Using Glycosyltransferases for the Development of Targeted Drug Delivery System

在目前流行的方法中，通常使用双功能交联剂将两个蛋白质在随机位点交联到一个蛋白质残基上，例如赖氨酸，赖氨酸分布在蛋白质表面的几个位置。这种交联方法通常会阻断蛋白质上的功能位点，从而降低蛋白质的生物功效。相比之下，项目Z01 BC 010742中描述的利用糖基转移酶通过聚糖残基连接两个糖蛋白或糖缀合物的方法具有特殊的优势，因为连接发生在两个伙伴之间的一个确定位点上，其中聚糖部分附着在糖缀合物上。例如，货物可以在远离抗原结合位点的fc片段中的天冬酰胺连接的聚糖链上附着到单克隆抗体上，并在不改变单克隆抗体的生物功效的情况下运送到作用位点。在这个项目中，我们使用项目Z01 BC 010742中描述的方法将凋亡分子HAMLET、半乳糖凝集素-1和半乳糖凝集素-3偶联到治疗性单克隆抗体上。<p>< p>< < >< >< >& gt;& gt;& gt;& gt;& gt；哈姆雷特（Human α -乳清蛋白（HLA） Made to Kill Tumor cell）由重组体外折叠HLA制备而成。有或没有c端融合多肽都显示出相似的肿瘤杀伤活性：&lt； >< & >& gt； / /生物杀伤分子哈姆雷特是载脂蛋白α -乳清蛋白和油酸的复合体，Catharina Svanborg博士已经证明它可以触发肿瘤细胞死亡，而健康细胞却能幸免。哈姆雷特治疗的肿瘤细胞发生凋亡。由于我们是第一个克隆、表达和体外折叠蛋白质的公司，确定了α -乳清蛋白与半乳糖转移酶复合物的结构，并在20多年的时间里对该分子进行了广泛的研究，我们拥有生产HAMLET的专业知识，并在特定位点将其与单克隆抗体或单链抗体结合，以实现其靶向递送。在FY 08-09中，我们在大肠杆菌中表达了含和不含c端融合多肽的重组人α -乳清蛋白（HLA），并在体外折叠该蛋白，通过去除Ca2+将其转化为熔融球状蛋白，与油酸络合形成HAMLET。这些HAMLET制剂对包括SKBR3、MDA-MB68、MCF-7和A549在内的多种人类癌细胞具有杀瘤活性。通过台泛蓝法和Annexin-V法测定，来源于HLA的油酸复合物具有和不具有c端融合多肽的具有相当的杀肿瘤活性。在项目Z01 BC 010742中使用ppGalNAc-T2将hla融合多肽与2-酮-半乳糖在一个独特的位点进行糖基化后，将该蛋白与Alexa Fluor 488偶联。在共聚焦活显微镜下，标记的哈姆雷特、标记的熔融HLA球与油酸的复合物，而不是标记的熔融HLA球单独在细胞膜、细胞质中染色，并在细胞核中积聚。将来我们计划与anti-Her2夫妇哈姆雷特单链抗体通过多糖一半和研究针对Her2阳性乳腺癌肿瘤并评估其bioefficacy灵活;/ p> & lt; p> & lt; p> & lt; i> & lt; b>制备Galectin-1 Galectin-3,有或没有一个c端标记为糖基化与ppGalNAc-T2链接到治疗性单克隆抗体:</i></b&gt；半乳糖凝集素包括一个聚糖结合蛋白家族，具有保守的碳水化合物识别结构域（CRD），与β -半乳糖苷具有亲和力。许多凝集素通过凝集素-碳水化合物相互作用与细胞表面聚糖相互作用，从而影响各种细胞过程。其中，Galectin-1和Galectin-3已被广泛研究并证实可诱导细胞凋亡。半乳糖凝集素-1已被证明在许多恶性细胞系中诱导细胞凋亡，并被提出具有治疗价值。由于半乳糖凝集素-1和半乳糖凝集素-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，我们将设计这些分子具有c端肽标签，可以与ppgalac - t2糖基化，如项目Z01 BC 010742所述。如Z01 BC 010742项目所述，这些分子将通过聚糖残基与治疗性单克隆抗体通过n -链聚糖链连接，并评估其生物功效。

<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>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:</i></b> 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.</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>