Facile Generation of Protein-Protein Conjugates Using Enzymatic Oxidative Coupling Reactions

利用酶促氧化偶联反应轻松生成蛋白质-蛋白质缀合物

基本信息

批准号：
10227116
负责人：
MATTHEW B FRANCIS
金额：
$ 29.72万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10227116
关键词：
Address Amino Acids Antibodies Antibody Formation Antibody-drug conjugates Biological Assay Biological Products Biology Biotechnology Bispecific Antibodies C-terminal Cell membrane Cell-Matrix Junction Cells Cellular biology Chemicals Chemistry Chimeric Proteins Complex Coupling Cysteine Cytoplasm Data Development Drug Delivery Systems Drug Targeting Engineering Enzymes Excision Flow Cytometry Generations Genetic Genetic Engineering Goals Immobilization Immunobiology Immunoglobulin G Immunoglobulins Immunotherapy Individual Libraries Ligation Location Membrane Proteins Methods Modeling Modification Monophenol Monooxygenase N-terminal Nature Outcome Oxidants Oxides Oxygen Peptides Pharmaceutical Preparations Positioning Attribute Post-Translational Protein Processing Production Property Proteins Protocols documentation Quinones Reaction Reagent Research Series Site Solid Solvents Specificity Sulfhydryl Compounds Surface T-Lymphocyte Techniques Temperature Testing Therapeutic Tyrosine Variant Water antibody conjugate cancer cell cancer therapy chemical group crosslink design flexibility functional group immunological synapse light microscopy novel strategies programs protein function screening small molecule success synthetic peptide uptake vaccine development water treatment

项目摘要

PROJECT SUMMARY/ABSTRACT Chimeric protein-protein conjugates provide a wide variety of successful platforms for immunotherapy, targeted drug delivery, cell biology studies, and vaccine development. However, many desirable constructs cannot be produced using genetic methods alone, and the targeted coupling of two proteins using chemical methods is still very challenging. In this program, a new approach will be explored for the rapid and site- specific coupling of proteins using native amino acids. Tyrosinase enzymes will be used to oxidize solvent- exposed tyrosine residues on protein and peptide substrates to generate ortho-quinones that react rapidly with strategically placed cysteine residues in other proteins. Preliminary data have confirmed that this approach can generate complex, multifunctional constructs from individual proteins in under 1 h at room temperature despite the high degree of steric interactions that are inherent in these reactions. The tyrosinase enzymes are inexpensive, can be immobilized on solid supports to facilitate removal, and require only adventitious oxygen to function. Tyrosine residues that extend from the N- or C-terminal positions on proteins are oxidized readily, but internal tyrosine residues are unaffected during the reactions. The cysteine residues can be placed anywhere on the surface of the second protein target. Due to these features, this method stands alone in its simplicity and flexibility for making complex, multidomain constructs, and thus it will greatly expand the range of bioconjugates that can be accessed for biotechnology applications. The first Specific Aim of the proposed research will explore the attachment of cell penetrating peptides to scFv constructs as useful model proteins, with the goal of increasing their ability to cross cell membranes and access the cytoplasm. Cysteines will be introduced into several locations on the scFv surfaces, and cell entry peptides bearing exposed tyrosine residues will be attached using the tyrosinase method. The uptake and intracellular localization of these constructs will be evaluated using 96-well plate assays, light microscopy and flow cytometry. Specific Aim 2 will explore the use of this approach for producing multifunctional proteins for immunotherapy applications. Tyrosine tags will be introduced in strategic locations on a panel of biomolecules, including scFv domains and IgG antibodies. Following tyrosinase activation, the ability of drug cargo molecules to couple to these sites will be evaluated, and the asymmetric nature of the protein-protein coupling chemistry will be explored for the production of bispecific and trispecific cell engagers. The success of this Aim would provide a highly efficient way to make and screen these therapeutically valuable constructs. The third Specific Aim will focus on identifying new tyrosinase enzymes with desirable properties, and the engineering of these enzymes to achieve the activation of different tyrosine-containing sequences.

项目概要/摘要嵌合蛋白-蛋白缀合物为免疫治疗提供了多种成功的平台，靶向药物输送、细胞生物学研究和疫苗开发。然而，许多理想的结构单独使用遗传方法无法产生，并且使用化学方法有针对性地偶联两种蛋白质方法还是很有挑战性的。在这个项目中，将探索一种新的方法来快速和现场使用天然氨基酸进行蛋白质的特异性偶联。酪氨酸酶将用于氧化溶剂- 将蛋白质和肽底物上的酪氨酸残基暴露出来，生成邻醌，并与策略性地将半胱氨酸残基放置在其他蛋白质中。初步数据证实该方法可以在室温下 1 小时内从单个蛋白质生成复杂的多功能结构尽管这些反应中固有的高度空间相互作用。酪氨酸酶是价格便宜，可以固定在固体支持物上以方便去除，并且仅需要外来氧发挥作用。从蛋白质 N 或 C 末端位置延伸的酪氨酸残基很容易被氧化，但内部酪氨酸残基在反应过程中不受影响。半胱氨酸残基可以放置在任何地方在第二个蛋白质靶标的表面上。由于这些特征，该方法以其简单性而独树一帜以及构建复杂的多域结构的灵活性，因此它将极大地扩展其范围可用于生物技术应用的生物共轭物。拟议研究的第一个具体目标将探索细胞穿透肽的附着将 scFv 构建为有用的模型蛋白，目的是提高其穿过细胞膜的能力并进入细胞质。半胱氨酸将被引入到 scFv 表面的多个位置，并且细胞将使用酪氨酸酶方法连接带有暴露的酪氨酸残基的进入肽。吸收率和这些构建体的细胞内定位将使用 96 孔板测定、光学显微镜和流式细胞术。具体目标 2 将探索使用这种方法生产多功能蛋白质免疫治疗应用。酪氨酸标签将被引入生物分子组的战略位置，包括 scFv 结构域和 IgG 抗体。酪氨酸酶激活后，药物货物分子的能力将评估与这些位点的偶联，以及蛋白质-蛋白质偶联化学的不对称性质将探索双特异性和三特异性细胞接合器的生产。这一目标的成功将提供了一种高效的方法来制造和筛选这些具有治疗价值的构建体。第三个具体目标将侧重于识别具有理想特性的新酪氨酸酶，以及对这些酶进行工程改造以实现不同含酪氨酸序列的激活。