&lt;p&gt;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.&lt;p&gt;&lt;P&gt;&lt;i&gt;&lt;b&gt;HAMLET (Human Alpha-lactalbumin (HLA) Made to Kill Tumor cell) prepared from recombinant in vitro folded HLA, with or without a C-terminal fusion polypeptide show comparable tumoricidal activities:&lt;/i&gt;&lt;/b&gt;The 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 and 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 conjugate it to a monoclonal antibody or a single chain antibody at a specific site for its targeted delivery. In the FY 08-09 we have expressed the recombinant human alpha-Lactalbumin (HLA), with and without C-terminal fusion polypeptide in E. coli and folded the protein in vitro and converted it to molten globule proteins by removal of Ca2+, and complexed with oleic acid to form HAMLET. These HAMLET preparations show tumoricidal activity against several human cancer cell lines including SKBR3, MDA-MB68, MCF-7 and A549. Oleic acid complexes derived from HLA with and without a C-terminal fusion polypeptide had comparable tumoricidal activities as measured by Trypan blue assay and Annexin-V method. Following glycosylation of HLA-fusion polypeptide at a unique site with 2-keto-galactose, using ppGalNAc-T2 as described in the project Z01 BC 010742, the protein was conjugated with Alexa Fluor 488. Using confocal live microscopy, the labeled HAMLET, the complex of labeled molten globule HLA with oleic acid, and not the labeled molten globule HLA alone shows staining in the cell membrane, cytoplasm and latter accumulation in the cell nuclei. In future we plan to couple HAMLET with the anti-Her2 single chain antibody via the glycan moiety and study its targeting to Her2 positive breast tumors and evaluate its bioefficacy.&lt;/p&gt;&lt;p&gt;&lt;P&gt;&lt;i&gt;&lt;b&gt;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: &lt;/i&gt;&lt;/b&gt;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.&lt;/p&gt;
NIH ExPorter
NIH ExPorter