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

• 批准号: 7733240
• 负责人: Pradman K Qasba
• 金额: $ 7.49万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/7733240
• 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

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. 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 re [summary truncated at 7800 characters]

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地方。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。人α -乳白蛋白的制备，带或不带c端标签，与ppGalNAc-T2糖基化，形成油酸复合物，使HAMLET (human α -乳白蛋白Made to Kill Tumor cell)：生物杀伤分子HAMLET是载脂蛋白α -乳白蛋白和油酸的复合物，Catharina Svanborg博士已经证明，它能触发肿瘤细胞死亡，而健康细胞却能存活。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，体外折叠蛋白质，确定α -乳清蛋白与半乳糖转移酶复合物的结构，并在20多年来广泛研究该分子的公司，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，该标签可以与项目Z01 BC 010742中描述的ppGalNAc-T2糖基化。折叠形式的载脂蛋白α -乳清蛋白，带或不带c末端肽标签，与油酸复合物将首先用于Jurkat和L1210细胞系，乳腺癌细胞系MCF-7和早幼粒细胞白血病细胞系HL-60的凋亡测试。在使用Annexin-FTIC流式细胞术检测细胞凋亡后，我们将把HAMLET与Herceptin单克隆抗体偶联用于靶向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，带或不带c端标签，用于与ppGalNAc-T2糖基化，以连接到治疗性单克隆抗体：半乳糖凝集素包括一个糖结合蛋白家族，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通过n -链聚糖链连接到治疗性单克隆抗体上，并评估其生物功效。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地方。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。人α -乳白蛋白的制备，带或不带c端标签，与ppGalNAc-T2糖基化，形成油酸复合物，使HAMLET (human α -乳白蛋白Made to Kill Tumor cell)：生物杀伤分子HAMLET是载脂蛋白α -乳白蛋白和油酸的复合物，Catharina Svanborg博士已经证明，它能触发肿瘤细胞死亡，而健康细胞却能存活。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，体外折叠蛋白质，确定α -乳清蛋白与半乳糖转移酶复合物的结构，并在20多年来广泛研究该分子的公司，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，该标签可以与项目Z01 BC 010742中描述的ppGalNAc-T2糖基化。折叠形式的载脂蛋白α -乳清蛋白，带或不带c末端肽标签，与油酸复合物将首先用于Jurkat和L1210细胞系，乳腺癌细胞系MCF-7和早幼粒细胞白血病细胞系HL-60的凋亡测试。在使用Annexin-FTIC流式细胞术检测细胞凋亡后，我们将把HAMLET与Herceptin单克隆抗体偶联用于靶向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，带或不带c端标签，用于与ppGalNAc-T2糖基化，以连接到治疗性单克隆抗体：半乳糖凝集素包括一个糖结合蛋白家族，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。然后这些分子将通过糖链连接起来[摘要截断为7800个字符]