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

项目概要： 由于抗体介导的药物递送的重要性迅速增长，因此迫切需要简单且可靠的方法 高效的位点特异性缀合技术，不需要大量的抗体工程工作。而且， 非常需要确定免受血浆酶影响并且可用于 疏水性有效负载的附着。该提案的目标是优化缀合技术 为了应对这些挑战，我们保留了 Q295 残留物。与大多数缀合位点相比，Q295 位点是 包含在一个大的疏水空腔内，该空腔在空间上与等离子体隔离，并且特别适合 大型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很大程度上尚未被探索 迄今为止，我们的初步研究表明，还有大量尚未开发的机会来利用独特的 本网站的特点。该项目的目标是展示该位点特异性结合的广泛实用性 通过制备和评估用于肿瘤学、免疫学和成像的抗体偶联物的技术 应用程序。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 对 Q295 残基周围的局部环境进行全面的分子了解并优化连接子， 将有效负载置于相关的疏水袋内。这一目标的目的是彻底了解 Q295 部分周围的疏水口袋的化学性质。目标#2 侧重于展示 通过制备可提供广泛有效负载的 ADC，该技术的治疗效用 – 特别关注通过传统（“随机”）连接时表现出困难的有效载荷 共轭方法。选择了四个特定的有效负载：MMAE（由于其临床相关性和已知的链接器 稳定性问题）、Tubulysin（由于对具有低 PGP 流出量的有效负载以及具有不稳定酯功能的兴趣） 导致了传统方法的问题）、Thailanstatin A（由于其独特的作用机制和不稳定的 结构中的官能团）和 Brequinar（由于其作为免疫抑制剂的效力及其非常高的 迄今为止，疏水性阻碍了 ADC 的输送）。由此产生的 B 细胞靶向 ADC 将得到彻底评估 其在 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 站点 开发可用于生成活细胞延时图像的拉曼成像探针。重要的是， 目前还没有尝试使用拉曼成像来研究 ADC 贩运的报道。传统 ADC 结合 由于拉曼标签具有非常高的疏水性，因此不能采用该方法来附着拉曼标签。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，用于针对特定地点 结合有问题的有效负载，并将建立新的成像技术来研究 ADC 贩运和 分解代谢。

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.