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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：
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.

在目前流行的方法中，通常使用双功能交联剂将两种蛋白质在随机位点交联到分布在蛋白质表面上多个位置的蛋白质残基，例如赖氨酸。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定的优势，因为连接发生在两个伙伴之间的确定位点，其中聚糖部分附着在糖缀合物上。例如，货物可以在天冬酰胺连接的聚糖链处附着到单克隆抗体上，该聚糖链位于Fc片段中，远离抗原结合位点，并被递送到作用位点，而不改变单克隆抗体的生物功效。在该项目中，我们使用项目 Z01 BC 010742 中描述的方法将凋亡分子 HAMLET、Galectin-1 和 Galctin-3 与治疗性单克隆抗体偶联。形成油酸复合物以制造 HAMLET（用于杀死肿瘤细胞的人类 α-乳白蛋白）：生物杀伤分子 HAMLET 是 apo α-乳白蛋白和油酸的复合物，Catharina Svanborg 博士已证明可以触发肿瘤细胞死亡，而健康细胞则幸免于难。 HAMLET 处理的肿瘤细胞发生凋亡。由于我们是第一个克隆、表达、体外折叠该蛋白质，确定了与半乳糖基转移酶复合物的 α-乳清蛋白的结构，并在二十年来对该分子进行了广泛研究，因此我们拥有生产 HAMLET 的专业知识，现在将其与单克隆抗体偶联以进行靶向递送。我们已经表达并折叠了带有 C 端标签的人 α-乳清蛋白，该标签可以与 ppGalNAc-T2 进行糖基化，如项目 Z01 BC 010742 中所述。apo α-乳清蛋白的折叠形式（带有或不带有 C 端肽标签）与油酸复合，将首先测试 Jurkat 和 L1210 细胞系的细胞凋亡，乳腺癌细胞系MCF-7和早幼粒细胞白血病细胞系HL-60。使用膜联蛋白-FTIC 通过流式细胞术检测细胞凋亡后，我们将 HAMLET 与赫赛汀单克隆抗体结合，用于靶向 Her2 阳性乳腺肿瘤。 半乳糖凝集素-1和半乳糖凝集素-3的制备，带有或不带有C端标签，用于与ppGalNAc-T2进行糖基化以连接治疗性单克隆抗体：半乳糖凝集素包含具有亲和力的保守碳水化合物识别域（CRD）的聚糖结合蛋白家族对于β-半乳糖苷。许多半乳糖凝集素通过凝集素-碳水化合物相互作用与细胞表面聚糖相互作用，从而影响多种细胞过程。其中Galectin-1和Galectin-3已被广泛研究并显示可诱导细胞凋亡。 Galectin-1 已被证明可诱导许多恶性细胞系凋亡，并被认为具有治疗价值。由于 Galectin-1 和 Galectin-3 在大肠杆菌中以可溶和折叠形式产生，已被证明具有生物活性，因此我们将设计这些分子，使其具有可与 ppGalNAc-T2 糖基化的 C 端肽标签，如项目 Z01 BC 010742 中所述。然后，这些分子将通过聚糖残基连接至治疗药物通过 N 连接聚糖链制备单克隆抗体，如项目 Z01 BC 010742 中所述，并评估其生物功效。