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Using Glycosyltransferases for Conjugation of Single-Chain Antibodies and Lipids

使用糖基转移酶缀合单链抗体和脂质

基本信息

批准号：
7965701
负责人：
Pradman K Qasba
金额：
$ 25.38万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7965701
关键词：
Affinity Alkynes Antibodies Antigens Binding Binding Sites Biological Assay Breast Cancer Cell C-terminal CD22 gene Cancer cell line Chemicals Chimeric Proteins Complex Contrast Media Development Drug Delivery Systems Drug Formulations ERBB2 gene Engineering Enzyme-Linked Immunosorbent Assay Escherichia coli Fluorescence Galactose Glycoproteins Glycosylphosphatidylinositols Goals Human Image Immunoglobulin G Immunoliposome In Vitro Inclusion Bodies Label Length Link Lipids Liposomes Magnetic Resonance Imaging Methodology Methods Modification Mucins Peptide Hydrolases Peptides Polysaccharides Protein Binding Proteins Recombinants Side Site Techniques Threonine Uridine Diphosphate Sugars Work antibody conjugate antigen binding cancer diagnosis glycosyltransferase hydroxyl group insight phosphoethanolamine polypeptide protein aminoacid sequence receptor sugar

项目摘要

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 fusion polypeptide containing 1, 3, or 17 threonine (Thr) residues. These scFv antibodies were expressed in E. coli 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, the C-terminal extended fusion polypeptides of these recombinant scFv fusion proteins are used as the acceptor substrate for human polypeptide-R-&#925;-acetylgalactosaminyltransferase II (h-ppGalNAc- T2) that transfers either GalNAc or 2-keto-Gal, a modified galactose with a chemical handle, from their respective UDP-sugars to the side-chain hydroxyl group of the Thr residue(s). Upon protease cleavage, the MALDI-TOF spectra of the glycosylated C-terminal fusion polypeptides showed that the glycosylated scFv fusion protein with a single Thr residue is fully glycosylated with a single 2-keto-Gal, whereas the glycosylated scFv fusion protein with 3 and 17 Thr residues is found as an equal mixture of 2-3 and 5-8 2-keto-Gal glycosylated fusion proteins, respectively. These fusion scFv proteins with the modified galactose are then conjugated with a fluorescence probe, Alexa488, that carries an orthogonal reactive group. The fluorescence labeled scFv proteins bind specifically to a human breast cancer cell line (SK-BR-3) that over expresses the HER2 receptor, indicating that the in Vitro folded scFv fusion proteins are biologically active and the presence of conjugated multiple Alexa488 probes in their C-terminal end does not interfere with their binding to the antigen.A large mucin protein like, muc6, has been expressed as a soluble protein in E. coli and has been in Vitro glycosylated with more than 50 sugars with GalNAc, using ppGalNAc-T1. Therefore, using our present site-specific and multiple site conjugating method, scFv proteins with a C-terminal muc6 fusion protein can be glycosylated with modified sugars and conjugated with bioactive molecules. Such complexes are expected to carry not just a few but several tens of bioactive molecules conjugated to scFv molecules. The methodology described here can generate site-specific and multiple site conjugated antibody-bioactive molecules that are in great need for the development of targeted MRI image contrast agents and 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. Blumenthals and Dr. Dimitrovs groups), for the formulation of liposomes for the targeted drug delivery.