Expressing and functionally evaluating anticancer bispecific antibody fragments using cyanobacterium Synechocystis sp. PCC6803 for sustainable production

Abstract

Anticancer bispecific antibody (BsAb) fragments are effective therapeutic agents because of their high tissue penetration and cytotoxic effects, which can be produced in cost-effective bacterial hosts. We used the cyanobacterium Synechocystis sp. PCC6803 (PCC6803) as an alternative host for sustainably producing BsAb fragments from CO₂. Cyanobacteria are sustainable and independent of external carbon sources such as glucose, because of producing substances via photosynthesis. Cyanobacteria can be cultured using wastewater or seawater, reducing freshwater consumption and the environmental impact of the process, suggesting that cyanobacteria can be used as a sustainable BsAb production platform. We examined the production of various antibody fragments, namely, single-chain bispecific diabody (scBsDb), single-chain fragment variable (scFv), variable domains of the heavy-chain antibody VHH, and bispecific tandem VHH (BsVHH), using PCC6803. scBsDb showed partial antigen-binding ability and no cytotoxic effects. In contrast, all scFvs and VHH had binding abilities and we prepared BsVHH up to 2.4 mg/L culture. BsVHH showed binding activity with the targets and cytotoxic effects against carcinoma, unlike scBsDb. Our results highlight the suitability of VHH and VHH-based engineered antibody fragments for using PCC6803 and indicate their potential for producing other therapeutic proteins. We report producing functional BsAb fragments using cyanobacteria. Our findings contribute to developing cyanobacterial bioprocesses for producing therapeutic BsAb fragments, expanding the possibilities for sustainably producing valuable materials from CO₂.