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 ~ 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.

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.

Hypoxic Conditions in Crown Galls Induce Plant Anaerobic Responses That Support Tumor Proliferation.

Agrobacterium tumefaciens infection of wounded plant tissues causes the formation of crown gall tumors. Upon infection, genes encoded on the A. tumefaciens tumor inducing plasmid are integrated in the plant genome to induce the biosynthesis of auxin and cytokinin, leading to uncontrolled cell division. Additional sequences present on the bacterial T-DNA encode for opine biosynthesis genes, which induce the production of opines that act as a unique carbon and nitrogen source for Agrobacterium. Crown galls therefore become a very strong sink for photosynthate. Here we found that the increased metabolic demand in crown galls causes an increase in oxygen consumption rate, which leads to a steep drop in the internal oxygen concentration. Consistent with this, plant hypoxia-responsive genes were found to be significantly upregulated in crown galls compared to uninfected stem tissue. Following this observation, we aimed at understanding whether the low-oxygen response pathway, mediated by group VII ethylene response factor (ERF-VII) transcription factors, plays a role in the development of crown galls. We found that quintuple knock-out mutants of all ERF-VII members, which are incapable of inducing the hypoxic response, show reduced crown gall symptoms. Conversely, mutant genotypes characterized by constitutively high levels of hypoxia-associated transcripts, displayed more severe crown gall symptoms. Based on these results, we concluded that uncontrolled cell proliferation of crown galls established hypoxic conditions, thereby requiring adequate anaerobic responses of the plant tissue to support tumor growth.