Tumor-specific drug activation by pericellular proteases

摘要 异常的细胞周蛋白分解是正常上皮细胞恶变的主要驱动力 对癌细胞来说。肿瘤微环境中蛋白分解活性的增强已被用于药物开发 发展目的。例如，抗体-药物结合物(ADC)由一种高度有效的抗癌药物组成 有效载荷与肿瘤靶向抗体相结合。这种联轴器使货物处于非活动状态，直到它被释放 在细胞周隙或靶细胞内抗体降解时的蛋白水解性裂解。ADC 仍然受到剂量限制毒性的限制，很可能是由于靶细胞结合或药物过早释放所致 通过流通中的蛋白酶。非靶标结合可通过工程化的蛋白酶激活的PRO来克服。 抗体(PADC)只有在疾病部位被蛋白酶激活后才能结合，而 通过利用在存在的情况下稳定的多肽连接物序列可以改善过早的药物释放 血液中的蛋白水解酶，但有效地被疾病部位的蛋白水解酶切割。我们的假设是PADDC 需要两个蛋白分解激活步骤，可能会提高我们选择性靶向抗癌药物的能力 肿瘤微环境。PADC将被细胞周蛋白酶转化为ADC，并随后 与肿瘤细胞结合。有效载荷将由胞外或胞内的蛋白酶释放。 (内体或溶酶体)蛋白酶。本提案的目标是设计具有以下特性的ADC和PADC 卓越的血浆稳定性和癌症相关酶改善的释放动力学。我们的长期合作 目的是开发与目前的治疗方法相比具有更强的抗癌效果的pADCs。 在目标1中，我们将为患者来源的器官分泌的蛋白水解酶生成底物特异性图谱。 乳腺癌。此外，我们还将分离溶酶体和内切体，并鉴定其蛋白水解性。 这些细胞器。最后，我们将评估血清和血浆中的蛋白酶活性，以了解循环情况。 蛋白水解性。在目标2中，我们将开发可被癌症蛋白酶选择性激活的adc和padc。 在血浆和血清中稳定存在。

ABSTRACT Dysregulated pericellular proteolysis is a major driver of the malignant transformation of normal epithelial cells to cancer cells. The increased proteolytic activity in the tumor microenvironment has been exploited for drug development purposes. For example, antibody-drug conjugates (ADCs) consist of a highly potent anti-cancer payload conjugated to a tumor-targeting antibody. This coupling renders the cargo inactive until it is released by proteolytic cleavage in the pericellular space or upon degradation of the antibody within the target cell. ADCs are still handicapped by dose-limited toxicities, most likely due to off-target cell binding or premature drug release by proteases in circulation. The off-target binding can be overcome by engineering protease-activated pro- antibodies (pADCs) that can bind only after being activated at the site of disease by a protease while the premature drug release can be improved by utilizing peptide linker sequences that are stable in the presence of blood proteases but are efficiently cleaved by proteases at the site of disease. Our hypothesis is that pADCs that require two proteolytic activation steps, may improve our ability to selectively target anti-cancer drugs to the tumor microenvironment. The pADC will be converted into an ADC by a pericellular protease and subsequently bind to the tumor cell. The payload will be released by either an extracellular protease or an intracellular (endosomal or lysosomal) protease. The objectives of this proposal are to design ADCs and pADCs that have superior plasma stability and improved release kinetics by cancer-associated proteases. Our long-term objectives are to develop pADCs with enhanced anti-cancer efficacy compared to current therapeutics. In Aim 1, we will generate a substrate specificity profile for proteases secreted by patient-derived organoids of breast cancer. In addition, we will isolate lysosomes and endosomes and characterize the proteolytic activity in these organelles. Finally, we will evaluate protease activity in serum and plasma to understand the circulating proteolytic activity. In Aim 2, we will develop ADCs and pADCs that are selectively activated by cancer proteases and stable in plasma and serum.