Glycoconjugation of single chain antibodies (scFv) with various molecules for cancer diagnosis and treatment: To extend our bioconjugation work towards the immunoliposome formulation, we have taken up single chain antibodies (scFv) against CD22 and HER-2 proteins. The cDNAs of scFv against CD22 and HER-2 proteins were obtained from Dr. Dimitrovs group. These cDNAs were manipulated such that the single chain antibodies expressed in E. coli have a C-terminal 17 amino acid tag peptide. We have now expressed in E. coli single-chain antibodies (scFv) against CD22 and HER-2 proteins with a 17 amino acid C-terminal tag peptide that has one or several O-glycosylation sites in large amounts. Both scFv antibodies, anti HER-2 and anti CD22, were expressed mainly in inclusion bodies and very poorly as a soluble protein, and only micro gram quantities were obtained from the soluble fraction. Therefore an in vitro folding method has been developed that produced nearly 20 mgs of each from a one liter bacterial culture. Competitive ELISA assay indicated that the in vitro folded anti HER-2 scFv is correctly folded active protein. Furthermore, we have now successfully glycosylated the O-GalNAc site present in the C-terminal end of the in vitro folded anti HER-2 scFv protein using ppGalNAc-T2 and UDP-GalNAc or UDP-2-keto-Gal. The fusion peptide at the C-terminal end of the scFv molecule that has been glycosylated with a modified sugar at a unique site can be next conjugated with a bio-molecule having a orthogonal reactive group, such as aminooxy or alkynes. This technique makes it possible to conjugate scFv, which are becoming very popular therapeutic molecules, with fluorophores for ELISA-based assays, radionuclides for imaging and immunotherapy applications, cytotoxic drugs, cytokines, or toxins for antibody-based cancer therapy and lipids for the assembly of immunoliposomes, and the development of a targeted drug-delivery system. Synthesis of lipids carrying aminooxy or alkyne group for linking with a glycoprotein that has a sugar moiety linked with an orthogonal reactive group: The lipid molecule 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) was converted into either DPPE-aminooxy or DPPE-alkyne derivatives for conjugation with scFV that carry sugar moiety with an orthogonal reactive group. The scFV molecules carrying lipid molecules will next used (in a collaborative project with Dr. Blumenthal's and Dr. Dimitrov's groups), for the formulation of liposomes for the targeted drug delivery.
