسیون استفاده گردید. برای تعیین حضور رونوشت‌ تراژن‌ها از آنالیز RT-PCR استفاده شد. تولید و میزان تجمع آنتی‌بادی نوترکیب از طریق آنالیز وسترن بلات مورد ارزیابی قرار گرفت.
نتایج: ساختار مولکولی وکتور نوترکیب ساخته شده از طریق آنالیز‌های PCR، هضم آنزیمی و توالی‌یابی تایید شد. آنالیز RT-PCR نشان داد تراژن‌ها به‌صورت یک رونوشت واحد بیان می‌شوند. آنالیز وسترن بلات تولید آنتی‌بادی هتروتترامر کامل در برگ‌های آگرواینفیلتر شده را تایید نمود. سطح تجمع آنتی‌بادی به طور متوسط ۲۰/۶۷ میلی‌‌گرم بواسیزوماب به ازای هر کیلوگرم وزن تر (معادل ۳۶/۳ درصد پروتئین محلول کل) برآورد شد.
نتیجه‌گیری: نتایج نشان داد وکتور دو‌سیسترونی مبتنی بر IntF2A بیان همزمان کارآمد ژن‌های رمزکنندة زنجیره‌های پپتیدی آنتی‌بادی و تجمع بالای mAb با فرم کامل را در سلول‌های گیاهی فراهم می‌کند. از وکتور ساخته شده می‌توان برای امکان توسعه و ساخت آنتی‌بادی بواسیزوماب در سیستم بیان گیاهی استفاده کرد.

کلیدواژه‌ها


عنوان مقاله [English]

Construction of the IntF2A fusion domain-based bicistronic vector for simultaneous expression of the polypeptide chains of bevacizumab antibody and investigating its transient expression in tobacco plants

نویسندگان [English]

  • Pari Andaz 1
  • Morad Jafari 2
1 Department of Plant Production and Genetics, Faculty of Agriculture, Urmia University, Urmia, Iran
2 Department of Plant Production and Genetics, Faculty of Agriculture, Urmia University, Urmia, Iran.
چکیده [English]

Objective
Bevacizumab (trade name Avastin®) is a humanized monoclonal antibody (mAb) that is widely used in the treatment of various cancers. It is one of the most expensive and best-selling cancer drugs in the world. Bevacizumab is produced by recombinant DNA technology in a mammalian expression system, the Chinese hamster ovary. In recent years, plant expression systems have attracted global attention due to their various benefits for producing recombinant pharmaceutical proteins. The successful expression of the mAbs is dependent on the proper folding and assembly of the light chains (LC) and heavy chains (HC). The efficient and controlled co-expression of the peptide chains is an essential consideration in designing antibody expression vectors. This study aimed to construct a plant expression vector for the simultaneous expression of HC and LC peptide chains of bevacizumab mAb using the IntF2A fusion domain.
Materials and methods
In order to construct the recombinant vector, the sequences of genes encoding bevacizumab and IntF2A were codon-optimized and artificially synthesized for expression in tobacco plants. The bicistronic recombinant vector was constructed by the routine restriction digestion-ligation method. Finally, the recombinant construct was used for the transient expression of bevacizumab in tobacco plants through agroinfiltration approach. RT PCR analysis was used to determine the presence of transgene transcripts. The production and accumulation level of the recombinant antibody was evaluated through western blotting analysis.
Results
Recombinant plasmid construction is verified by colony PCR, restriction digestion, and sequencing analyses. Expression of the transgenes as a single-transcript unit was confirmed by RT-PCR analysis. Western blot analysis showed the production of the full-length heterotetrameric antibody in agroinfiltrated leaves. The level of antibody accumulation was estimated at an average of 67.20 mg of bevacizumab per kg of fresh weight (3.36% TSP).
Conclusions
The results showed that the IntF2A-based bicistronic vector provides an efficient simultaneous expression of genes encoding the polypeptide chains of antibody and high accumulation of the full-length mAb in plant cells. The constructed vector can be used for the possibility of development and manufacturing of bevacizumab antibody in plant expression system.

کلیدواژه‌ها [English]

  • Agroinfiltration
  • Bevacizumab
  • Bicistronic vector construction
  • IntF2A
  • Tobacco
جعفری مراد، دلیرژ نوروز، حبیبی مهدی (۱۴۰۱) تولید آزمایشگاهی ریتوکسی‌ماب، آنتی‌بادی ضد CD20، در گیاه توتون (Nicotiana tabacum L.). گزارش نهایی طرح تولید محور،‌ دانشگاه ارومیه، ۱۴۲-۱.
References
Bang YH, Kim JE, Lee JS et al. (2021) Bevacizumab plus capecitabine as later-line treatment for patients with metastatic colorectal cancer refractory to irinotecan, oxaliplatin, and fluoropyrimidines. Sci Rep 11, 7118.
Barazandeh A, Mohammadabadi MR, Ghaderi-Zefrehei M, Nezamabadipour H (2016) Genome-wide analysis of CpG islands in some livestock genomes and their relationship with genomic features. Czech J Anim Sci 61, e487.
Bashandy H, Jalkanen S, Teeri TH (2015) Within leaf variation is the largest source of variation in agroinfiltration of Nicotiana benthamiana. Plant Methods 11, e47.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.
Brenner AJ, Floyd J, Fichtel L et al. (2021) Phase 2 trial of hypoxia activated evofosfamide (TH302) for treatment of recurrent bevacizumab-refractory glioblastoma. Sci Rep 11, 2306.
Chan CE, Lim AP, Chan AH et al. (2010) Optimized expression of full-length IgG1 antibody in a common E. coli strain. PLoS One 20, e10261. 
Chen L, Yang X, Luo D, Yu W (2016) Efficient production of a bioactive bevacizumab monoclonal antibody using the 2A self-cleavage peptide in transgenic rice callus. Front Plant Sci 7, 1156.
Chellappan DK, Leng KH, Jia LJ et al. (2018) The role of bevacizumab on tumour angiogenesis and in the management of gynaecological cancers: A review. Biomed Pharmacother 102, 1127-1144.
Chu G, Liu X, Yu W et al. (2021) Cisplatin plus paclitaxel chemotherapy with or without bevacizumab in postmenopausal women with previously untreated advanced cervical cancer: a retrospective study. BMC Cancer 21, 133.
Clarke JL, Paruch L, Dobrica MO et al. (2017) Lettuce‐produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination. Plant Biotechnol J 15, 1611-1621.
Diamos AG, Rosenthal SH, Mason HS (2016) 5′ and 3′ untranslated regions strongly enhance performance of geminiviral replicons in Nicotiana benthamiana leaves. Front Plant Sci 7, e200.
Dudek AZ, Liu LC, Gupta S et al. (2020) Phase Ib/II clinical trial of pembrolizumab with bevacizumab for metastatic renal cell carcinoma: BTCRC-GU14-003. J Clin Oncol 38, 1138-1146.
Feldman DR, Ged Y, Lee CH et al. (2020) Everolimus plus bevacizumab is an effective first‐line treatment for patients with advanced papillary variant renal cell carcinoma: Final results from a phase II trial. Cancer 126, 5247-5255.
François IE, Broekaert WF, Cammue BP (2002) Different approaches for multi-transgene-stacking in plants. Plant Sci 163, 281-295.
Frenzel A, Hust M, Schirrmann T (2013) Expression of recombinant antibodies. Front Immunol 4, 217.
Halpin C, Cooke SE, Barakate A et al. (1999) Self‐processing 2A‐polyproteins–a system for co‐ordinate expression of multiple proteins in transgenic plants. Plant J 17, 453-459.
Haryadi R, Ho S, Kok YJ et al. (2015) Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells. PloS One 10, e0116878.
Holsters M, de Waele D, Depicker A et al. (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163, 181-187.
Huang Z, Phoolcharoen W, Lai H et al. (2010) High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system. Biotechnol Bioeng 106, 9-17.
Jafari M, Delirezh N, Habibi M (2022) Lab-scale production of Rituximab, an anti-CD20 monoclonal antibody, in tobacco plant (Nicotiana tabacum L). Final report of project, Urmia University, 1-142 (in Persian).
Jugler C, Sun H, Nguyen K et al. (2023) A novel plant‐made monoclonal antibody enhances the synergetic potency of an antibody cocktail against the SARS‐CoV‐2 Omicron variant. Plant Biotechnol J 21(3), 549-559.
Jutras PV, Marusic C, Lonoce C et al. (2016) An accessory protease inhibitor to increase the yield and quality of a tumour-targeting mAb in Nicotiana benthamiana leaves. PLoS One 11, e0167086.
Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236, 1299-1302.
Kolotilin I, Topp E, Cox E et al. (2015) Plant-based solutions for veterinary immunotherapeutics and prophylactics. Vet Res 45, 117.
Lin Y, Hung CY, Bhattacharya C et al. (2018) An effective way of producing fully assembled antibody in transgenic tobacco plants by linking heavy and light chains via a self-cleaving 2A peptide. Front Plant Sci 9, 1379.
Liu S, Kasherman L, Fazelzad R et al. (2021) The use of bevacizumab in the modern era of targeted therapy for ovarian cancer: A systematic review and meta-analysis.
Gynecol Oncol 161, 601-612.
Lu RM, Hwang YC, Liu IJ et al. (2020) Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27, 1.
Lugano R., Ramachandran M, Dimberg A. (2020) Tumor angiogenesis: causes, consequences, challenges and opportunities. CMLS 77, 1745-1770.
Luke GA, Roulston C, Tilsner J, Ryan MD (2015) Growing uses of 2A in plant biotechnology. In: Biotechnology (1st edn). Ekinci D (ed). InTech, Croatia. pp. 165-193.
Ma JKC, Drossard J, Lewis D et al. (2015) Regulatory approval and a first‐in‐human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants. Plant Biotechnol J 13, 1106-1120.
Ma L, Lukasik E, Gawehns F, Takken FL (2012) The use of agroinfiltration for transient expression of plant resistance and fungal effector proteins in Nicotiana benthamiana leaves. Plant fungal pathogens: Methods Mol Biol, 835, 61–74.
Marcos JF, Beachy RN (1994) In vitro characterization of a cassette to accumulate multiple proteins through synthesis of a self-processing polypeptide. Plant Mol Biol 24, 495-503.
Masoudzadeh SH, Mohammadabadi MR, Khezri A et al. (2020) Dlk1 gene expression in different tissues of lamb. Iran J Appl Anim Sci 10, 669-677.
Merlin M, Gecchele E, Capaldi S et al. (2014) Comparative evaluation of recombinant protein production in different biofactories: the green perspective. Biomed Res Int 2014, e136419.
Mett V, Chichester JA, Stewart ML et al. (2011) A non-glycosylated, plant-produced human monoclonal antibody against anthrax protective antigen protects mice and non-human primates from B. anthracis spore challenge. Hum Vaccin 7, 183-190.
Mohammadinejad F, Mohammadabadi M, Roudbari Z, Sadkowski T (2022) Identification of key genes and biological pathways associated with skeletal muscle maturation and hypertrophy in Bos taurus, Ovis aries, and Sus scrofa. Animals 12(24):3471.
Pettitt J, Zeitlin L, Kim DH et al. (2013) Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail. Sci Transl Med 5(199):199ra113.
Peyret H, Brown JK, Lomonossoff GP (2019) Improving plant transient expression through the rational design of synthetic 5′ and 3′ untranslated regions. Plant Methods 15, 108.
Phan HT, Pham VT, Ho TT et al. (2020) Immunization with plant-derived multimeric H5 hemagglutinins protect chicken against highly pathogenic avian influenza virus H5N1. Vaccines 8, 593.
Qiu X, Wong G, Audet J et al. (2014) Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature 514, 47-53.
Ralley L, Enfissi EM, Misawa N et al. (2004) Metabolic engineering of ketocarotenoid formation in higher plants. Plant J 39, 477-486.
Rasband, WS (2020) ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/
Robert S, Khalf M, Goulet MC et al. (2013) Protection of recombinant mammalian antibodies from development-dependent proteolysis in leaves of Nicotiana benthamiana. PLoS One 8, e70203. 
Sainsbury F, Lomonossoff GP (2008) Extremely high-level and rapid transient protein production in plants without the use of viral replication. Plant Physiol 148, 1212-1218.
Schlatter S, Stansfield SH, Dinnis DM et al. (2005) On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells. Biotechnol Prog 21, 122-33.
Schillberg S, Finnern R (2021) Plant molecular farming for the production of valuable proteins-Critical evaluation of achievements and future challenges. J Plant Physiol, 258-259, 153359.
Shahsavari M, Mohammadabadi M, Khezri A et al. (2022) Effect of Fennel (Foeniculum Vulgare) Seed Powder Consumption on Insulin-like Growth Factor 1 Gene Expression in the Liver Tissue of Growing Lambs. Gene Express 21 (2), 21-26.
Sil B, Jha S (2014) Plants: the future pharmaceutical factory. Am J Plant Sci 5, 319-327.
Stevens LH, Stoopen GM, Elbers IJ et al. (2000) Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco. Plant Physiol 124, 173-182.
Taylor P (2022) The top 15 best-selling cancer drugs in 2022. www.fiercepharma.com.
Topp E, Irwin R, McAllister T et al. (2016) The case for plant-made veterinary immunotherapeutics. Biotechnol Adv 34, 597-604.
Torres E, Vaquero C, Nicholson L et al. (1999) Rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies. Transgenic Res 8, 441-449.
Tremblay R, Wang D, Jevnikar AM, Ma S (2010) Tobacco, a highly efficient green bioreactor for production of therapeutic proteins. Biotechnol Adv 28, 214-221.
Tsai JS, Su PL, Yang SC et al. (2021) EGFR-TKI plus bevacizumab versus EGFR-TKI monotherapy for patients with EGFR mutation-positive advanced non-small cell lung cancer-A propensity score matching analysis. J Formos Med Assoc 120, 1729-1739.
Urwin P, Yi L, Martin H et al. (2000) Functional characterization of the EMCV IRES in plants. Plant J 24, 583-589.
Urwin PE, McPherson MJ, Atkinson HJ (1998) Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs. Planta 204, 472-479.
van der Veen SJ, Hollak CE, van Kuilenburg AB, Langeveld M (2020) Developments in the treatment of Fabry disease. J Inherit Metab Dis 43, 908-921.
Ward BJ, Makarkov A, Séguin A et al. (2020) Efficacy, immunogenicity, and safety of a plant-derived, quadrivalent, virus-like particle influenza vaccine in adults (18–64 years) and older adults (≥ 65 years): Two multicentre, randomised phase 3 trials. Lancet 396, 1491-1503.
Yang M, Sun H, Lai H et al. (2018) Plant‐produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice. Plant Biotechnol J 16, 572-580.
Zhang B, Rapolu M, Kumar S et al. (2017) Coordinated protein co‐expression in plants by harnessing the synergy between an intein and a viral 2A peptide. Plant Biotechnol J 15, 718-728.
Zimran A, Gonzalez-Rodriguez DE, Abrahamov A et al. (2018) Long-term safety and efficacy of taliglucerase alfa in pediatric Gaucher disease patients who were treatment-naive or previously treated with imiglucerase. Blood Cells Mol Dis 68, 163-172.