Expression and characterization of bispecific single-chain Fv fragments produced in transgenic plants

We describe the expression of the bispecific antibody biscFv2429 in transge nic suspension culture cells and tobacco plants. biscFv2429 consists of two single-chain antibodies, scFv24 and scFv29, connected by the Trichoderma r eesi cellobiohydrolase I linker. biscFv2429 binds two epitopes of tobacco m osaic virus (TMV): the scFv24 domain recognizes neotopes of intact virions, and the scFv29 domain recognizes a cryptotope of the TMV coat protein mono mer. biscFv2429 was functionally expressed either in the cytosol (biscFv242 9-cyt) or targeted to the apoplast using a murine leader peptide sequence ( biscFv2429-apoplast). A third construct contained the C-terminal KDEL seque nce for retention in the ER (biscFv2429-KDEL). Levels of cytoplasmic biscFv 2429 expression levels were low. The highest levels of antibody expression were for apoplast-targeted biscFv2429-apoplast and ER-retained biscFv2429-K DEL that reached a maximum expression level of 1.65% total soluble protein in transgenic plants. Plant-expressed biscFv2429 retained both epitope spec ificities, and bispecificity and bivalency were confirmed by ELISA and surf ace plasmon resonance analysis. This study establishes plant cells as an ex pression system for bispecific single-chain antibodies fur use in medical a nd biological applications.