TRIM21-mediated degradation of antibody-targeted cytosolic proteins

TRIM21 介导的抗体靶向胞浆蛋白降解

• 批准号: 10006659
• 负责人: Feifan Yu
• 金额: $ 27.09万
• 依托单位: ALPHATHERA, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-04 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10006659
• 关键词: Amino Acid Substitution; Antibodies; Binding; Binding Proteins; Binding Sites; Biological; Cations; Cells; Chimeric Proteins; Complex; Cytosol; Degradation Pathway; Doxorubicin; Drug Targeting; Electrostatics; Engineering; Ensure; Enzymes; Epitopes; Exhibits; Fc domain; G-substrate; Goals; Immunoglobulin G; Label; Lipids; Mediating; Multidrug Resistance-Associated Proteins; Pennsylvania; Pharmaceutical Preparations; Phase; Proteins; Reagent; Signal Transduction; Site; Small Business Innovation Research Grant; System; Technology; Testing; Transfection; Universities; Variant; benzoylphenylalanine; crosslink; design; neonatal Fc receptor; nucleic acid delivery; polypeptide; protein degradation; protein function; small molecule; tool; ubiquitin-protein ligase; unnatural amino acids

The overall goal of this proposal is to develop a biological reagent that not only enables the facile cytosolic delivery of any ‘off-the-shelf’, native immunoglobulin G (IgG) antibody, but also leads to degradation of any intracellular protein bound by the IgG. We envision that the ability to cytosolically deliver IgGs and degrade target proteins would have a transformative impact, since it would dramatically expand the pool of viable drug targets and provide a powerful new tool to study protein function and intracellular signaling. Recently, the Tsourkas lab, at the University of Pennsylvania, developed photoreactive antibody-binding domains (pAbBDs) derived from protein G that can be used for the rapid and site-specific covalent labeling of the IgG Fc domain with small molecules, polypeptides, proteins, or enzymes. This technology has been licensed to AlphaThera. Using this technology, the Tsourkas lab has labeled IgGs with highly anionic polypeptides (ApPs) and complexed them with cationic lipids designed for nucleic acid delivery via electrostatic interactions. These complexes enabled the efficient delivery of IgG into the cytosol of cells with up to 90% delivery efficiency. Cytosolically delivered IgGs are functional and were used to successfully inhibit multidrug resistance-associated protein 1 (MRP1), leading to a reduction in the export of the drug doxorubicin, resulting in a significant improvement in its EC50. Despite the exciting potential of this approach, direct inhibition of target proteins with antibodies is highly epitope-dependent and requires a stoichiometric amount of antibody to be delivered, with each antibody only able to inhibit up to two targets. It has recently been demonstrated that a native intracellular system exists whereby cytosolic IgG can naturally engage TRIM21 E3 ubiquitin ligase to degrade antibody-bound targets. By harnessing TRIM21, protein degradation is epitope agnostic and only a sub-stoichiometric amount of cytosolically delivered antibody can catalyze multiple rounds of target protein degradation. Since pAbBDs derived from protein G bind to the same IgG site as TRIM21, i.e. the FcRn binding site, the TRIM21-mediated degradation pathway is not triggered when using pAbBD-ApP fusions to facilitate cytosolic IgG delivery. A major goal of this SBIR Phase I application is to engineer a new pAbBD that is derived from TRIM21, through the incorporation of a photocrosslinker, and by fusing it with an ApP to enable cytosolic delivery. This will ensure that every antibody is bound by TRIM21, regardless of endogenous TRIM21 expression levels, to facilitate degradation of the target protein. The specific aims for this proposal are: Aim 1. Identify the optimal site for incorporating a photocrosslinker into TRIM21; Aim 2. Demonstrate the effective degradation of target intracellular proteins.

该提案的总体目标是开发一种易于胞质的生物试剂 递送任何“现成”，本地Imunoglobulin g（IgG）抗体，但也导致任何抗体 由IgG结合的细胞内蛋白质。 蛋白质w是什么具有变革性影响的，因为它会巨大扩展紫罗兰色药物靶标的 并提供一个强大的新工具来研究蛋白质功能和细胞内信号传导。 最近，宾夕法尼亚大学的Tsourkas Lab开发了光反应性抗体结合 源自蛋白质G的结构域（PABBD），可用于快速，特定的共价标记 具有小分子，多肽，蛋白质或酶的IgG FC结构域已获得许可。 对于Alphathera，Tsourkas实验室使用了高度的IGG（应用程序） 并通过静电相互作用将它们与核酸递送的capids相吻合。 复合物使IgG有效地递送到细胞的细胞质中，并提高了90％的递送功效。 胞质递送的IgG是功能性的，并且被允许成功抑制与多药相关的抑制 蛋白1（MRP1），导致药物阿霉素的出口进行了修订，导致显着 EC50的改善是直接吸入的令人兴奋的潜力吗？ 抗体高度依赖于表位，需要递送化学计量的抗体，并使用 每种抗体能够抑制两个靶标。 最近已经证明存在一个天然的细胞内系统，胞质IgG自然可以 通过利用TRIM21，蛋白质，使TRIM21 E3泛素连接酶降低抗体结合靶标。 降解是表位不可知的，只有亚化学计量量的胞质输送抗体可以 催化靶蛋白降解的多个回合。 IgG位点为TRIM21，即FCRN结合位点，Trim21介导的降解途径不会触发 使用PABBD-APP融合来促进胞质IgG递送。 SBIR I期应用程序的主要目标是设计一种从TRIM21派生的新PABBD， 光叠链链接的插入，并用应用程序填充它，以确保胞质传递 每种抗体都受TRIM21的约束，无论内源性TRIM21表达水平如何 靶蛋白的降解。 将光叠链链接纳入TRIM21； AIM 2。 蛋白质。