Development and plant-based expression of recombinant immunotoxins for the targeted treatment of myelomonocytic leukemia

• Entwicklung und pflanzen-basierte Expression rekombinanter Immuntoxine für die gezielte Behandlung der myelomonozytären Leukämie

Knödler, Matthias; Buyel, Johannes Felix (Thesis advisor); Blank, Lars M. (Thesis advisor)

Aachen : RWTH Aachen University (2023)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2023

Abstract

Immunotoxins are highly potent biopharmaceutical anti-cancer drugs consisting of an antibody and a drug that can improve therapeutic indices compared to monoclonal antibodies (mAbs). However, the synthesis of antibody-drug conjugates (ADCs) is complex due to the individual production and purification of the antibody and toxin components in Chinese hamster ovary (CHO) cells or by chemical synthesis, accordingly. Moreover, a chemical conjugation process and additional purification steps are required, which increases the production and therapy costs compared to conventional mAbs. In contrast, the production of a recombinant fusion protein consisting of an antibody (fragment), a linker, and a protein-based toxin can be achieved in only one expression host. However, the production of such recombinant immunotoxins (RITs) in bacteria can be problematic due to the limited repertoire of bacterial post-translational modifications (PTMs). In contrast, plants are capable of all relevant PTMs required for efficient RIT folding and have been successfully used to produce various biopharmaceutical proteins such as mAbs, enzymes and active toxins (e.g., the recombinant mistletoe lectin viscumin).In this work, plants of the genus N. benthamiana and N. tabacum as well as tobacco BY-2 suspension cells which allow the preparation of plant cell packs (PCPs) were used to produce active RITs that selectively bind CD64 antigens for the therapy of myelomonocytic leukemia. Various immunotoxin candidates with different scFv, protein linkers, and protein-based toxins were expressed under control of three different untranslated regions (UTRs) and directed into different subcellular plant organelles. The in planta accumulation of selected RIT candidates was optimized to ~40 mg kg-1 biomass in PCPs, and isolated with an overall yield of 84% and purity of > 80% after optimization of a protein L affinity chromatography. For one drug candidate based on the mistletoe toxin viscumin, the stability as well as in vitro activity towards human acute myelomonocytic leukemia (AML M4/5) cell lines was verified. With an intermediate inhibitory concentration (IC50) in the lower μM range, the drug candidate showed satisfactory activity against CD64+ leukemia cells for a proof of concept. A preliminary cost calculation indicated that the plant-based RIT expression process could become economic feasible with further improvements and when compared to current ADC sale prices. Based on this study, we identified potential bottlenecks of the plant-based expression platform that require further improvement. In addition, this thesis provided the basis for evaluating critical process parameters to be considered during a Quality by Desgin (QbD)-based process development for the plant-based RIT manufacturing.

Institutions

• Department of Biology [160000]
• Chair of Molecular Biotechnology [162910]