In the currently prevailing methods, generally two proteins are cross-linked, using a bi-functional cross-linker, at random sites to a protein residue, e.g., lysine, which is distributed at several places on the protein surface. This method of cross-linking often blocks the functional sites on the protein and thus reduces the bioefficacy of the protein. In contrast, the advantage of the method of linking two glycoproteins or glycoconjugates via glycan residues utilizing glycosyltransferases, described in the project Z01 BC 010742, has specific advantage since the linkage occurs between the two partners at a defined site, where the glycan moiety is attached to the glycoconjugates. For example, a cargo can be attached to a monoclonal antibody at the asparagine-linked glycan chain, which resides in the Fc-fragment, away from the antigen binding site, and delivered to the site of action without altering the bioefficacy of the monoclonal antibody. In this project we are coupling the apoptotic molecules HAMLET, Galectin-1 and Galctin-3, to the therapeutic monoclonal antibodies using the method described in the project Z01 BC 010742.
Preparation of human alpha-lactalbumin with and without a C-terminal tag for glycosylation with ppGalNAc-T2 for the formation of an oleic acid complex to make HAMLET (Human Alpha-lactalbumin Made to Kill Tumor cell): The biological killer molecule HAMLET, a complex of apo alpha-lactalbumin and oleic acid, has been shown by Dr. Catharina Svanborg to trigger tumor cell death while healthy cells are spared. HAMLET treated tumor cells undergo apoptosis. Since we have been the first to have cloned, expressed, in vitro folded the protein, determined the structure of alpha-lactalbumin in complex with galactosyltransferase and extensively studied the molecule over two decades, we have the expertise to produce the HAMLET and now to couple it to a monoclonal antibody for its targeted delivery. We have expressed and folded the human alpha-lactalbumin with a C-terminal tag that can be glycosylated with the ppGalNAc-T2 as described in the project Z01 BC 010742. A folded form of apo alpha-lactalbumin, with and without C-terminal peptide tag, in complex with oleic acid will be first tested for apoptosis of Jurkat and L1210 cell lines, breast cancer cell line MCF-7 and promyelocytic leukaemia cell line HL-60. After detecting apoptosis by flow cytometry using Annexin-FTIC we will couple HAMLET with the Herceptin monoclonal antibody for targeting to Her2 positive breast tumors.
Preparation of Galectin-1 and Galectin-3, with and without a C-terminal tag for glycosylation with ppGalNAc-T2 for linking to therapeutic monoclonal antibodies: Galectins comprise a family of glycan binding proteins having a conserved carbohydrate recognition domain (CRD) with affinity for beta-galactosides. A number of galectins interact with cell surface glycans via lectin-carbohydrate interactions thus affecting a variety of cellular processes. Among them, Galectin-1 and Galectin-3 have been extensively studied and shown to induce apoptosis. Galectin-1 has been shown to induce apoptosis in many malignant cell lines and has been proposed to have therapeutic value. Since Galectin-1 and Galectin-3 are produced in E. coli in a soluble and folded form, which have been shown to be bioactive, we will engineer these molecules to have the C-terminal peptide tag that can be glycosylated with the ppGalNAc-T2, as described in the project Z01 BC 010742. These molecules will be then linked via the glycan residue to therapeutic monoclonal antibodies via N-linked glycan chains, as described in the project Z01 BC 010742, and evaluated for their bioefficacy.
