TRIM21-mediated degradation of antibody-targeted cytosolic proteins

这项提议的总体目标是开发一种生物试剂，它不仅能够使胞浆中的易溶物质 传递任何现成的、本地免疫球蛋白G(IgG)抗体，但也会导致任何 与免疫球蛋白结合的细胞内蛋白。我们设想，通过细胞裂解递送免疫球蛋白和降解靶标的能力 蛋白质将产生革命性的影响，因为它将极大地扩大可行的药物靶点的池 为研究蛋白质功能和细胞内信号转导提供了有力的新工具。 最近，宾夕法尼亚大学的图尔卡斯实验室开发出了光反应性抗体结合 来源于蛋白G的结构域(PAbBds)，可用于快速和位点特异性的共价标记 含有小分子、多肽、蛋白质或酶的Ig G Fc结构域。这项技术已获得许可 敬阿尔法塞拉。使用这项技术，图尔卡斯实验室已经用高度阴离子多肽(APP)标记了IGG 并通过静电作用将它们与专为核酸输送而设计的阳离子脂类络合。这些 复合体能够有效地将免疫球蛋白输送到细胞的胞浆中，输送效率高达90%。 细胞递送的免疫球蛋白具有功能性，并被成功地用于抑制与多药耐药相关的 蛋白1(MRP1)，导致药物阿霉素出口减少，导致显著 其EC50有所改善。尽管这种方法具有激动人心的潜力，但通过直接抑制靶蛋白 抗体是高度依赖于表位的，需要传递化学计量的抗体， 每个抗体最多只能抑制两个靶点。 最近已经证明，存在一种天然的细胞内系统，在这种系统中，胞浆中的免疫球蛋白可以自然地 利用TRIM21 E3泛素连接酶降解抗体结合的靶标。通过利用TRIM21，蛋白质 降解是表位不可知的，只有亚化学计量比的胞浆递送抗体才能 催化多轮目标蛋白质降解。由于来源于蛋白G的pAbBD与相同的 作为TRIM21，即FcRN结合位点，TRIM21介导的降解途径在以下情况下不被触发 利用pAbBD-APP融合促进胞质免疫球蛋白递送。 此SBIR第一阶段应用程序的一个主要目标是设计一个从TRIM21派生的新pAbBD，通过 加入光交联剂，并将其与APP融合以实现胞质递送。这将确保 每个抗体都与TRIM21结合，无论内源性TRIM21的表达水平如何，以促进 目的蛋白的降解。这项建议的具体目标是：目标1.确定 在TRIM21中加入光交联剂；目标2.展示目标细胞内的有效降解 蛋白质。

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.