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(例如赖氨酸)交联，赖氨酸分布在蛋白质表面的几个位置。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。从体外折叠的人类α-乳白蛋白(人类α-乳白蛋白)制备的HAMLET(人类α-乳白蛋白)，无论是否含有C端融合多肽，都显示出类似的杀瘤活性：凯瑟琳娜·斯万伯格博士已经证明，生物杀伤分子HAMLET是由载脂蛋白α-乳白蛋白和油酸组成的复合体，可以触发肿瘤细胞死亡，而健康细胞则可以幸免。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、表达和体外折叠该蛋白、确定α-乳清蛋白与半乳糖基转移酶复合体的结构并对该分子进行了20多年的广泛研究的人，我们拥有生产Hamlet并将其连接到特定位置的单抗或单链抗体以进行靶向传递的专业知识。在2009-11财年，我们在大肠杆菌中表达了不含重组人乳清蛋白(RhLA)或含C端融合多肽(rhLA-ext)的重组人乳清蛋白，并在体外折叠蛋白，通过去除钙离子将其转化为熔融的球状蛋白。熔融的球状蛋白与油酸络合形成哈姆雷特。这些哈姆雷特制剂，而不仅仅是熔融的球蛋白或油酸，对包括SKBR3、MDA-MB68、MCF-7和A549在内的几种人类癌细胞株显示出杀瘤活性。台盼蓝法和Annexin-V法检测表明，rhLA和rhLA-ext的油酸络合物具有相似的杀瘤活性。如Z01 BC 010742项目所述，使用ppGalNAc-T2将重组人乳酸-EXT的融合多肽与2-酮基-半乳糖在一个独特的位置糖基化。该蛋白通过半乳糖的化学手柄C2-酮基团与Alexa Fluor 488偶联。在共聚焦显微镜下，标记的HAMLET，即标记的熔球rhLA-ext与油酸的络合物，而不是仅标记的熔球，染色在细胞膜、细胞质中，后者聚集在细胞核中。因此，我们已经成功地在不影响哈姆雷特生物活性的情况下向哈姆雷特添加了一个化学句柄，使我们能够在细胞表面或细胞内追踪复合体。在抗肿瘤复合体上增加位点特异性手柄可以进一步用于分离配对/靶分子，从而深入了解该复合体的肿瘤特异性。重组人乳白蛋白α结构域(ad-rhLA)与OA的结合具有与Hamlet相似的杀瘤活性：α-乳清蛋白有4个螺旋，位于α结构域中，β-折叠形成一个β结构域。Hla(Ad-Hla)的α结构域在C-末端带有多肽标记，可与ppGalNAc-T2特异性糖基化，转移带有化学手柄的修饰半乳糖(C2-酮-半乳糖)，该半乳糖可进一步用氨氧基-AlexaFluor 488标记。我们现在已经证明，来自位点特异性标记的ad-Hla-ext和油酸的杀瘤复合体也可以杀死许多肿瘤细胞，并且标记的蛋白内化在肿瘤细胞中。孵育3h后，只有与油酸络合的Ad-Hla-ext蛋白内化。这些结果表明，我们已经成功地表达、标记和成像，通过共聚焦显微镜，较小形式的α-乳清蛋白与油酸络合，油酸是一种仍具有杀瘤活性的Hamlet样分子。在抗肿瘤复合体Hamlet中添加位置特异性手柄正被进一步用于分离伴侣/目标分子，从而提供对该复合体的肿瘤特异性的洞察，这项工作正在进行中。

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. 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: 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 09-11 we have expressed the recombinant human alpha-Lactalbumin without (rhLA) or with a C-terminal fusion polypeptide (rhLA-ext) in E. coli and folded the proteins in vitro and converted each to molten globule protein by removal of Ca2+. The molten globule proteins are complexed with oleic acid to form HAMLET. These HAMLET preparations, and not the molten globule proteins or oleic acids alone, show tumoricidal activity against several human cancer cell lines including SKBR3, MDA-MB68, MCF-7 and A549. Oleic acid complexes derived from rhLA and rhLA-ext had comparable tumoricidal activities as measured by Trypan blue assay and Annexin-V method. Following glycosylation of the fusion polypeptide of rhLA-ext 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 via the chemical handle, C2-keto group, of galactose. Using confocal live microscopy, the labeled HAMLET, the complex of labeled molten globule rhLA-ext with oleic acid, and not the labeled molten globule rhLA-ext alone shows staining in the cell membrane, cytoplasm and latter accumulation in the cell nuclei. Thus we have been successful in adding a chemical handle to HAMLET without affecting its biological activity, allowing us to trace the complex at the cell surface or inside the cell. The addition of a site-specific handle to a tumoricidal complex could be further exploited for isolation of partner/target molecules, providing insight into the tumor specificity of the complex. Alpha-domain of rhLA (aD-rhLA) complexed with OA has tumoricidal activity comparable to HAMLET: Alpha-lactalbumin has 4 helices that are contained in the alpha-domain and beta-sheets that form a beta-domain. The alpha-domain of hLA (aD-hLA) with a polypeptide tag at the C-terminal end can be specifically glycosylated with ppGalNAc-T2, transferring a modified galactose with a chemical handle (C2-keto-galactose) that can be further labeled with aminooxy-AlexaFluor 488. We have now shown that the tumoricidal complex derived from the site-specific-labeled aD-hLA-ext and oleic acid also kills many tumor cells and the labeled protein is internalized in tumor cells. Only aD-hLA-ext protein complexed with oleic acid was internalized after 3 h of incubation. These results indicate that we have been successful in expressing, labeling, and imaging, by confocal microscopy, a smaller form of alpha-lactalbumin complexed with oleic acid, a HAMLET-like molecule that still exhibits tumoricidal activity. The addition of a site-specific handle to a tumoricidal complex HAMLET is being further exploited for the isolation of partner/target molecules, providing insight into the tumor specificity of the complex, the work that is in progress.