Conjugation of folate via gelonin carbohydrate residues retains ribosomal-inactivating properties of the toxin and permits targeting to folate receptor positive cells

Conjugation of folate to proteins permits receptor-mediated endocytosis via the folate receptor (FR) and delivery of the conjugate into the cytoplasm of cells. Since many cancers up-regulate the FR it has enabled the targetin g of toxins to tumor cells resulting in specific cell death. However, curre nt conjugation methods rely on chemistries that can affect certain catalyti c subunits, such as the A-chain of the plant toxin gelonin. As a result man y folate-targeted tons are a compromise between receptor/ligand interaction and toxin activity. We describe the first example of folate conjugated to a protein via carbohydrate residues, using a novel SH-folate intermediate. The folate-gelonin conjugate retains over 99% of toxin activity in a cell-f ree translational assay compared with unmodified gelonin and is able to bin d the FR at the same affinity as free folic acid (10(-10) M). Additionally, the conjugate exhibits prolonged inhibition of protein synthesis in FR pos itive cell lines in vitro. Folate linked to gelonin via amino conjugation e xhibits the same affinity for FR as free folic acid but the toxin is 225-fo ld less active in a cell-free translational assay. The effect of different conjugation methods on toxin activity and the implications for folate targe ting of other glycoproteins are discussed.