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Using Glycosyltransferases for the Development of Targeted Drug Delivery System

使用糖基转移酶开发靶向药物递送系统

基本信息

批准号：
7965699
负责人：
Pradman K Qasba
金额：
$ 20.3万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7965699
关键词：
A549 Affect Affinity Antibodies Apoptosis Apoptotic Asparagine Binding Proteins Binding Sites Biological Biological Assay Biological Process C-terminal Cancer cell line Carbohydrates Cell Death Cell Line Cell Nucleus Cell membrane Cell physiology Cell surface Cells Chelating Agents Chemicals Complex Contrast Media Coupled Coupling Cytoplasm Development Dose Drug Delivery Systems Engineering Escherichia coli Excision Family Fc Immunoglobulins Future Gadolinium Galactose Galactose Binding Lectin Galactosyltransferases Galectin 1 Galectin 3 Glycoconjugates Glycoproteins Goals Human Image Immunoglobulin G In Vitro Label Laboratories Lectin Life Ligands Link Location Lysine MCF7 cell Mammary Neoplasms Measures Membrane Proteins Methods Microscopy Monoclonal Antibodies Nucleotides Oleic Acids Oligosaccharides Peptides Pharmaceutical Preparations Polysaccharides Preparation Proteins Recombinants SKBR3 Site Staining method Stains Structure System Therapeutic Therapeutic Monoclonal Antibodies Tissues Toxic effect Trypan Blue alpha Lactalbumin annexin A5 antigen binding apo-alpha-lactalbumin beta-galactoside cancer cell cell preparation crosslink functional group glycosylation glycosyltransferase killings mutant neoplastic cell polypeptide protein aminoacid sequence protein folding receptor sugar targeted delivery tool

项目摘要

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 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.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.

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.&lt；p&gt；&lt；P&gt；&lt；i&gt；&lt；b&gt；HAMLET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。生物杀手分子哈姆雷特是载脂蛋白α-乳清蛋白和油酸的复合体，凯瑟琳娜·斯万伯格博士已经证明，它可以触发肿瘤细胞死亡，而健康细胞则幸免于难。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08-09财年，我们在大肠杆菌中表达了带有和不带有C端融合多肽的重组人α-乳清蛋白(HLA)，并将其体外折叠，通过去除钙离子将其转化为熔融的球状蛋白，并与油酸络合形成Hamlet。这些哈姆雷特制剂对包括SKBR3、MDA-MB68、MCF-7和A549在内的几种人类癌细胞具有杀瘤活性。台盼蓝比色法和Annexin-V法表明，含和不含C端融合多肽的人类白细胞抗原油酸复合体具有相似的杀瘤活性。在人类白细胞抗原融合多肽与2-酮-半乳糖在一个独特的位置糖基化后，使用项目Z01BC 010742中描述的ppGalNAc-T2，该蛋白与Alexa Fluor488偶联。在共聚焦显微镜下，标记的HAMLET，即标记的熔球与油酸的络合物，而不是标记的熔球，仅在细胞膜、胞浆中染色，后者在细胞核中聚集。在未来，我们计划将Hamlet与抗Her2单链抗体通过糖链部分偶联，研究其对Her2阳性乳腺肿瘤的靶向性，并评估其Galectin-1和Galectin-3的bioefficacy.&lt；/p&gt；&lt；p&gt；&lt；P&gt；&lt；i&gt；&lt；b&gt；Preparation，有或没有C末端标记与ppGalNAc-T2糖基化以连接治疗性单抗：&lt；/i&gt；&lt；/b&gt；Galectins是一类糖链结合蛋白，具有与β-半乳糖苷有亲和力的保守碳水化合物识别结构域(CRD)。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。