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：
7592951
负责人：
Pradman K Qasba
金额：
$ 8万
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/7592951
关键词：
Acute Promyelocytic Leukemia Affect Affinity Annexins Apoptosis Apoptotic Asparagine Binding Proteins Binding Sites Biological Biological Process Breast Cancer Cell C-terminal Cancer cell line Carbohydrates Cell Death Cell Line Cell physiology Cell surface Cells Chelating Agents Chemicals Complex Contrast Media Coupled Coupling Development Dose Drug Delivery Systems Engineering Escherichia coli Family Fc Immunoglobulins Flow Cytometry Gadolinium Galactose Binding Lectin Galactosyltransferases Galectin 1 Galectin 3 Glycoconjugates Glycoproteins Goals HL60 Human Image Immunoglobulin G In Vitro Laboratories Lectin Ligands Link Location Lysine MCF7 cell Mammary Neoplasms Membrane Proteins Methods Monoclonal Antibodies Nucleotides Numbers Oleic Acid Oleic Acids Oligosaccharides Peptides Pharmaceutical Preparations Polysaccharides Preparation Proteins Roche brand of trastuzumab Site Structure System Testing Therapeutic Therapeutic Monoclonal Antibodies Tissues Toxic effect alpha Lactalbumin antigen binding apo-alpha-lactalbumin beta-galactoside cancer cell crosslink functional group glycosylation glycosyltransferase killings mutant neoplastic cell 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. 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): 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. 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.

在当前流行的方法中，通常在随机位点使用双官能交联的交联，通常将两种蛋白质交联至蛋白质残基，例如赖氨酸，赖氨酸在蛋白质表面的多个位置分布。这种交联方法通常会阻止蛋白质上的功能部位，从而降低蛋白质的生物效能。相反，在Z01 BC 010742项目中描述的使用糖基转移酶通过聚糖残基连接两种糖蛋白或糖缀合物的方法的优势具有特定的优势，因为这些链接发生在两个定义的位点的两个伴侣之间，在该位点附加了糖果，在该位点附加了glycocococococococococococococococonjaugates glycoconjaugates。例如，可以将货物连接到驻留在FC碎片中的天冬酰胺连接的聚糖链的单克隆抗体上，它远离抗原结合位点，并在不改变单克隆抗体的生物效能的情况下递送到作用部位。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ham，galectin-1和Galctin-3耦合到治疗性单克隆抗体。形成油酸复合物以使小村庄（人α-乳糖蛋白杀死肿瘤细胞）： 生物杀伤分子小村庄，一种apoα-乳乳蛋白和油酸的综合体，已被Catharina svanbor博士触发肿瘤细胞死亡，同时触发了健康的细胞。小村庄治疗的肿瘤细胞患凋亡。由于我们是第一个在体外折叠蛋白质的克隆，表达的人，因此确定了与半乳糖基转移酶复合体中α-乳蛋白酶的结构，并在二十年中广泛研究了该分子，我们拥有产生小村庄的专业知识，现在可以将其搭配到单细胞抗体中，以送给单细胞抗体。 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,乳腺癌细胞系MCF-7和临时细胞性白血病细胞系HL-60。在使用膜联蛋白-FTIC检测流式细胞仪的凋亡后，我们将Hamlet与Herceptin单克隆抗体将靶向HER2阳性乳腺肿瘤靶向。 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用于β-半乳糖苷。许多甲状腺蛋白通过凝集素 - 碳水化合物相互作用与细胞表面糖相互作用，从而影响了多种细胞过程。其中，Galectin-1和Galectin-3已进行了广泛的研究并证明会诱导凋亡。 Galectin-1已被证明会诱导许多恶性细胞系中的凋亡，并被提议具有治疗价值。由于Galectin-1和Galectin-3以大肠杆菌的形式以大肠杆菌的形式产生，已显示出具有生物活性的形式，因此我们将设计这些分子以具有可以用PPGALNAC-T2糖基化的C末端肽标签，如PPGALNAC-T2，如Project Z01 BC 010742所述。如项目Z01 BC 010742中所述，通过N连接的聚糖链单克隆抗体进行了评估。