Exploiting the Hydrophobic Glycosyl Pocket of IgG1 for Imaging and Drug Delivery Applications

利用 IgG1 的疏水性糖基口袋进行成像和药物输送应用

• 批准号: 10619285
• 负责人: Lawrence Tumey
• 金额: $ 7.01万
• 依托单位: STATE UNIVERSITY OF NY,BINGHAMTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10619285
• 关键词: Achievement; Antibodies; B-Lymphocytes; Catabolism; Cells; Communities; Development; Drug Delivery Systems; Drug Kinetics; Environment; Enzymes; Esters; Evaluation; Exhibits; Goals; Hydrophobicity; IgG1; Image; Imaging Techniques; Immunoglobulin G; Immunology; Immunosuppressive Agents; Mediating; Methods; Molecular; Oncology; Plasma; Preparation; Property; Raman Spectrum Analysis; Reporting; Research; Site; Structure; Techniques; Technology; Therapeutic; Time; Xenograft Model; antibody conjugate; antibody engineering; chemical property; clinically relevant; functional group; imaging probe; interest; prevent; tool; trafficking

Project Summary: Due to the rapidly growing importance of antibody-mediated drug delivery, there is a critical need for simple and efficient site-specific conjugation techniques that do not require extensive antibody engineering efforts. Moreover, there is significant need to identify sites of conjugation that are protected from plasma enzymes and are useful for the attachment of hydrophobic payloads. The goal of this proposal is to optimize conjugation technology at the conserved Q295 residue in order to meet these challenges. In contrast to most sites of conjugation, the Q295 site is contained within a large hydrophobic cavity that is sterically shielded from plasma and is particularly amenable to the conjugation of large nonpolar payloads. Remarkably, the properties of this hydrophobic pocket are largely unexplored to-date and our preliminary research shows that there are significant untapped opportunities for exploiting the unique features of this site. The goal of this project is to demonstrate the broad utility of this site-specific conjugation technology through the preparation and evaluation of antibody conjugates for oncology, immunology, and imaging applications. We will accomplish this goal through the achievement of three aims. Aim#1 focuses on developing a thorough molecular understanding of the local environment around the Q295 residue and optimizing linkers that can place the payload within the associated hydrophobic pocket. The goal of this aim is to thoroughly understand the chemical properties of the hydrophobic pocket that surrounds the Q295 moiety. Aim#2 focuses on demonstrating the therapeutic utility of this technology through the preparation of ADCs that deliver a wide range of payloads – particularly focusing on payloads that have exhibited difficulties when attached through traditional (“stochastic”) conjugation approaches. Four particular payloads were selected: MMAE (due to its clinical relevance and known linker stability issues), Tubulysin (due to interest in payloads with low PGP efflux and also a labile ester functionality that has caused problems with traditional approaches), Thailanstatin A (due to its unique mechanism of action and to the labile functional groups in its structure), and Brequinar (due to its potency as an immunosuppressive agent and its very high hydrophobicity that has so-far prevented ADC delivery). The resulting B-cell targeting ADCs will be thoroughly evaluated for their pharmacokinetic profile and efficacy in a B-cell xenograft model. Aim#3 focuses on using the Q295 site for the development of Raman imaging probes that can be used for generating live-cell time lapse images. Importantly, there have been no reported attempts to use Raman imaging to study ADC trafficking. Traditional ADC conjugation methods cannot be employed for the attachment of the Raman tags due to their very high hydrophobicity. Successful achievement of these aims will provide the drug-delivery community with a valuable new tool for site-specific conjugation of problematic payloads and will establish new imaging techniques for the study of ADC trafficking and catabolism.

项目概要： 由于抗体介导的药物递送的重要性迅速增长，迫切需要简单且 有效的位点特异性缀合技术，其不需要大量的抗体工程努力。此外，委员会认为， 非常需要鉴定被保护不受血浆酶影响并可用于 疏水有效载荷的附着。该提案的目标是优化在最佳条件下的缀合技术。 保守的Q295残基以应对这些挑战。与大多数缀合位点相反，Q295位点是 包含在空间上与等离子体屏蔽的大的疏水空腔内， 大的非极性有效载荷的共轭。值得注意的是，这种疏水口袋的特性在很大程度上是未开发的 到目前为止，我们的初步研究表明，有显着的未开发的机会，利用独特的 这个网站的特点。这个项目的目标是展示这种位点特异性结合的广泛用途 通过制备和评估用于肿瘤学、免疫学和成像的抗体缀合物的技术 应用.我们将通过实现三个目标来实现这一目标。目标#1专注于开发一个 对Q295残基周围的局部环境进行彻底的分子理解，并优化可以 将有效载荷置于相关的疏水袋内。这一目标的目标是彻底了解 Q295部分周围的疏水口袋的化学性质。目标#2重点展示了 该技术通过制备递送广泛有效载荷的ADC的治疗效用- 特别关注通过传统（“随机”） 共轭方法选择了四种特定的有效载荷：MMAE（由于其临床相关性和已知的连接物） 稳定性问题）、微管溶素（由于对具有低PGP流出的有效载荷的兴趣以及具有不稳定酯官能团的不稳定酯官能团， 引起的问题与传统的方法），Thailanstatin A（由于其独特的作用机制和不稳定的 其结构中的官能团）和布喹那（由于其作为免疫抑制剂的效力和其非常高的 疏水性，其迄今为止阻止ADC递送）。将对所得的B细胞靶向ADC进行全面评价 在B细胞异种移植模型中的药代动力学特征和功效。目标#3侧重于使用Q295网站， 拉曼成像探针的发展，可用于产生活细胞的时间推移图像。重要的是， 还没有报道尝试使用拉曼成像来研究ADC运输。传统ADC结合 由于拉曼标记物的疏水性非常高，因此不能采用这些方法来附着这些标记物。成功 这些目标的实现将为药物输送界提供一个有价值的新工具， 结合有问题的有效载荷，并将建立新的成像技术，用于研究ADC贩运和 猫