Design, optimization, production and activity testing of recombinant immunotoxins expressed in plants and plant cells for the treatment of monocytic leukemia

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 similar to 40 mg kg(-1) wet biomass using a design of experiments approach, and corresponding proteins were isolated with a purity of similar to 80% using an optimized affinity chromatography method with an overall yield of similar to 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. Highlights Toxin type and domain sequence affect accumulation of recombinant immunotoxins. Transient expression in plant cell packs and intact plants correlates well. IC50 values of toxicity correlate with the cell surface receptor concentration

Automated summary

Antibody-drug conjugates (ADCs) have higher therapeutic indices compared to plain monoclonal antibodies (mAbs), but are more complex and costly to synthesize. Recombinant immunotoxins (RITs) consisting of antibody derivatives, linkers, and proteinaceous toxins can be produced more easily in plants. In this study, effective RITs targeting CD64 for the treatment of myelomonocytic leukemia were produced in Nicotiana benthamiana and N. tabacum plants and tobacco BY-2 cell-based plant cell packs (PCPs). The accumulation of RITs was optimized to a high yield using a design of experiments approach and an optimized affinity chromatography method. One RIT candidate targeting CD64 was characterized for storage stability and cytotoxicity. Bottlenecks in the plant-based expression platform were identified and critical process parameters for plant-made RITs were assessed.