Antibody-drug conjugates (ADCs) can improve therapeutic indices compared to plain
monoclonal antibodies (mAbs). However, ADC synthesis is complex because the components are produced separately in CHO cells (mAb) and often by chemical synthesis (
drug). They are individually purified, coupled, and then the ADC is purified, increasing production costs compared to regular mAbs. In contrast, it is easier to produce
recombinant fusion proteins consisting of an antibody derivative, linker and proteinaceous
toxin, i.e. a recombinant
immunotoxin (RIT). Plants are capable of the post-translational modifications needed for functional
antibodies and can also express active
protein toxins such as the recombinant mistletoe
lectin viscumin, which is not possible in prokaryotes and mammalian cells respectively. Here, we used Nicotiana benthamiana and N. tabacum plants as well as tobacco BY-2 cell-based plant cell packs (PCPs) to produce effective RITs targeting CD64 as required for the treatment of myelomonocytic
leukemia. We compared RITs with different subcellular targeting signals, linkers, and proteinaceous toxins. The accumulation of selected candidates was improved to ~ 40 mg kg-1 wet biomass using a design of experiments approach, and corresponding
proteins were isolated with a purity of ~ 80% using an optimized affinity chromatography method with an overall yield of ~ 84%. One anti-CD64 targeted
viscumin-based
drug candidate was characterized in terms of storage stability and cytotoxicity test in vitro using human myelomonocytic
leukemia cell lines. We identified bottlenecks in the plant-based expression platform that require further improvement and assessed critical process parameters that should be considered during process development for plant-made RITs.