In planta transient recombinant production of a bispecific T-cell engager: Blinatumomab

Document Type : Research Paper

Authors

1 Institute of Biotechnology, Shiraz University, Shiraz, I. R. Iran

2 Institute of Plant Biotechnology and Cell Biology, Department of Biotechnology and Food Sciences, BOKU University, Vienna, Austria

3 Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, I. R. Iran & Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, I. R. Iran

4 Research Center of Plant Virology, School of Agriculture, Shiraz University, Shiraz, I. R Iran

Abstract

Bispecific T-cell engagers (BiTEs) are increasingly being recognized as promising therapeutic molecules in tumor management. However, these largely artificial and complex antibody-based molecules are often difficult to express in conventional platforms. Here, we evaluated the recombinant expression of the approved dual-targeting bispecific T-cell engager Blinatumomab (BLIN) for its production in plants. A highly potent viral vector carrying the coding sequence of BLIN with a His tag was used for transiently expressing Blinatumomab in Nicotiana benthamiana. Four days post-DNA construct delivery, immunoblotting exhibited BLIN-specific signals in total soluble proteins (TSP) as well as in the intercellular fluid (IF), indicating secretion of the recombinant protein to the extracellular space. Co-expression of an RNA silencing suppressor (p19) increased BLIN-specific signals. While Ni-NTA purification from TSP resulted in insufficient BLIN quality, IF-derived BLIN was eluted as a single band at the correct molecular weight. The yield of IF-BLIN was approximately 15 µg/g fresh leaf weight, which is ten times lower than usually obtained for monoclonal antibodies (mAbs). Chromatographic profiling of IF-BLIN exhibited significant amounts of unwanted high molecular weight forms next to the targeted monomeric fraction. After purification of this fraction, the functional activity of IF-BLIN was confirmed by binding to cells expressing the targeted antigens CD3 or CD19. Collectively, we showed the successful production of functionally active BLIN in plants. Further optimization steps are required to improve the yield and quality of plant-produced BLIN. 

Graphical Abstract

In planta transient recombinant production of a bispecific T-cell engager: Blinatumomab

Keywords

References

Amirzadeh, N., Moghadam, A., Niazi, A., & Afsharifar, A. (2023). Recombinant anti-HIV MAP30, a ribosome inactivating protein: Against plant virus and bacteriophage. Scientific Reports, 13(1), 2091. https://doi.org/10.1038/s41598-023-29365-7.
Asghar, N., Melik, W., Paulsen, K. M., Pedersen, B. N., Bø-Granquist, E. G., Vikse, R., Stuen, S., Andersson, S., Strid, Å., Andreassen, Å. K., & Johansson, M. (2022). Transient expression of flavivirus structural proteins in Nicotiana benthamiana. Vaccines, 10(10), 1667. https://doi.org/10.3390/vaccines10101667.
Balieu, J., Jung, J. W., Chan, P., Lomonossoff, G. P., Lerouge, P., & Bardor, M. (2022). Investigation of the N-glycosylation of the SARS-CoV-2 S protein contained in VLPs produced in Nicotiana benthamiana. Molecules, 27(16). https://doi.org/10.3390/molecules27165119
Benchabane, M., Goulet, C., Rivard, D., Faye, L., Gomord, V., & Michaud, D. (2008). Preventing unintended proteolysis in plant protein biofactories. Plant Biotechnology Journal, 6(6), 633–648. https://doi.org/10.1111/j.1467-7652.2008.00344.x
Bornhorst, J. A., & Falke, J. J. (2000). Purification of proteins using polyhistidine affinity tags. Methods in Enzymology, 326, 245-254.
Burt, R., Warcel, D., & Fielding, A. K. (2019). Blinatumomab, a bispecific B-cell and T-cell engaging antibody, in the treatment of B-cell malignancies. Human Vaccines & Immunotherapeutics, 15(3), 594-602. https://doi.org/10.1080/21645515.2018.1540828
Castilho, A., Neumann, L., Gattinger, P., Strasser, R., Altmann, F., & Steinkellner, H. (2014). Proteolytic and N-glycan processing of human alpha1-antitrypsin expressed in Nicotiana benthamiana. Plant Physiology, 166(4), 1839–1851. https://doi.org/10.1104/pp.114.250720
Castilho, A., Neumann, L., Gattinger, P., Strasser, R., Vorauer-Uhl, K., Sterovsky, T., Altmann, F., & Steinkellner, H. (2013). Generation of biologically active multi-sialylated recombinant human EPOFc in plants. PLoS ONE, 8(1). https://doi.org/10.1371/journal.pone.0054836
Castilho, A., Strasser, R., Stadlmann, J., Grass, J., Jez, J., Gattinger, P., Kunert, R., Quendler, H., Pabst, M., Leonard, R., Altmann, F., & Steinkellner, H. (2010). In planta protein sialylation through overexpression of the respective mammalian pathway. Journal of Biological Chemistry, 285(21), 15923.15930. https://doi.org/10.1074/jbc.M109.088401
Chen, X., Zaro, J. L., & Shen, W. C. (2013). Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, 65(10), 1357-1369. https://doi.org/10.1016/j.addr.2012.09.039
Chen, Q. (2022). Development of plant-made monoclonal antibodies against viral infections. Current Opinion in Virology, 52, 148-160. https://doi.org/10.1016/j.coviro.2021.12.005
Coates, R. J., Young, M. T., & Scofield, S. (2022). Optimising expression and extraction of recombinant proteins in plants. Frontiers in Plant Science, 13, 1074531. https://doi.org/10.3389/fpls.2022.1074531
Dhillon, S. (2023). Elranatamab: First approval. Drugs, 83(17), 1621-1627. https://doi.org/10.1007/s40265-023-01954-w
Dreier, T., Lorenczewski, G., Brandl, C., Hoffmann, P., Syring, U., Hanakam, F., Kufer, P., Riethmuller, G., Bargou, R., & Baeuerle, P. A. (2002). Extremely potent, rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody. International Journal of Cancer, 100(6), 690-697. https://doi.org/10.1002/ijc.10557
Eidenberger, L., Eminger, F., Castilho, A., & Steinkellner, H. (2022). Comparative analysis of plant transient expression vectors for targeted N-glycosylation. Frontiers in Bioengineering and Biotechnology, 10, 1073455. https://doi.org/10.3389/fbioe.2022.1073455
Eidenberger, L., Kogelmann, B., & Steinkellner, H. (2023). Plant-based biopharmaceutical engineering. Nature Reviews Bioengineering, 1(6), 426-439. https://doi.org/10.1038/s44222-023-00044-6
Galeffi, P., Lombardi, A., Donato, M. D., Latini, A., Sperandei, M., Cantale, C., & Giacomini, P. (2005). Expression of single-chain antibodies in transgenic plants. Vaccine, 23(15), 1823-1827. https://doi.org/10.1016/j.vaccine.2004.11.025
Goldstein, R. L., Goyos, A., Li, C. M., Deegen, P., Bogner, P., Sternjak, A., Thomas, O., Klinger, M., Wahl, J., Friedrich, M., & Rader, C. (2020). AMG 701 induces cytotoxicity of multiple myeloma cells and depletes plasma cells in cynomolgus monkeys. Blood Advances, 4(17), 4180-4194. https://doi.org/10.1182/bloodadvances.2020002565
Gomes, M., Alvarez, M. A., Quellis, L. R., Becher, M. L., Castro, J. M. de A., Gameiro, J., Caporrino, M. C., Moura-da-Silva, A. M., & Santos, M. de O. (2019). Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper. Toxicon, 160, 38–46. https://doi.org/10.1016/j.toxicon.2019.02.011
Hutchings, M., Morschhauser, F., Iacoboni, G., Carlo-Stella, C., Offner, F. C., Sureda, A., Salles, G., Martínez-Lopez, J., Crump, M., Thomas, D. N., Morcos, P. N., Ferlini, C., Bröske, A.-M. E., Belousov, A., Bacac, M., Dimier, N., Carlile, D. J., Lundberg, L., Perez-Callejo, D., Umaña, P., Moore, T., Weisser, M., & Dickinson, M. J. (2021). Glofitamab, a novel, bivalent CD20-targeting T-cell-engaging bispecific antibody, induces durable complete remissions in relapsed or refractory B-cell lymphoma: A phase I trial. Journal of Clinical Oncology, 39(18), 1959–1970. https://doi.org/10.1200/JCO.20.03175
Kallolimath, S., Hackl, T., Gahn, R., Grunwald-Gruber, C., Zich, W., Kogelmann, B., Lux, A., Nimmerjahn, F., & Steinkellner, H. (2020). Expression profiling and glycan engineering of IgG subclass 1-4 in Nicotiana benthamiana. Frontiers in Bioengineering and Biotechnology, 8, 825.
Kallolimath, S., Sun, L., Palt, R., Föderl-Höbenreich, E., Hermle, A., Voss, L., Kleim, M., Nimmerjahn, F., Gach, J. S., Hitchcock, L., Chen, Q., Melnik, S., Eminger, F., Lux, A., & Steinkellner, H. (2024). IgG1 versus IgG3: Influence of antibody-specificity and allotypic variance on virus neutralization efficacy. Frontiers in Immunology, 15, 1490515. https://doi.org/10.3389/fimmu.2024.1490515
Keam, S. J. (2023). Talquetamab: First approval. Drugs, 83(15), 1439-1445. https://doi.org/10.1007/s40265-023-01945-x
Keshvari, T., Melnik, S., Sun, L., Niazi, A., Aram, F., Moghadam, A., Kogelmann, B., Wozniak-Knopp, G., Kallolimath, S., Ramezani, A., & Steinkellner, H. (2024). Efficient expression of functionally active aflibercept with designed N-glycans. Antibodies, 13(2), 29. https://doi.org/10.3390/antib13020029
Lai, H., He, J., Hurtado, J., Stahnke, J., Fuchs, A., Mehlhop, E., Gorlatov, S., Loos, A., Diamond, M. S., & Chen, Q. (2014). Structural and functional characterization of an anti-West Nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants. Plant Biotechnology Journal, 12(8), 1098–1107. https://doi.org/10.1111/pbi.12217
Liu, H., & Timko, M. P. (2022). Improving protein quantity and quality—The next level of plant molecular farming. International Journal of Molecular Sciences, 23(3), 1326. https://doi.org/10.3390/ijms23031326.
Loffler, A., Kufer, P., Lutterbuse, R., Zettl, F., Daniel, P. T., Schwenkenbecher, J. M., Riethmuller, G., Dorken, B., & Bargou, R. C. (2000). A recombinant bispecific single-chain antibody, CD19 x CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes. Blood, 95 (6), 2098-2103.
Maghsoodi, N., Zareinejad, M., Golestan, A., Mahmoudi Maymand, E., & Ramezani, A. (2023). Anti-CD19/CD8 bispecific T cell engager for the potential treatment of B cell malignancies. Cellular Immunology, 393–394, Article 104787. https://doi.org/10.1016/j.cellimm.2023.104787
Moghadam, A., Niazi, A., Afsharifar, A., & Taghavi, S. M. (2016). Expression of a recombinant anti-HIV and anti-tumor protein, MAP30, in nicotiana tobacum hairy roots: A pH-stable and thermophilic antimicrobial protein. PloS one, 11(7), e0159653. https://doi.org/10.1371/journal.pone.0159653
Montero-Morales, L., & Steinkellner, H. (2018). Advanced plant-based glycan engineering. Frontiers in Bioengineering and Biotechnology, 6, 81. https://doi.org/10.3389/fbioe.2018.00081
Moreau, P., Garfall, A. L., van de Donk, N. W. C. J., Nahi, H., San-Miguel, J. F., Oriol, A., Nooka, A. K., Martin, T., Rosinol, L., Chari, A., Karlin, L., Benboubker, L., Mateos, M.-V., Bahlis, N., Popat, R., Besemer, B., Martínez-López, J., Sidana, S., Delforge, M., &Usmani, S. Z. (2022). Teclistamab in relapsed or refractory multiple myeloma. New England Journal of Medicine, 387(6), 495–505. https://doi.org/10.1056/NEJMoa2203478
Naddafi, F., Shirazi, F. H., Talebkhan, Y., Tabarzad, M., Barkhordari, F., Aliabadi Farahani, Z., Bayat, E., Moazzami, R., Mahboudi, F., & Davami, F. (2018). A comparative study of the bispecific monoclonal antibody, blinatumomab expression in CHO cells and E. coli. Preparative Biochemistry & Biotechnology, 48(10), 961–967. https://doi.org/10.1080/10826068.2018.1525562
Nicholson, I. C., Lenton, K. A., Little, D. J., Decorso, T., Lee, F. T., Scott, A. M., Zola, H., & Hohmann, A. W. (1997). Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma. Molecular Immunology, 34 (16–17), 1157-1165. https://doi.org/10.1016/s0161-5890(97)00144-2
Phoolcharoen, W., Prehaud, C., van Dolleweerd, C. J., Both, L., da Costa, A., Lafon, M., & Ma, J. K. (2017). Enhanced transport of plant-produced rabies single-chain antibody-RVG peptide fusion protein across an in cellulo blood-brain barrier device. Plant Biotechnology Journal, 15(10), 1331-1339. https://doi.org/10.1111/pbi.12719
Pirkalkhoran, S., Grabowska, W. R., Kashkoli, H. H., Mirhassani, R., Guiliano, D., Dolphin, C., & Khalili, H. (2023). Bioengineering of antibody fragments: Challenges and opportunities. Bioengineering, 10(2), https://doi.org/10.3390/bioengineering10020122
Ravanrouy, F., Niazi, A., Moghadam, A., & Taghavi, S. M. (2021). MAP30 transgenic tobacco lines: from silencing to inducing. Molecular Biology Reports, 48(10), 6719-6728. https://doi.org/10.1007/s11033-021-06662-w
Safonov, A. M., Altunina, A. V., Kolpashnikov, I. S., Solovyeva, D. O., & Oleynikov, V. A. (2025). Application of 3D imaging in biomedical research. Russian Journal of Bioorganic Chemistry, 51(4), 1453-1470. https://doi.org/10.1134/S1068162024607043
Sanford, M. (2015). Blinatumomab: First global approval. Drugs, 75(3), 321-327. https://doi.org/10.1007/s40265-015-0356-3
Seber Kasinger, L. E., Dent, M. W., Mahajan, G., Hamorsky, K. T., & Matoba, N. (2019). A novel anti-HIV-1 bispecific bNAb-lectin fusion protein engineered in a plant-based transient expression system. Plant Biotechnology Journal, 17(8), 1646-1656. https://doi.org/10.1111/pbi.13090
Seigner, J., Zajc, C. U., Dötsch, S., Eigner, C., Laurent, E., Busch, D. H., Lehner, M., Traxlmayr, M. W. (2023). Solving the mystery of the FMC63-CD19 affinity. Scientific Reports, 13(1), 23024. https://doi.org/10.1038/s41598-023-48528-0
Stanciu-Herrera, C., Morgan, C., & Herrera, L. (2008). Anti-CD19 and anti-CD22 monoclonal antibodies increase the effectiveness of chemotherapy in Pre-B acute lymphoblastic leukemia cell lines. Leukemia Research, 32(4), 625-632.
Hutchings, M., Morschhauser, F., Iacoboni, G., Carlo-Stella, C., Offner, F. C., Sureda, A., Salles, G., Martínez-Lopez, J., Crump, M., Thomas, D. N., Morcos, P. N., Ferlini, C., Bröske, A.-M. E., Belousov, A., Bacac, M., Dimier, N., Carlile, D. J., Lundberg, L., Perez-Callejo, D., Umaña, P., Moore, T., Weisser, M., & Dickinson, M. J. (2021). Glofitamab, a novel, bivalent CD20-targeting T-cell-engaging bispecific antibody, induces durable complete remissions in relapsed or refractory B-cell lymphoma: A Phase I trial. Journal of Clinical Oncology, 39(18), 1959–1970. https://doi.org/10.1200/JCO.20.03175
Thoreau, F., & Chudasama, V. (2022). Enabling the next steps in cancer immunotherapy: from antibody-based bispecifics to multispecifics, with an evolving role for bioconjugation chemistry. RSC Chemical Biology, 3(2), 140-169.
Wang, Q., Chen, Y., Park, J., Liu, X., Hu, Y., Wang, T., McFarland, K., & Betenbaugh, M. J. (2019). Design and production of bispecific antibodies. Antibodies (Basel, Switzerland), 8(3), 43.
Wu, J., Fu, J., Zhang, M., & Liu, D. (2015). Blinatumomab: A bispecific T cell engager (BiTE) antibody against CD19/CD3 for refractory acute lymphoid leukemia. Journal of Hematology & Oncology, 8, 104. https://doi.org/10.1186/s13045-015-0195-4
Zaninelli, S., Panna, S., Tettamanti, S., Melita, G., Doni, A., D’Autilia, F., Valgardsdottir, R., Gotti, E., Rambaldi, A., Golay, J., & Introna, M. (2024). Functional activity of cytokine-induced killer cells enhanced by CAR-CD19 modification or by soluble bispecific antibody blinatumomab. Antibodies (Basel), 13(3), 71. https://doi.org/10.3390/antib13030071.
Zhang, J., Carter, J., Siu, S., W O''Neill, J., H Gates, A., Delaney, J., & Mehlin, C. (2010). Fusion partners as a tool for the expression of difficult proteins in mammalian cells. Current pharmaceutical biotechnology, 11(3), 241-245. https://doi.org/10.2174/138920110791111898
Iran Agricultural Research
Volume 44, Issue 2
In Progress
2025
Pages 88-96

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