Modular approach for the delivery of antibodies into the cytoplasm of cells

摘要 许多细胞内靶点对小分子药物不敏感，因为它们缺乏天然配体，甚至 配基结合部位。此外，即使一种小分子药物可以结合理想的靶蛋白，它也可能无法结合。 有效抑制蛋白质功能。小分子药物能够破坏两者之间的相互作用 蛋白质尤其难以识别。此外，即使在小分子药物是 经鉴定，它必须能够到达其目标部位，具有良好的药代动力学特性和最小的副作用。 靶标毒性。这些严格的要求导致了很长的开发时间和很小一部分 小分子药物已经成功地转化为临床，尽管经过几十年的研究和 数不清的高通量屏幕。 治疗性单抗在癌症治疗方面取得了相当大的成功，但它们无法 跨细胞膜将它们的靶标限制在分泌或膜相关的抗原上。如果抗体 可以有效地输送到活细胞的胞浆中，它将显著增加可能的 可下药的目标。抗体可以与几乎任何暴露的蛋白表位结合，具有高度的特异性。 和亲和力。有无数种治疗的可能性，如果抗体可以 有效地传递到细胞中，从抑制蛋白质功能，到推动蛋白质相互作用，再到标记 蛋白酶体降解的蛋白质。不足为奇的是，已经进行了无数次尝试来传递抗体 进入细胞，但一个强大和有效的方法尚未确定。这项提案的总体目标是 开发一种模块化的方法，将抗体高效地输送到活细胞的细胞质中。 最近，我们开发了一种新的生物偶联策略，使位置特异性和共价连接成为可能 小分子、蛋白质和酶与免疫球蛋白结合。利用这项技术，我们筛选出了多种免疫球蛋白 交联物能够被输送到活细胞的胞浆中，并鉴定出可以 在亚微摩尔浓度下以~60%的效率在细胞质中传递免疫球蛋白 细胞毒性。我们方法的模块化性质不仅允许轻松交换任何现成的免疫球蛋白 进入我们的系统，还保留了与免疫球蛋白可变区的结合亲和力。 在这项计划中，我们计划进一步优化我们的抗体传递技术，并评估抗体的能力 进入细胞质以抑制正常的细胞内功能或以细胞内蛋白质为目标进行降解。 本方案的具体目标是：目标1.优化免疫球蛋白结合物和获得最大胞质的条件 提供；目标2.开发能够针对细胞内蛋白进行降解的免疫球蛋白偶联物；目标3.开发 并优化体内抗体胞浆递送的配方。

ABSTRACT Many intracellular targets are not susceptible to small molecule drugs because they lack natural ligands or even ligand binding sites. Moreover, even if a small molecule drug can bind a desirable target protein, it may not be effective in inhibiting protein function. Small molecules drugs capable of disrupting interactions between two proteins have been particularly difficult to identify. Further, even in cases where a small molecule drug is identified, it must be capable of reaching its target site with good pharmacokinetic properties and minimal off- target toxicity. These stringent requirements have led to long development times and a very small fraction of small molecule drugs that have been successfully translated into the clinic, despite decades of research and countless high-throughput screens. Therapeutic monoclonal antibodies have had considerable success as cancer therapeutics, but their inability to cross cell membranes has restricted their targets to secreted or membrane-associated antigens. If antibodies could be efficiently delivered into the cytosol of living cells, it would significantly increase the number of possible druggable targets. Antibodies can be developed to bind nearly any exposed protein epitope, with high specificity and affinity. There are a countless number of therapeutic possibilities that could be pursued if antibodies could be effectively delivered into cells, from inhibiting protein function, to driving proteins interactions, to tagging proteins for proteosomal degradation. Not surprisingly, numerous attempts have been made to deliver antibodies into cells, but a robust and efficient approach has yet to be identified. The overall goal of this proposal is to develop a modular approach to efficiently deliver antibodies into the cytoplasm of living cells. Recently, we developed a novel bioconjugation strategy that enables the site-specific and covalent attachment of small molecules, proteins, and enzymes to IgG. Utilizing this technology, we screened a variety of IgG conjugates in their ability to be delivered into the cytosol of living cells and identified conjugates that could be cytoplasmically delivered with a ~60% efficiency at sub-micromolar concentrations of IgG with minimal cytotoxicity. The modular nature of our approach not only allows for any `off-the-shelf' IgG to be easily swapped into our system, but also preserves the binding affinity of the IgG variable region. In this proposal, we plan to further optimize our antibody-delivery technology and evaluate the ability of antibodies delivered into the cytoplasm to inhibit normal intracellular function or target intracellular proteins for degradation. The specific aims for this proposal are: Aim 1. Optimize IgG conjugates and conditions for maximum cytoplasmic delivery; Aim 2. Develop IgG conjugates that can target intracellular proteins for degradation; Aim 3. Develop and optimize formulations for cytoplasmic delivery of antibodies in vivo.