Using Glycosyltransferases for the Development of Targeted Drug Delivery System

在目前流行的方法中，通常使用双官能交联剂将两个蛋白质在随机位置与蛋白质残基(例如，分布在蛋白质表面的几个位置的赖氨酸)进行交联。这种交联法通常会阻断蛋白质上的功能部位，从而降低蛋白质的生物效力。相反，在项目Z01 BC 010742中描述的利用糖基转移酶通过糖基残基连接两个糖蛋白或糖共轭化合物的方法的优点具有特定的优点，因为连接发生在两个伙伴之间的限定位置，在该位置，多聚糖部分连接到糖共轭化合物。例如，货物可以附着在天冬酰胺连接的糖链上的单抗上，该单抗位于Fc片段中，远离抗原结合部位，并被输送到作用部位，而不改变单抗的生物效力。在这个项目中，我们使用Z01 BC 010742项目中描述的方法将凋亡分子Hamlet、Galectin-1和Galctin-3偶联到治疗性单抗上。<p><p><i><b>制备带有和不带有C-末端标签的人α-乳白蛋白与ppGalNAc-T2糖基化形成油酸复合体，形成HAMLET(人α-乳白蛋白用于杀死肿瘤细胞)：</I></b>生物杀手分子HAMLET是载脂蛋白α-乳蛋白和油酸的复合体，凯瑟琳娜·斯万伯格博士已经证明，在健康细胞幸免于难的情况下，它可以触发肿瘤细胞死亡。哈姆雷特治疗的肿瘤细胞发生了凋亡。由于我们是第一个克隆、表达、体外折叠蛋白质、确定α-乳清蛋白与半乳糖基转移酶复合体的结构并在20多年内对该分子进行了广泛研究的人，我们拥有生产Hamlet的专业知识，现在将其偶联到单抗上进行靶向递送。我们已经表达并折叠了带有C-末端标签的人α-乳清蛋白，该标签可以与ppGalNAc-T2糖基化，如项目Z01BC 010742中所述。一种折叠形式的载脂蛋白α-乳清蛋白，带有和不带有C端肽标签，与油酸形成复合体，将首先检测Jurkat和L1210细胞系、乳腺癌MCF-7细胞系和早幼粒细胞白血病细胞系HL-60的凋亡情况。在用Annexin-FTIC流式细胞仪检测细胞凋亡后，我们将Hamlet与Herceptin单抗偶联，以靶向Her2阳性乳腺肿瘤。Galectin-1和Galectin-3的制备，具有和不带有用于与ppGalNAc-T2糖基化的C-末端标签，用于连接治疗性单抗：Galectins包括具有与β-半乳糖苷亲和力的保守的碳水化合物识别结构域(CRD)的糖链结合蛋白家族。许多Galectins通过凝集素-碳水化合物相互作用与细胞表面的多聚糖相互作用，从而影响多种细胞过程。其中，Galectin-1和Galectin-3已被广泛研究并显示出诱导细胞凋亡的作用。Galectin-1已被证明可诱导许多恶性细胞系的凋亡，并已被提出具有治疗价值。由于Galectin-1和Galectin-3在大肠杆菌中以可溶和折叠的形式产生，已被证明具有生物活性，我们将改造这些分子，使其具有可与ppGalNAc-T2糖基化的C-末端多肽标签，如Z01 BC 010742项目中所述。然后，这些分子将通过糖链残基通过N-连接的糖链连接到治疗性的单抗上，如Z01 BC 010742项目中所述，并对它们的生物有效性进行评估。

<p>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. <p><P><i><b>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): </i></b> 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. </p><p><P><i><b>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: </i></b> 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.</p>