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

• 批准号: 10033745
• 负责人: MATTHEW B FRANCIS
• 金额: $ 29.5万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 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/10033745
• 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

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.

项目摘要/摘要 嵌合蛋白质-蛋白质结合物为免疫治疗提供了各种成功的平台， 靶向给药、细胞生物学研究和疫苗开发。然而，许多理想的结构 不能单独使用遗传方法来产生，而使用化学物质来定向偶联两种蛋白质 方法仍然非常具有挑战性。在这个项目中，将探索一种新的方法，用于快速和现场- 使用天然氨基酸的蛋白质的特定偶联。酪氨酸酶将用来氧化溶剂- 暴露在蛋白质和多肽底物上的酪氨酸残基产生邻苯二酚，与之迅速反应 在其他蛋白质中战略性地放置半胱氨酸残基。初步数据证实，这种方法 在室温下，可以在不到1小时的时间内从单个蛋白质中产生复杂的、多功能的构建体 尽管这些反应中存在高度的空间相互作用。酪氨酸酶是 价格低廉，可以固定在固体载体上以便于移除，并且只需要外来氧气 才能发挥作用。从蛋白质的N-末端或C-末端延伸的酪氨酸残基很容易被氧化， 但在反应过程中，体内的酪氨酸残基不受影响。半胱氨酸残基可以放在任何地方 在第二个蛋白质靶点的表面。由于这些特点，这种方法在简单性上独树一帜 和灵活性来构建复杂的多域结构，因此它将极大地扩展 可用于生物技术应用的生物偶联物。 拟议研究的第一个具体目标是探索细胞穿透性多肽的附着。 将scFv构建为有用的模型蛋白，目的是提高它们穿越细胞膜的能力 并进入细胞质。半胱氨酸将被引入到scFv表面的几个位置，细胞 含有暴露的酪氨酸残基的进入肽将使用酪氨酸酶方法附着。摄取和 这些构建物的细胞内定位将使用96孔板分析，光学显微镜和 流式细胞术。 特殊目标2将探索使用这种方法来生产多功能蛋白质 免疫疗法的应用。酪氨酸标签将被引入生物分子面板上的战略位置， 包括单链抗体结构域和抗体。酪氨酸酶激活后，载药分子的能力 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 将探索用于生产双功能和三功能电池接合剂。这一目标的成功将是 提供一种高效的方法来制造和筛选这些具有治疗价值的结构。 第三个具体目标将集中在识别具有理想特性的新的酪氨酸酶，以及 通过改造这些酶来实现不同酪氨酸序列的激活。