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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010805-02
Application #
7733241
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2008
Total Cost
$187,282
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code