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

将抗体递送到细胞质中的模块化方法

基本信息

批准号：
9975797
负责人：
Andrew Tsourkas
金额：
$ 36.65万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-10 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9975797
关键词：
ABCC1 gene Affinity Amino Acids Antibodies Antigens Aspartic Acid Automobile Driving Binding Binding Sites Cations Cell membrane Cells Charge Chimeric Proteins Clinic Cytoplasm Cytosol Dependence Development Doxorubicin Drug Kinetics Electroporation Engineering Enzymes Epitopes Excipients Exhibits Formulation Gene Delivery Glutamic Acid Goals Goat Heavy-Chain Immunoglobulins Human Immunoglobulin G Immunoglobulin Variable Region Libraries Ligand Binding Ligands Lipids Lipofectamine Liposomes Malignant Neoplasms Mediating Membrane Microinjections Mus Nature Oryctolagus cuniculus Pharmaceutical Preparations Property Proteins RAS inhibition Rattus Reagent Reporting Research Safety Site Specificity Structure System Tacrolimus Binding Protein 1A Techniques Technology Testing Therapeutic Therapeutic Monoclonal Antibodies Time Toxic effect Transfection Translating base cytotoxicity druggable target fluorexon high throughput screening in vivo nanoformulation nanoparticle novel polypeptide preservation protein degradation protein function small molecule success therapeutic target tumor unnatural amino acids

项目摘要

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.

抽象的许多细胞内靶标对小分子药物不敏感，因为它们缺乏天然配体，甚至配体结合位点。而且，即使小分子药物能够结合所需的靶蛋白，也可能不有效抑制蛋白质功能。能够破坏两者之间相互作用的小分子药物蛋白质尤其难以识别。此外，即使在小分子药物是确定后，它必须能够以良好的药代动力学特性和最小的副作用到达其目标位点。目标毒性。这些严格的要求导致了较长的开发时间和很小的一部分经过数十年的研究和研究，小分子药物已成功转化为临床无数的高通量屏幕。治疗性单克隆抗体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其靶标限制为分泌的或膜相关的抗原。如果有抗体可以有效地传递到活细胞的细胞质中，这将显着增加可能的数量可药物靶标。可以开发抗体来结合几乎任何暴露的蛋白质表位，具有高特异性和亲和力。如果抗体能够实现，那么就有无数的治疗可能性可以实现。有效地传递到细胞中，从抑制蛋白质功能，到驱动蛋白质相互作用，再到标记用于蛋白酶体降解的蛋白质。毫不奇怪，人们已经进行了许多尝试来递送抗体进入细胞，但尚未确定一种稳健且有效的方法。该提案的总体目标是开发一种模块化方法，有效地将抗体递送到活细胞的细胞质中。最近，我们开发了一种新颖的生物共轭策略，可以实现位点特异性和共价连接将小分子、蛋白质和酶转化为 IgG。利用这项技术，我们筛选了多种IgG 缀合物被递送到活细胞的细胞质中的能力，并鉴定了可以被以亚微摩尔浓度的 IgG 进行细胞质递送，效率约为 60% 细胞毒性。我们方法的模块化性质不仅允许轻松更换任何“现成”IgG 进入我们的系统，但也保留了 IgG 可变区的结合亲和力。在这个提案中，我们计划进一步优化我们的抗体递送技术并评估抗体的能力递送到细胞质中以抑制正常的细胞内功能或靶向细胞内蛋白质进行降解。该提案的具体目标是：目标 1. 优化 IgG 缀合物和条件以获得最大细胞质送货;目标 2. 开发可靶向细胞内蛋白质进行降解的 IgG 缀合物；目标 3. 发展并优化体内抗体细胞质递送的制剂。