Legumain to the rescue: A new ADC linker strategy to address the limitations of cathepsin cleavage

项目总结： 现在市场上有8种FDA批准的抗体药物结合物(ADC)。ADC技术正在得到广泛的应用 致力于肿瘤学、自身免疫学、眼科和传染病的应用。尽管有巨大的 然而，成功的ADC开发过程充满了与ADC聚合、链接器- 稳定性问题、意外毒性和较差的药代动力学。虽然不可能将这一点归咎于 单一现象的脚，绝大多数ADC共有的一个共同因素是依赖于 疏水性多肽连接物(即ValCit-PABC)，在血浆中稳定，但被溶酶体组织蛋白快速切割。 最近的各种研究表明，这种连接物在循环中比以前认为的更不稳定， 容易被血浆酯酶(Ces1C)和淋巴细胞相关的蛋白酶(中性粒细胞弹性蛋白酶)切割。 现在人们普遍认为，中性粒细胞弹性蛋白酶的切割是导致剂量限制性中性粒细胞减少症的原因，并 血小板减少症在许多ADC中都很常见。迫切需要新一代可切割连接子 来克服这些债务。在此，我们建议对重新设计的ADC链接器进行优化和评估 含有天冬酰胺(ASN)基序，可被天冬酰胺内切酶(又称豆类)迅速切割。我们的 研究小组已经确定了几个含有ASN的序列，这些序列可以被溶酶体豆蛋白快速切割，同时保留 在小鼠和人血浆中的稳定性。我们的初步数据显示，合并了这些链接器的ADC显示 体外细胞毒性与经典的ValCit-PABC连接物相当，但不容易被Ces1C或 中性粒细胞弹性蛋白酶。我们设计了三个具体目标来优化此链接器，并演示ADC模型 与传统的ValCit-PABC接头相比，使用这些接头的患者具有更好的治疗潜力。目标1是 专注于多肽连接物本身的优化。我们将准备一个基于FRET的包含ASN的库 多肽，以便确定可以整合到各种癌症靶向ADC中的强健连接体。目标2是 重点开发了适合豆类可裂解多肽的自焚间隔基。间隔物将 进行优化，以促进溶酶体的快速切割和血浆中的高稳定性。目标#3将确立 豆类可裂解adcs在胰腺癌模型中的治疗作用。具体地说，我们将把 我们的新接头进入了抗GCC的ADC，这对胰腺癌的治疗很有意义。ADC将是 将面对面与传统ADC(TAK-264)进行比较，后者因剂量限制而未能通过2期临床试验 中性粒细胞减少症。我们预计，我们的新型连接器将赋予该ADC更好的耐受性，同时保持 功效。这些目标的实现将坚定地确立豆类卵裂用于治疗的适宜性。 应用-并将使这项技术稳定在临床应用中。新型胰腺癌靶向治疗的研究进展 ADC将为在一个巨大的未得到满足的医疗需求领域进行临床评估铺平道路。

Project Summary: There are now 8 FDA-approved antibody-drug-conjugates (ADCs) on the market. ADC technology is being widely pursued for oncology, autoimmunity, ophthalmology, and infectious disease applications. In spite of tremendous success, however, the ADC development process is riddled with challenges associated with ADC aggregation, linker- stability concerns, unexpected toxicity, and poor pharmacokinetics. While it is impossible to lay the blame for this at the feet of a single phenomenon, one common factor shared by the vast majority of ADCs is the reliance on a hydrophobic peptide linker (i.e. ValCit-PABC) that is stable in plasma but rapidly cleaved by lysosomal cathepsins. Various studies have recently shown that this linker is more unstable in circulation was than previously believed, being susceptible to cleavage by both plasma esterases (Ces1C) and lymphocyte-associated proteases (neutrophil elastase). It is now widely believed that cleavage by neutrophil elastase is responsible for the dose-limiting neutropenia and thrombocytopenia so commonly seen for many ADCs. A new generation of cleavable linkers is urgently needed in order to overcome these liabilities. Herein, we propose the optimization and evaluation of “redesigned” ADC linkers that contain asparagine (Asn) motifs which are rapidly cleaved by asparaginyl endoprotease, also known as legumain. Our team has identified several Asn-containing sequences that are rapidly cleaved by lysosomal legumain while retaining stability in mouse and human plasma. Our preliminary data shows that ADCs that incorporate these linkers exhibit comparable in vitro cytotoxicity to classical ValCit-PABC linkers – but are not susceptible to cleavable by Ces1C or neutrophil elastase. We have designed three specific aims to optimize this linker and to demonstrate that model ADCs that use these linkers have improved therapeutic potential as compared to traditional ValCit-PABC linkers. Aim #1 is is focused on the optimization of the peptide linker itself. We will prepare a FRET-based library of Asn-containing peptides in order to identify robust linkers that can be incorporated into various cancer-targeting ADCs. Aim #2 is focused on the development of suitable self-immolative spacers for the legumain cleavable peptides. The spacer will be optimized in order to facilitate rapid cleavage in the lysosome and high stability in plasma. Aim #3 will establish the therapeutic utility of legumain-cleavable ADCs in a model of pancreatic cancer. Specifically, we will incorporate our novel linkers into anti-GCC ADCs that are of interest for the treatment of pancreatic cancer. The ADCs will be compared head-to-head with a traditional ADC (TAK-264) that failed phase 2 clinical trials due to dose limiting neutropenia. We anticipate that our novel linkers will impart an improved tolerability to this ADC while maintaining the efficacy. Accomplishment of these aims will firmly establish the suitability of legumain-cleavage for therapeutic applications – and will poise the technology for clinical application. Development of novel pancreatic-cancer targeting ADCs will pave the way towards clinical evaluation in an area of tremendous unmet medical need.