Quanta BioDesign, 7470 Montgomery Drive, Plain City, Ohio 43064, United States.
Bioconjug Chem. 2021 Oct 20;32(10):2257-2267. doi: 10.1021/acs.bioconjchem.1c00429. Epub 2021 Sep 29.
Protease-activated prodrugs leverage the increased activity of proteases in the tumor microenvironment and the tight regulation in healthy tissues to provide selective activation of cytotoxins in the tumor while minimizing toxicity to normal tissues. One of the largest classes of protease-activated prodrugs are composed of therapeutic agents conjugated to macromolecular carriers via peptide motifs that are substrates for cathepsin B, and antibody-drug conjugates are one of the most successful designs within this class. However, many of these peptide motifs are also cleaved by extracellular enzymes such as elastase and carboxylesterase 1C. Additionally, some peptide sequences have little selectivity for other lysosomal cathepsins, which have also been found to have extracellular activity in normal physiological processes. A lack of selectivity or oversensitivity to other extracellular enzymes can lead to off-target release of the cytotoxic payload and subsequent toxicities. In this report, we describe an approach for modulating cathepsin-mediated release of the cytotoxic payload through steric shielding provided by the synergistic effects of appropriately designed hydrophilic linkers and the conjugated carrier. We prepared a fluorogenic model payload with a Val-Cit cleavable trigger and attached the trigger-payload to a variety of PEG-based linker architectures with different numbers of PEG arms (), different numbers of ethylene oxide units in each arm (), and different distances between the cleavable trigger and PEG branch point ('). These linker-payloads were then used to prepare DAR2 conjugates with the cleavable triggers at three different distances () from the antibody, and cathepsin-mediated payload release was monitored with assays. The results show that structural variables of the linker architectures can be manipulated to effectively shield enzymatically labile trigger-payloads from enzymes with readily accessible binding sites, and may offer an additional strategy for balancing off-target and tumor-targeted payload release.
蛋白酶激活前药利用蛋白酶在肿瘤微环境中的活性增加和在健康组织中的紧密调节，提供细胞毒素在肿瘤中的选择性激活，同时最大限度地减少对正常组织的毒性。最大的蛋白酶激活前药类别之一是由通过肽基序与大分子载体缀合的治疗剂组成的，这些肽基序是组织蛋白酶 B 的底物，抗体药物偶联物是该类别中最成功的设计之一。然而，许多这些肽基序也被弹性蛋白酶和羧基酯酶 1C 等细胞外酶切割。此外，一些肽序列对其他溶酶体组织蛋白酶几乎没有选择性，这些组织蛋白酶在正常生理过程中也具有细胞外活性。缺乏对其他细胞外酶的选择性或过度敏感性会导致细胞毒性有效载荷的非靶向释放和随后的毒性。在本报告中，我们描述了一种通过适当设计的亲水性接头的协同作用提供的空间位阻来调节组织蛋白酶介导的细胞毒性有效载荷释放的方法。我们制备了具有 Val-Cit 可切割触发的荧光模型有效载荷，并将触发有效载荷附着到具有不同 PEG 臂数（）、每个臂中的不同环氧乙烷单元数（）和可切割触发与 PEG 分支点之间不同距离（'）的各种 PEG 基接头架构上。然后，使用这些接头-有效载荷来制备具有三个不同距离（）的可切割触发的 DAR2 缀合物，并且使用 测定法监测组织蛋白酶介导的有效载荷释放。结果表明，接头架构的结构变量可以被操纵以有效地将酶不稳定的触发有效载荷与具有易于接近的结合位点的酶屏蔽，并且可能为平衡非靶标和肿瘤靶向有效载荷释放提供另一种策略。
